- Volume 1, Issue 1A, 2019

The genus Streptomyces is comprised of soil-dwelling Gram-positive Actinobacteria that are widely used for the industrial production of antibiotics. S. clavuligerus is used for the industrial production of clavulanic acid, which is a potent b-lactamase inhibitor, and is, therefore, able to restore the sensitivity of b-lactamase-producing bacteria penicillins and cephalosporins. In fermentations, the carbon sources available for utilisation by the producing organism have profoundly impact central carbon and specialised metabolic pathways. We have a long-term goal of using carbon sources from food waste to produce clavulanic acid with a view to developing more sustainable fermentations. To achieve this, the carbon utilisation profile of S. clavuligerus has to be diversified. Wildtype S. clavuligerus is a natural glucose auxotroph and has adapted to utilise glycerol most efficiently. It has been shown that the lack of glucose utilisation by S. clavuligerus is due to the insufficient expression of genes whose products are required for glucose uptake (glcP) and phosphorylation (glk). To enable glucose utilisation by S. clavuligerus strains, we have constructed strains for heterologous expression of either glcP or glk from different Streptomyces species. Further, the range of utilisable carbon sources for growth and clavulanic acid production has been investigated. Growth on solid media has revealed interplay between carbon and nitrogen metabolism, with extracellular protease production being regulated in a carbon source-dependent manner. Therefore, the role of protease secretion and its relationship with clavulanic acid production has been examined, revealing a complex role between carbon catabolite repression, protease production and clavulanic acid biosynthesis.

Acetogens are microbes which produce acetate as a fermentation by-product. They have diverse phylogeny but a metabolic feature in common called the Woods-Ljungdahl Pathway (WLP), which confers the ability to fix carbon dioxide via a non-photosynthetic route. Electrons for this process are derived from diverse substrates including molecular hydrogen and carbon monoxide. The ability of acetogens to utilise components of syngas (H₂, CO, CO₂) make them an attractive target for metabolic engineering for industrially relevant products. We have previously reported the construction of a genome-scale metabolic model of the model acetogen Acetobacterium woodii using a sequenced and annotated genome of strain DSM1030. The model consists of 836 metabolites, 909 reactions and 84 transporters and can account for growth on diverse substrates reported in the literature. We identified the reactions used to catabolise fifteen single substrates and 121 substrate pair combinations, and used this to construct a sub-model representing a core set of energy producing catabolic pathways. We then introduced heterologous reactions to allow for the production of chemical of interest. Elementary modes analysis of this extended sub-model was applied to further decompose it into unique sets of the smallest functioning sub-networks. With CO₂ and H₂ as substrates, we find routes for the production of several chemicals where small amounts of excess ATP are produced simultaneously. Repeated analysis with alternative renewable feedstocks such as methanol and formate, indicate a wider potential in producing compounds of interest while also maintaining energy generation and co-factor conservation.

The rapid expansion of global plastic production in the last number of decades (>355 million tonnes in 2017), coupled with poor waste management, has resulted in an estimated 5–12 million metric tonnes of plastic waste entering our oceans. Packaging applications account for much of the current waste production, and commonly include polyethylene- and polyethylene terephthalate (PET)-based materials that are resistant to natural degradation processes, particularly in marine environments. In response to this global marine pollution issue and the continuing demand for effective treatment of such plastics in terrestrial environments (e.g. 27.1 million tons of annual, post-consumer plastic waste in Europe alone), researchers have focused on addressing the biodegradation of recalcitrant plastic waste such as PET. To this end, we screened 20 Streptomyces spp. strains isolated from marine sponges for polycaprolactone (PCL)-degrading activity, which is considered a model substrate for PET. Although the Streptomyces genus is commonly explored for natural products discovery, little is still known about its potential for polymer degradation. Genomic analysis of two of the Streptomyces isolates which screened positive for PCL-degrading activity were found to have PET-hydrolase gene homologs that shared 41 % identity to the well-characterised PETase from Ideonella sakaiensis 201 F6. One of these genes was subsequently heterologous expressed in E. coli in order to further characterise the enzymatic activity and other biochemical properties of the enzyme.

The global brewing industry produces a large amount of waste, 85 % of this is composed of spent brewers’ grain. One use for this waste product is in the bioethanol industry where the yeast, S. cerevisiae uses the spent grain as a feedstock. Due to the nature of the feedstock, there is a lack of utilisable carbon for S. cerevisiae. To obtain optimum utilisation of the waste product in conjunction with high process efficiency, enhanced carbon metabolism of the production strain is required. As well as expanded nutrient utilisation there is also a requirement to maintain high ethanol production and ethanol tolerance that industrial strains have acquired in a preferred growth medium. We are using high-throughput phenotypic arrays to rapidly identify strains best able to grow in a wide range of conditions, including various carbon and nitrogen sources and multiple stress inducing conditions. This method has shown small but measurable differences between production strains in industrially relevant growth conditions. In collaboration with an industrial partner, both targeted and random chromosomal integration of transgenes have been made to multiple candidate production strains to improve recycled feedstock utilisation and process efficiency. In addition, whole genome sequencing is being utilised to interrogate the genetic basis for phenotypic differences between production strains. It has been found that some important null phenotypes are at the transcription level, this information is now in use to drive future rounds of genetic manipulation.

Municipal solid waste (MSW) production is projected to reach 3.4 billion tonnes per annum by 2050. The majority of MSW produced globally is incinerated or diverted to landfill, both methods which pollute the environment and contribute substantially to climate change. The organic fraction of MSW (OMSW) typically comprises ∼50 % lignocellulosic material and presents an abundant renewable feedstock for producing biofuels and chemicals. An important step toward OMSW valorisation is the identification of suitable microorganisms capable of fermenting this highly inconsistent, heterogeneous and complex feedstock. We have characterised the fermentation performance of eight biotechnologically relevant microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis) on enzymatic hydrolysate of OMSW fibre produced by a commercial autoclave pre-treatment. S. cerevisiaewas the most efficient ethanol producer, followed closely by Z. mobilis. Both species produced ethanol to high titre within 24 h, but neither could ferment xylose. The most effective performance was demonstrated by R. opacus, which consumed all available glucose and xylose concurrently over 72 h and produced a remarkably large yield of triacylglycerol (a biodiesel and aviation fuel precursor). This work demonstrates that OMSW is a promising renewable feedstock capable of supporting the growth several industrially useful microorganisms to high product titres. The best performing species identified here are interesting candidates to study further for application in a MSW biorefinery.

Biosurfactants produced from microbial sources are increasingly viewed by industry as more sustainable and less toxic alternatives to their chemically derived counterparts. One major class of biosurfactant that has the potential for commercial exploitation are the rhamnolipids. Rhamnolipids are composed of one or two rhamnose monosaccharides covalently bonded to fatty acid chains of varying molecular weights. The major microbial producer of rhamnolipid is Pseudomonas aeruginosa, however as this is a known human pathogen many industries are reluctant to utilise rhamnolipids synthesised by this bacterium. In order to avoid this problem a consortium of both academic and industrial partners have been screening marine bacteria for their ability to synthesis biosurfactants in a project called MARISURF. Here we report our findings of rhamnolipid production by two marine bacterial strains. Rhamnolipid production by these strains was first identified via the phenotypic screening of surface tension reduction. Rhamnolipid synthesis was then confirmed and characterised via HPLC-MS and NMR. Both 16S rDNA and subsequent genomic sequencing revealed these strains to be Marinobacter sp. and Pseudomonas mendocina, both species where rhamnolipid production was previously un-reported. Finally, both strains were assessed for potential pathogenicity using the Galleria mellonella model. Importantly for commercial exploitation, neither strain was shown to be harmful to G. mellonella over a 72 h infection period. Confirmed identification of rhamnolipid production in bacterial strains isolated from the marine environment highlights global oceans as an untapped resource in the ocean for the discovery of novel sources of biosurfactants.

Microbially-induced calcite precipitation (MICP) is ubiquitous in nature and has become an area of interest for environmental, geotechnical, and civil engineering applications. These include bioremediation, soil engineering, and self-healing of cementitious materials. To date, ureolytic bacteria have been favoured due to their ability to rapidly increase the pH of the environment through the hydrolysis of urea and thereby induce precipitation of calcite. However, the requirement for urea can contribute to nitrogen-loading in the environment and prove to be incompatible in certain applications, such as in self-healing concrete where it delays setting. Non-ureolytic bacteria are thought to be less efficient at MICP as they lack the ability to hydrolyze urea and thus to induce rapid increases in pH. Profiling of environmental bacteria has revealed the fundamentally different mechanisms that ureolytic and non-ureolytic bacteria utilize to precipitate calcite. These affect the timing of MICP and morphology of the crystals, but not necessarily the overall quantity of calcite precipitated. Furthermore, we show that MICP facilitated by non-ureolytic bacteria results in precipitates that contain significant organic components. These precipitates appear to have increased volume and cohesiveness, which may prove advantageous in application. Our findings offer important new insights into the use of MICP for geotechnical and environmental engineering and will enable us to create a toolbox of microbial precipitators tailored for specific applications.

Antimicrobial resistant bacteria can become harboured in sediments of post-industrial estuaries. Subsequently, their resistance traits could be enriched by pollutants deposited in the sediments. Recent evidence strongly suggests this may pose hazards that not only affects the health care sector, but could also impact tourism and the aquaculture industries. The River Clyde, UK was chosen for this study due to its extensive industrial history, and three sites were chosen to sample from representing different levels and types of industrial activities—two highly polluted and one relatively ‘pristine’ site. We extracted and analysed for metal pollutants (or ‘potentially toxic elements’, PTE), and other geochemical characteristics for all sediment cores. Gram-negative, enteric bacteria were isolated from all sediment cores from the three sites. Their susceptibilities to antibiotics and metals were assayed—determining minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The results indicate that co-selection of PTEs and antibiotic resistance does occur, and this impacts bacteria that are potential human pathogens. Higher concentrations of metals in the environment correlated to antibiotic resistance and higher MICs to metals than among bacteria found in less polluted sites. To continue to protect human health, the interactions between environmental and human health must be fully understood. This study provides critical information behind the specific causes of antibiotic resistance due to a legacy of pollution.

Predatory myxobacteria have an antimicrobial nature that dominates their interactions with neighbouring organisms. They are abundantly found in soil, water, dung of herbivores, and have the potential to significantly affect the microbiome of an environment. In this project, we hypothesize that potential prey organisms evolve in response to the selective pressure exerted by predatory microbes. Using a variety of nutrient media, we isolated bacteria from soil samples to test their susceptibility and resistance to the laboratory strain Myxococcus xanthus DK1622 and its predatory secreted outer membrane vesicles (OMVs). Soil (with and without heat treatment) was spread onto plates which had been pre-inoculated with myxobacteria, OMVs, or no pre-treatment. Plates with myxobacteria lawns or OMVs exhibited reduced diversity of isolates compared to control plates. The yield and diversity of isolates obtained also depended on the media used. Heat pre-treated soil gave rise to distinct morphologies and fewer slime producers. Co-existence and competition were exhibited by soil isolates, which were identified using 16S rRNA gene sequencing and phylogenetic analysis. Purified isolates were also characterised for their resistance and/or susceptibility to predatory attack by a variety of myxobacteria. The bacterial isolates obtained varied when exposed to seven different myxobacteria predators. Our data suggest that the addition of myxobacteria to isolation plates biases isolation towards relatively predation-resistant prey organisms. Our next goal is to isolate myxobacterial predators from the same soil samples (on different prey isolates) and test predator-prey interactions quantitative using pure strains. The genetic basis of differential predatory activity and prey susceptibility can then be investigated.

Currently, the surface of Mars cannot sustain liquid water, but there is evidence suggesting that water was present in the Noachian. Although water might exist in the subsurface of Mars, it could not sustain in the present day unless it was highly saline. Thus, saline springs in polar desert environments are analogues with which to investigate martian conditions. An example of this is Axel Heiberg Island, located in a region of continuous permafrost in the Canadian High Arctic, which hosts sulfidic and highly saline springs. In this study, cultivation-dependant and independent techniques were used to investigate the microbial diversity of a sediment sample collected from a saline cold (3–8 °C) pool at Colour Peak Springs on Axel Heiberg. Both DNA and RNA were extracted from the samples, and the microbial community was characterised using the 16S rRNA gene from the extracted nucleic acids. The metabolic profile was characterised by screening DNA and cDNA for functional genes relating to the cycling of carbon (coxL, xoxF, cbbL), nitrogen (nifH, nosZ, nod) and sulfur (dsrB, soxB). The community profiles were used to inform enrichment strategies, allowing for the isolation and characterisation of several halophilic isolates including strains of Marinobacter, Halomonas, Halanaerobium and Loktanella. Through this work we have been able to develop an in-depth characterisation of the metabolic and phylogenetic diversity that is present and viable within this analogue site. This allows us to start building an understanding of the underlying mechanisms and strategies that enable organisms to persist in these environments.

Understanding distribution patterns at various spatial scales is a central issue in microbial ecology. Beyond the lone identification of biogeographical patterns, understanding the environmental drivers behind community diversity and structure is key. While many studies identify pH as a major parameter structuring microbial communities at large spatial scales, many other variables impact distribution patterns on smaller scales. Here, we investigated the biogeographical patterns of Arctic soil microbial communities from 1 m to 500 m, within Adventdalen, Svalbard, using 16S sequencing, gravimetric measurements and X-ray fluorescence. Multivariate analyses identified key environmental variables shaping microbial communities and revealed the importance of soil moisture, organic carbon and elements such as aluminium, calcium and potassium in structuring distribution patterns. The indicator species analyses identified key associations between environmental variables and OTUs. Using geostatistical kriging, we mapped the biodiversity and distribution of key OTUs across the landscape. Overall, our results highlight the spatial heterogeneity in Arctic soils and identifies the sampling scale needed to characterize microbial communities within an area of interest with seemingly homogeneous landscape.

Commercial mushroom crops (Agaricus bisporus) are susceptible to a disease causedby a complex of 18 viruses known collectively as mushroom virus X (MVX). Symptoms of MVX infection vary from bare patches in crops to mushroom discolouration (browning). To understand the dynamic interaction between A. bisporus and MVX, we have studied five strains; including the globally-cultivated commercial strain, one wild strain, and one commercial-wild hybrid strain. Our transmission experiments using ‘healthy’ mycelium challenged with MVX-infected mycelium, detected MVX in the first day of hyphal anastomosis in all five strains. However, our commercial-scale crop experiment revealed varying degrees of disease tolerance in the fruiting body, the commercial strain being most susceptible and the hybrid strain most resistant to MVX. LC-MS/MS proteomics and RNA-seq analyses have elucidated key differences in response to both early and late crop inoculation of MVX. Quantitative shotgun proteomics of the susceptible commercial strain at late MVX inoculation revealed striking levels of proteins relating to mechanical membrane damage via detection of myo-inositol-associated biosynthetic proteins. Defense proteins relating to chalcone isomerase activity were also detected exclusively in the MVX-infected commercial strain. MVX-infected wild strain isolates showed significantly greater abundance of proteins related to fundamental cellular processes such as RNA polymerase activity and cell-redox homeostasis. Our findings show that although vegetative transmission of MVX is prevalent in all five strains, the fruiting body may oppose the infection in certain strains. Our findings of dynamic host response of A. bisporus to MVX, provides novel insights for this economically important, globally cultivated crop.

Ruminants depend on the highly diverse microbial community that resides in the rumen, the first and largest chamber of their digestive system, to gain nutrients from their herbivorous diet. The Hyper Ammonia Producers (HAPs) are obligate amino acid fermenting bacteria found in low numbers in this community. This break down of amino acids and peptides results in excessive ammonia production, as well as hydrogen and carbon dioxide, resulting in loss of nitrogen from the host and contribution to environmental emissions from enteric fermentation. Despite their large impact, little is known about the genomic underpinnings of the HAP phenotype. Our study employed comparative genomics and transcriptomics approaches to address this question. A phylogenetic tree of 498 rumen prokaryotic microbial genomes from the Hungate 1000 project (including 12 known HAPs) identified the HAP phenotype as polyphyletic, indicating independent origins or a result of horizontal gene transfer (HGT). However, following construction of sequence similarity networks for all genomes, few uniquely shared homologous genes families were apparent in the HAPs, suggesting that HGT did not drive their evolution. Instead, independent evolution of the phenotype is supported by similar functional analog profiles in the genomes of organisms with the HAP phenotype. Genome-wide characterisation and expression of functional analogs in known HAPS will allow in silico prediction of novel HAPs from the rumen which can be confirmed in vitro.

It has been well documented that antibiotic resistance (AR) is a clinical concern that affects both human and animal health but AR in the environment and food-chain is not as well understood. AR bacteria can occur naturally in soil, water and organic fertilizers used in agriculture so there is a risk that AR can pass to humans via the food-chain. This study focuses on lettuce cultivation undergoing four treatments (Normal irrigation water+normal soil, normal irrigation water+manure, UV irrigation water+normal soil, UV irrigation water+manure)to determine the mechanisms by which the AR is transferred to the plants over the growth period of the lettuce (7 time-points – week 0 to week 6). Plasmids (n=318) have been isolated from irrigation water (n=36), soil (n=45) and lettuce (n=42) samples using the exogenous isolation method for week 0 and week 6 initially. Antibiotic susceptibility testingto amikacin, cefotaxime, ciprofloxacin, imipenem, kanamycin, tetracycline has been carried out. Multi-drug resistance profiles were established for soil taken at timepoint 0 and lettuce taken at timepoint 6. Extracted plasmid DNA was sent for metagenomic analysis to determine which genes are involved in the transfer of AR at the interfaces. The results of the sequencing showed that there are multiple AR genespresent, including Tet, Sme, Cmy, Oxa and ANT(4’)-Ib, that confer resistance to bacteria. The identification of multi-drug resistance in soil and lettuce samples is concerning and highlights the need to determine the mechanisms leading to antibiotic resistance in food.

One potential design for a geological disposal facility (GDF) for intermediate level radioactive waste (ILW) involves the use of a cement base grout which will establish a highly alkaline environment for extended time periods [1]. Methane generation by colonising microbes could impact the long-term performance of the facility by influencing gas pressures and potentially leading to the migration of 14C to the biosphere [1]. Sediments acquired from a wide-range of anthropogenic alkaline sites in the UK were used to develop acetoclastic and hydrogenotrophic methanogen enrichment cultures over a broad range of pH values (7.0–12.0). The generation of methane from hydrogen and acetate was assessed to determine the dominant methanogenic pathways. Archaeal community analysis via Illumina MiSeq was employed to describe the populations involved and the acetoclastic inhibitor methyl fluoride was utilised to confirm the lack of acetate-dependent methane generation under alkaline conditions. High pH (pH>9.0) microcosms employing alkaline sediments were dominated by hydrogen-consuming methanogens of the orders Methanobacteriales and Methanomicrobiales, with no acetate consumption detected under these conditions. In contrast, neutral pH microcosms employing control sediments were dominated by acetoclastic methanogens of the order Methanosarcinales and demonstrated high acetate consumption rates. The rate of acetate consumption and proportion of acetoclastic methanogens decreased in a linear fashion as the pH within cultures was increased, however hydrogen consumption rates remained stable up to pH 11.0. The data shown suggests hydrogenotrophic methanogenesis is the dominant methanogenic pathway at high pH which could have important consequences on gas pressures within a GDF.

Nuclear power is an important energy source that can compensate for carbon emissions from fossil fuel power plants. However, processing of radioactive waste from nuclear plants is a significant challenge. The current treatment prior to final geological disposal involves wet storage of spent fuel in designated ponds, and microbial colonisation of these ponds can complicate plant operation. To help identify the key microbes that colonise hydraulically interlinked spent fuel storage ponds at Sellafield, UK, a series of samples were collected and analysed using next generation (Illumina) sequencing. Samples were taken from the facility’s indoor Fuel Handling Plant (FHP) pond (feeding head tank, main and subponds), and also from the open-air First Generation Magnox Storage Pond (FGMSP). 16S rRNA gene sequencing revealed that the FHP is colonized mainly by Bacteria (99%), affiliated with species of Curvibacter, Rhodoferax, Sphingomonas and Roseococcus, in addition to the hydrogen-oxidising bacterium Hydrogenophaga. In contrast the open-air FGSMP pond contained species of Hydrogenophaga, Nevskia, and Roseococcus, and also photosynthetic cyanobacteria (Pseudanabaena). Biological function was also assessed by metagenomic sequencing and analyses. The most abundant genes were associated with carbohydrate and protein metabolism, cell wall and capsule synthesis, stress responses and respiration. Genes involved in respiration were also more abundant in the indoor pond microbiome, including genes underpinning hydrogen metabolism, whilst photosynthesis genes were more abundant in the open-air ponds. These datasets give valuable insight into the microbial communities inhabiting nuclear storage facilities, the metabolic processes that underpin their colonisation and can help inform appropriate control strategies.

The type VI secretion system (T6SS) is a bacterial nanomachine utilised by many Gram-negative bacteria, including Acinetobacter baumannii, to deliver toxic effectors for microbial warfare. These toxic effectors are often delivered via specific non-covalent interactions with cognate VgrG proteins, which form part of the T6SS tip. In A. baumannii, each vgrG gene is usually located in the same locus as two other genes, one encoding the cognate effector and one encoding an immunity protein that protects against self-intoxication. Bioinformatic searches of ninety seven A. baumannii genomes using a highly conserved domain found within the VgrG proteins, enabled the identification of more than 250 genes encoding putative effectors and, in most cases, the gene encoding the corresponding immunity protein. Phylogenetic analysis revealed that the predicted effectors clustered into 33 distinct groups, some of which contained predicted amidases, chitinases, lipases, nucleases and deaminases. Two effectors, Tse5Ab, containing no toxic domains and Tse6Ab, containing a Tox-GHH nuclease domain characteristic of nucleases, were chosen for functional analysis. The C-terminal region encoding the predicted toxic domain of each effector was cloned and expressed in E. coli. Expression of this region of Tse5Ab did not perturb E. coli growth. In contrast, expression of Tse6Ab was toxic but toxicity could be neutralised by the co-expression of the cognate immunity protein. However, Tse6Ab did not exhibit DNase activity and instead may function as an RNase. Further characterisation of the diverse A. baumannii T6SS effectors may lead to the identification of antibacterial molecules with novel activities.

The microbiota is crucial for gut homeostasis by aiding in nutrient uptake, and protecting against pathogens. Recent evidence suggests the benefits provided by the microbiota are not restricted to the intestine but also extend to systemic sites. Systemic benefits are hypothesized to be mediated by bacterial products, derived from the microbiota, such as peptidoglycan and lipopolysaccharide, entering the bloodstream and acting as novel signalling molecules at distal sites. However the precise way in which these microbial products enter the bloodstream remains largely unclear. Our data suggest bacterial products can cross the intestinal epithelium, and that routes across may vary between different bacterial products. Using in vitro and in vivo models, we find that host processing of cell wall molecules, by host antimicrobial lysozyme, promotes their translocation across the epithelium. Once they have traversed the intestinal barrier our preliminary data provide support that the liver plays a role in clearing bacterial products from the blood, as here we see a reservoir of peptidoglycan. This increased dissemination of cell wall molecules additionally enhances resistance to pulmonary infection. Therefore lysozyme treatment enhances bacterial product migration and increases host protection against systemic pathogens. Our work provides mechanistic insight into how the gut microbiota exerts systemic effects. Furthermore it provides a basis on which to launch further investigations, including examining the influence these aggregated cell wall proteins have over innate immune cells at these sites. Giving us greater insight into how the host controls microbial signalling and the benefits provided to our innate immune system.

Antibiotic resistance is a serious threat to human health and new treatments for bacterial infections are urgently needed. Bacteriophages, first used at the beginning of the 20th century, and the predatory bacterium Bdellovibrio bacteriovorus (discovered in 1962) are potential alternatives to antibiotics. We developed a mathematical predator prey model to explore the effects of Bdellovibrio and bacteriophage on prey bacterial numbers. Our system has an abiotic resource that is consumed by the E. coli prey following Monod kinetics and up to two predator species with Holling type I or type II functional responses. As Bdellovibrio spends considerable time in the periplasm of its prey as a ‘bdelloplast’, this stage is also modelled, giving a delay between prey removal and ‘birth’ of predators. We used the model to examine the effects of Bdellovibrio and a bacteriophage on prey populations and found a distinct difference in effectiveness between Bdellovibrio and bacteriophages. We also looked at how various biological factors change predation effeciency. We found that there is an optimal predator:prey ratio for the predator. We also discovered that there is an optimal attack rate and an optimal mortality for the predator.

As the consequences of increasing bacterial resistance to traditional antibiotics become evident, the importance of research and development of therapeutic alternatives is apparent. Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host. However, whether and how bacteriophages can kill intracellular bacteria in a human cell environment remains elusive. E. coli strains displaying the K1 polysaccharide capsule virulence factor, are nosocomial pathogens responsible for urinary tract infections (UTIs), neonatal meningitis and potentialprecursors for septicaemia. These different types of infections were modelled in vitro by infecting human bladder epithelial cells (T24 HTB-4) and humancerebral endothelial cells (hCMEC/D3) with E. coli EV36, a strain displaying the K1 capsule. The infected human cells then received in vitro phage therapy using bacteriophage K1F that specifically targets E. coli strains displaying the K1 capsule. The rate of bacterial infection and the molecular and cellular mechanisms of in vitro phage therapy was analysed by means of flow cytometry, confocal and live microscopy. We show that rfp-tagged E. coli EV36-RFP and gfp-tagged bacteriophage K1F-GFP, enter the human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP can efficiently kill intracellular E. coli EV36-RFP in human urinary bladder epithelial cells and humancerebral endothelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy. Collectively this data contribute evidence-based knowledge for the ongoing development of phage therapy.

Staphylococcus aureus is one of the dominant organisms isolated from the airways of cystic fibrosis (CF) patients, particularly early in life, and is usually regarded as pathogenic. However, there remains significant gaps in our understanding of the role of S. aureus in the progression of pulmonary infection and lung disease in CF. Mouse models of S. aureus lung infection, even in CF animals, frequently demonstrate pneumonia and abscesses of the lung, a phenomenon very rarely observed in people with CF. Furthermore, live host models are associated with high costs and are limited in duration and sample size for ethical reasons. Most in vitro models fail to consider the influence of host tissue interaction or spatial structure on the development and persistence of infection. We have previously described an ex vivo pig lung model (EVPL) of cystic fibrosis for Pseudomonas aeruginosa lung infection. Here we show the progression of this model to support the growth of Staphylococcus aureus. Our data suggests that, in our model, S. aureus cells may preferentially aggregate in artificial sputum rather than adhere to lung tissue. In the context of historical case reports, this result potentially reflects the clinical situation in cystic fibrosis more accurately than mouse models and could have substantial clinical significance.

Aging can be defined as an accumulation of damage, or a loss of function, with increasing age. For bacteria, it has generally been assumed that the mechanism used to cope with aging is the asymmetric segregation of damage at division, so that all of the damage is inherited by one cell and the other is therefore rejuvenated. Another, often neglected, mechanism is to repair the damage; our previous computational modelling work has found that an optimized, fixed rate of repair is fitter than damage segregation in well-mixed environments such as chemostats. The predominant mode of growth for bacteria is in biofilms, however, and here we investigate aging in biofilms using the individual-based model iDynoMiCS. In addition to the previously used damage segregation and fixed repair strategies, we introduced adaptive repair: sensing the current damage levels within the cell and responding to this by investing in damage repair machinery. We found that the optimal method for dealing with cellular damage varies with the environment being investigated. The investment of additional resources into adaptive repair is only beneficial when competition is sufficiently strong, in the chemostat and in biofilms, and the speed at which the fittest strategy becomes apparent depends upon the initial density of cells. When the bacterial cells are dense initially, and thus the competition between strategies is stronger, the adaptive repair strategy emerges as the winner much more rapidly.

Candida species are commensal yeasts but are also responsible of life-threatening infection in at-risk populations, like new-born or immunocompromised patients. Candida albicans is the most common causative species, and the most studied. Moreover, non-albicans Candida species as Candida glabrata, Candida parapsilosis and Candida tropicalis cause a large proportion of infections. In our study we investigated the interaction between four Candida species and human vaginal epithelial cells A431 by using a dual RNA-seq method. Our aim is to identify the different transcriptomic response of each yeast, and of the host, during the infection of human cells. Gene Ontology analysis of up-regulated genes in the yeasts during infection implicated the ergosterol (ERG) pathway in C. parapsilosis only. We therefore investigated the role of the ERG pathway in the three Candida species in which it is currently possible to generate gene knockouts. The transcriptional factor Upc2 is the main regulator of ERG gene expression in C. albicans and C. parapsilosis. C. glabrata has two UPC2 orthologs, called CgUPC2A and CgUPC2B. We found that deleting CgUPC2A or CgUPC2B or both together does not reduce the damage inflicted by C. glabrata on host cells. However, deleting UPC2in C. albicans greatly reduces damage. Deleting UPC2 in C. parapsilosis appears to reduce damage of host cells; however further investigation is required. Our results show the that the role of ergosterol pathway in the host pathogen interaction differs between Candida species.

MGB-BP-3 (MGB) is a novel synthetic antibiotic inspired by Distamycin – a natural product that is capable of binding to the minor groove of DNA. MGB has a high bactericidal activity against a broad range of Gram-positive bacteria without the toxicity associated with the natural products that it was inspired by. Its oral formulation, developedfor the treatment of Clostridium difficile infections, is currently progressing through a phase 2 clinical trial. This study investigatesthe mode of action of this novel antibiotic. To allow better understanding of MGB’s mode of action, RNA-Seq analysis was undertaken on S. aureus following challenge with 0.5 x MIC (0.1 µg ml−1) MGB-BP-3. Triplicate samples of RNA were extracted at 10 min after challenge. RNA-Seq analysis identified 698 transcripts showing significant changes in expression profile, which were confirmed by quantitative RT-PCR. Amongst these, 62 essential genes showed transcriptional arrest. Glycolysis, pentose phosphate pathway and the TCA cycle were affected. In addition, biosynthesis of nucleotides and certain amino acids were altered and Biolog phenotype arrays were performed in the presence of MGB to confirm this. DNA binding assays demonstrated MGB binding to intergenic regions upstream of strongly down-regulated essential genes (mraY and dnaD). Attempts to evolve resistance to MGB have so far been unsuccessful unlike with the rifampicin control. In conclusion our findings are consistent with a bactericidal mode of action of MGB at the transcriptional level of multiple essential genes.

The human gut microbiota is complex, dense, and hugely influential to health. Imbalances in the microbiota have been associated with numerous disease states, in many cases due to the overgrowth of Enterobacteriaceae, such as Escherichia coli. We describe an oligonucleotide antimicrobial that selectively reduces levels of Enterobacteriaceae in vitro, in a model of the human colonic microbiota, and in vivo in a murine study, whilst leaving the core microbiota intact. The antimicrobials are Transcription Factor Decoys (TFDs) that bind to and competitively inhibit an identified transcription factor necessary for growth in the intestine. This is highly conserved amongst Enterobacteriaceae and controls anaerobic respiration and response to nitrosative stress caused by the innate immune response of the host. A nanoparticulate formulation delivers the TFDs to the cytoplasm of E. coli, as visualized by confocal microscopy, and rapidly kills the bacteria under microaerobic conditions. When applied to anin vitro model of the human intestinal microbiota the TFD produced a decrease up to log10 6 c.f.u. ml−1 in coliforms within the Enterobacteriaceae family while other families remain intact. When delivered orally to the intestines of mice similar results were seen: Enterobacteriaceae were decreased or cleared from the wild-type intestinal microbiota while the remaining bacteria were unaffected. This demonstrates that TFDs can be used to make precise changes to the microbiota and has utility in testing associations between dysbiosis and disease and developing microbiota targeted therapeutics.

The long-term reproductive success of a lineage depends on its ability to tolerate a wide range of environmental conditions and for a virus a substantial part of its environment is the host that it infects. Viruses may overcome limitations of a given host environment by switching to other available hosts. Experiments addressing host switching may pave the way for an improved understanding of the emergence of new viral diseases. Here, a model bacteriophage, φX174, its canonical laboratory bacterial host strain, Escherichia coli, and a novel host, Salmonella Typhimurium were employed. Bacteriophage φX174 adaptation was studied at population level in a bioreactor. We analyzed phenotypes and genotypes arising during continuous evolution of φX174 on alternating hosts for four consecutive periods of 10 days each. The fitness and adsorption of each viral population were measured using qPCR in liquid culture. Deep sequencing analysis of isolates was carried out to determine the genetic basis of pleiotropic costs and to characterize allelic variations occurring during growth. Some alleles specific to one host were lost or reduced in frequency in the alternative host while other alleles (not present in the ancestor) were shared between hosts. The fitness effects of specific alleles were examined in isolation through targeted mutagenesis. This work contributes to a better understanding of some of the general constraints, costs and benefits influencing the evolution of parasite populations as they adapt to the complexities of a novel host environment- a key consideration during the emergence of infectious diseases.

Campylobacter spp. is a leading cause of foodborne illness globally. The pathogen colonises the gastrointestinal tract of the host, where small concentrations of neuroendocrine hormones are also secreted. Epinephrine and norepinephrine are neuroendocrine hormones involved in the stress response that have been shown to promote the expression of virulence factors in pathogens including E. coli, Salmonella spp., and Campylobacter spp. In our study Campylobacter jejuni strains from human infection and broiler farms that were supplemented with epinephrine and norepinephrine showed increased growth characterised by shorter lag phases and higher maximum OD595, and enhanced pathogenicity characterised by increased motility, attachment to and invasion of Caco-2 cells. The data obtained suggests that host stress may promote C. jejuni proliferation and pathogenicity.

Streptomyces is a genus of soil dwelling actinomycetes that play an important role in plant health through association with plant roots. They provide an array of benefits to the plant host such as infectious disease prevention and plant growth promotion. We investigate the role of nitric oxide (NO), a ubiquitous signalling molecule used by plants and bacteria alike, in root colonisation by Streptomyces coelicolor. Plating studies were conducted for Arabidopsis thaliana and Triticum aestivum. Relative colonisation was determined by comparing selective recovery of marked mutant strains, alongside a marked control. The effect of increased endogenous NO was interrogated with a deletion mutant of nsrR-hmpA – genes responsible for NO detoxification in Streptomyces. Strains were also engineered to express recombinant NO synthase genes, to investigate the impact of NO production by the bacteria. We show that S. coelicolor ΔnsrR-hmpA is significantly more competent at colonising T. aestivum rhizosphere compared to the control (P<0.005). Endosphere colonisation is sporadic for both mutant and control, this observation is supported by fCLSM imaging. Preliminary data indicates that the Streptomyces strains engineered to express NO synthase at high levels, colonise poorly. This suggests that NO is a dynamic and finely tuned signalling molecule. We are excited to present promising new evidence to support an as yet undescribed link between NO and plant root colonisation by Streptomyces coelicolor. Understanding the mechanisms that underpin this process is the first step in exploiting these interactions for agricultural technology.

The study of animal digestive tracts reveals important information on the host’s health status. For livestock, being able to predict the effect of different treatments on the gut microbiome has important implications for increased sustainability, enhanced animal welfare and increased food safety. However, gut contents can be investigated only after the slaughter of the animal, but cloaca/rectal samples may be collected from live animals and reduce the number of animals killed for experimental purposes. The aim of this study is comparing the microbial communities of caecum and cloaca associated with eight poultry broiler flocks from two English farms. 16S amplicon libraries were run on a MiSeq with a 250 bp PE read metric. The data were evaluated with in qiime1 and qiime2. Comparisons of bacterial communities of cecum and cloaca revealed they are significantly different in terms of the number and types of bacterial species, as well as their abundance (P-value Indicator species analysis of cecum samples showed the class Bacilli were enriched, while Clostridia had greater prevalence in cloaca. Finally, no pathogenic bacterial species of poultry were identified in the analysed animals. Despite the fact sampling cloaca content could be a method to reduce cost and suffering for research purposes, this study reveals the limit of the use of cloacal microbiomes to provide a window into poultry alimentary canal microbiomes.

Anti-virulence strategies are an alternative approach to combat zoonotic bacterial pathogens. Although control measures are in place against E. coli O157, it still remains a global health concern with cattle being the most important reservoir. Different feed constituents have varying effects on the virulence of E. coli O157 present in the gut which then are transferred to meat processing surfaces and meat during slaughter. This study explored the anti-virulence properties of a mixture of natural plant extracts and organic acids (citrus, grape and oregano extracts, lactic and citric acid) before and after refrigerated storage as means of reducing the risk from E. coli O157 by reducing its virulence. Assessment of the effects of sub-inhibitory concentrations (0.1 and 0.5 %, v/v) of the antimicrobial mixture before refrigerated storage showed that the pathogen’s motility and adhesion onto HCT-8 cells was significantly reduced (P<0.05) in a dose-dependent manner. Shiga-toxin 2 production was also significantly reduced. Real-time PCR analysis revealed that the mixture of natural antimicrobials repressed expression of adhesion (eae) and shiga-toxin 2 (stx2) genes, which was consistent with the observed reduction in adhesion and toxin production. The same virulence factors were investigated after simulated storage of E. coli O157 in meat simulation medium. Results revealed that after exposure to the antimicrobial mixture, the virulence was significantly lower compared to non-treated control after refrigerated storage. The present work shows the potential of the antimicrobial mixture in reducing virulence and thus risk from E. coli O157 by applying it as a feed additive.

Excess application of Nitrogen (N) to agricultural soils can lead to environmental pollution. As nitrous oxide (N₂O) is a potent greenhouse gas, it is of critical importance to reduce its emission for climate change mitigation. Availability of nitrogen can facilitate denitrification, an anaerobic respiratory pathway carried out by microbial communities in which N₂O is an intermediate product. An understanding of soil, climatic and edaphic factors influencing microbial communities and their activity is key to reducing N₂O emissions. Soil pH strongly impacts microbial community structure, with a direct effect on NosZ, the pH-sensitive enzyme catalysing N₂O reduction. We thus expect that microbial communities in acidic soils have a reduced capacity to mitigate N₂O emissions. It is likely that other management factors, like phosphorus application, interact with pH; causing changes to chemical nutrient availability and direct effects on microbial composition. The complexity linking N₂O emissions and microbial activity (impacted by soil pH), and the interacting role phosphorus availability plays in this relationship, is not yet understood. In this study, the capacity of microbial communities to denitrify, as well as the functional microbial community impacted by soil pH, were analysed by potential denitrification assays, measuring N₂O fluxes and N₂O/(N₂O+N₂) ratios; and qPCR analysis of denitrification genes. This was investigated across two soil types with a pH gradient and range of phosphorus application rates. Understanding the link between the microbial communities and N₂O production can be applied in agricultural management to reduce emissions from fields.

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.

Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress conditions [1]. A phylogenetic analysis of multiple WHy homologues from different species suggested that the ancestral origin of this protein gene lies within the ancient archaea [1]. Our previous studies have shown that this bacterial protein elicits significant protection against freeze and cold stress in recombinant E. coli [2]. Expression of the WHy gene in Arabidopsis resulted in a wide range of statistically significant stress-tolerant phenotypic properties. These included an increase of up to 6-fold higher germination efficiency of transgenic recombinant seeds compared to the WT, and a 100 % survival rate of WHy gene-expressing plants compared to 0 % survival of adult WT plants after freeze shock. Similar improvements in survival rates were observed for recombinant plants in drought stress experiments.

Commercial poultry rearing systems often house successive flocks of birds with limited between-flock cleaning of the poultry houses. Previous research focused on opportunities for successive flocks to become colonised with pathogenic bacterial species. However, there is a paucity of information regarding the transfer and persistence of commensal bacterial between flocks, and if this might confer health benefits on subsequent flocks. The work presented here utilised 16S community sequencing to characterise the development of the microbial flora of commercially reared broiler chickens and turkeys to determine microbial environmental persistence. DNA was isolated from caecal contents, faeces, and various bedding samples collected from nine sites over a nine month period. Samples were taken from houses used for rearing chickens or turkeys or where alternating chicken and turkey flocks were reared. Measures of alpha diversity for the different samples suggested that both chickens and turkeys had a similar microbiota. Unsurprisingly, members of the microflora could also be found in the environmental samples tested, although survival was dependent on the phyla and bedding material. Further analysis of the samples is currently underway, in order to determine the extent, if any, of microbial transfer between flocks, with a particular focus on potential poultry microbiota species differences. This study demonstrated how commensal microbes are able to persist within poultry rearing sheds and if this transfer impacts on subsequent flock performance. Alongside increasing understanding of microbial environmental persistence, the work also shows how effective current biosecurity methods are in controlling the transfer of all microbes, including pathogens.

The overall value of Irelands beef exports is worth approximately €2.5bn which is an annual increase of 5 % in 2017. However, the beef industry faces many challenges to export products to distant markets including a short shelf life and other economic losses that are mainly caused by microbial contamination. One of the simplest approaches to limit this contamination on the surfaces of beef carcasses is to use alternative carcass chill regimes. Carcasses underwent an industry standard chill process (10 °C for 10 h followed by 0 °C for 38 h) and were compared with carcasses that underwent a more rapidly chilled process (0 °C for 5 h and −2 °C for 3 h). Bacterial concentrations (mesophilic and psychrophilic total viable counts, total Enterobacteriaceae counts, Lactic Acid Bacteria, Pseudomonas spp., Brochothrix thermosphacta, Clostridium spp.), physiochemical (pH, temperature, water activity (aw)) and organoleptic (colour, odour, texture) changes were monitored throughout the entire beef food chain (carcass → primal → retail steak) until end of shelf life. Rapidly chilled carcasses had significantly (P<0.05) less bacterial surface contamination compared to conventionally chilled carcasses. There was also significantly (P<0.05) less evaporative loss on carcasses and primals which will have a positive economic impact on the beef industry. This longitudinal study is one of the largest trials ever performed on beef shelf life extension.

Phoma stem canker is one of the most important diseases of winter oil seed rape (Brassica napus) world-wide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and Leptosphaeria biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double stranded (ds) RNA virus in L. biglobosa and no viruses in L. maculans. The virus forms isometric particles ca. 40–45 nm in diameter and has four genomic segments, each possessing a single open reading frame flanked by untranslated regions. Phylogenetic analysis revealed modest similarities to known and suspected members of the family Quadriviridaeand therefore the virus was nominated Leptosphaeria biglobosa quadrivirus-1. Following eradication of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance towards L. maculans in B. napus following lower leaf pre-inoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defences. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of phoma stem canker later in the growing season.

Recent landmark studies indicate that the nucleolus plays key roles in stress responses including the DNA-damage response (DDR). The latter involves interactions of components of the DDR machinery including NBS1 with the sub-nucleolar protein Treacle, a key mediator of ribosomal RNA (rRNA) transcription and processing, implicated in Treacher-Collins syndrome. Using comparative proteomics, confocal and single molecule super-resolution imaging, and infection under BSL-4 containment, we have shown for the first time that the nucleolar DDR pathway is targeted by infectious pathogens [1]. We found that the matrix (M) proteins of Hendra virus and Nipah virus, highly lethal viruses of the Henipavirus genus (order Mononegavirales), target Treacle to inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhanced henipavirus production. These data identify a novel mechanism for viral subversion of host cell biology by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intra-nucleolar function for proteins of any mononegavirus [1, 2]. For the presentation I will discuss our new data, which is advancing our understanding both of the mechanisms impacted by the Henipavirus-Treacle interaction, and potential roles of such interactions in infection by other viruses, including highly lethal lyssaviruses [3].

[1] Rawlinson et al. Nature Communications 2018.9 : 3057 (2018)

[2] Rawlinson et al. Cellular Microbiology 2015. 17(8):1108–20

[3] Oksayan et al. Journal of Virology 2015.89(3):1939–43

The mechanism by which non-enveloped RNA viruses, such as the caliciviruses, escape the endosome, is poorly understood. The Caliciviridae are a family of viruses which include many important human and animal pathogens, most notably norovirus which causes winter vomiting disease. We used cryoEM and asymmetric three-dimensional reconstruction to investigate structural changes in the capsid of feline calicivirus (FCV) that occur upon virus binding to its cellular receptor; feline junctional adhesion molecule-A (fJAM-A). We discovered that following receptor engagement substantial conformational changes in the FCV capsid lead to the assembly of a portal-like structure at a unique three-fold symmetry axis. Atomic models of the major capsid protein, VP1, in the presence and absence of fJAM-A were calculated, revealing the conformational changes induced by the interaction. In the course of this analysis we discovered a large portal-like structure which assembles at a unique three-fold axis. The portal-like complex comprises 12 copies of the minor capsid protein VP2. We calculated an atomic model of VP2 and revealed structural changes in VP1 that lead to the formation of a pore in the capsid shell at the portal vertex. VP2 is encoded by all caliciviruses although despite being critical for the production of infectious virus, its function and structure were, until now, undetermined. We hypothesise that the VP2 portal-like complex is the method by which the virus escapes the endosome during virus entry, allowing delivery of the viral genome into the cytoplasm for replication to then ensue.

Arboviruses constitute a major public health problem, in particular mosquito-borne arboviruses that continue to emerge and re-emerge. Arbovirus infection of mammals is enhanced by the presence of a mosquito bite at the inoculation site, in comparison to virus experimentally administered by needle inoculation in the absence of a bite. Inflammatory responses to bites appear to be a key factor in this enhancement. However, the experimental inoculation of mosquito saliva with virus inoculum by needle, in the absence of bite trauma, also has the ability to enhance viral infections. In this study, we have studied the mechanistic basis for these observations. We have studied whether saliva from different mosquito species can enhance virus infection. Interestingly, while saliva from Aedes genus enhanced virus infection, An. gambiae saliva did not. Instead, An. gambiae saliva actively inhibited infection compared to inoculation with virus alone. This could partly explain why An. gambiae mosquitos are unsuitable vectors for transmitting most arboviruses. By comparing the effects that saliva of these different mosquitoes have at the bite site we have further specified which inflammatory responses at the inoculation site modulate arbovirus infection in the skin. Using an in vivo mouse model we have shown that An. gambiae causes significantly less oedema but a higher up-regulation of key inflammatory genes in the skin than A. aegypti. As such, we are providing important insights into how mosquito saliva modulates infection. A better understanding of this will aid the development of anti-viral treatments by targeting factors within the mosquito bite that are common to many distinct infections.

Human cytomegalovirus (HCMV) rapidly mutates during in vitro passage, and this strongly alters the way the virus spreads. In vivo HCMV spreads by direct cell-cell contact, as do recent clinical isolates. In contrast, passaged strains spread via cell-free virions. Because of this, cell-cell spread remains largely uncharacterised. We have developed a strain (Merlin) that retains a full length, wildtype genome. As a result, it mimics clinical HCMV and spreads by direct cell-cell contact, a method of spread that is more resistant to neutralising antibodies, and innate and intrinsic immunity. We now show that each cell-cell transfer is equivalent to an extremely high MOI infection, with up to 300 genomes delivered to each cell, potentially providing an explanation for the ‘immune-evasive’ properties of cell-cell transfer. Furthermore, infectious virions accumulate at cell-cell contacts between cells. This may represent a ‘virological synapse’ that protects virions from neutralising antibodies. Not only does Merlin enable us to characterise cell-cell spread, but it enables us to infect a wide range of clinically relevant cells with a virus expressing the complete complement of virus genes. In vivo, HCMV infects dendritic cells (DCs), but is never cleared, implying that it is able to subvert DC function. Therefore, we performed quantitative proteomic analysis of infected primary immature DCs, following cell-cell transfer. This quantified 7992 intracellular proteins, and 703 plasma membrane proteins. Over 99 proteins were downregulated following infection. Many of these are DC-specific, and have roles in regulating adaptive immunity. These viral-manipulations may therefore dramatically impact DC function.

Zika virus (ZIKV) is an arbovirus (family Flaviviridae) mainly transmitted by Aedes mosquitoes causing febrile illness and Zika congenital syndrome in infants if mothers were infected during pregnancy. ZIKV manipulates its host’s cellular machinery in order to facilitate infection and evade antiviral responses. The identification of host and vector proteins involved in these processes may lead to novel antiviral strategies. In this study, Ae. aegypti cell lines (AF5) stably expressing V5-tagged ZIKV capsid (C) or anchored capsid (AC) proteins were developed to investigate virus-vector protein interactions. To identify interaction partners, immunoprecipitation (IP) of V5-tagged C or AC was performed and subjected to proteomic analyses using nLC-MS/MS under label-free quantification conditions. A total of 148 and 53 mosquito protein interactors unique to C and AC were identified, respectively. Protein network and gene ontology analyses showed biological processes possibly important for ZIKV infection. To investigate further the role of these proteins during infection, 25 were chosen for dsRNA-based knockdown screen and infection with reporter virus (ZIKV-Nluc) in AF5 cells. Significant reduction in reporter virus signal was observed during knockdown of 6 interactors suggesting a pro-viral role for these proteins during infection. This was corroborated by conducting the same knockdown experiments but infecting with a clinical isolate of ZIKV (PE243), which showed reduced virus RNA levels and titre. Interestingly, three of the six proteins are part of the ubiquitin-proteasome pathway (UPP). Currently, functional experiments are underway to investigate the role of UPP during ZIKV infection.

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.

Human Cytomegalovirus (HCMV) is a master immune regulator, encoding multiple proteins that modulate a variety of immune signalling pathways. We previously performed a systematic proteomic analysis of temporal changes in host and viral proteins throughout the course of infection and determined that HCMV downregulates >900 host proteins. HCMV is the largest human herpesvirus, potentially encoding hundreds of ORFs. Identification of which individual gene targets a given cellular factor can therefore be challenging. To facilitate the mapping of viral gene functions, we employed a panel of HCMV mutants, each deleted in contiguous gene blocks dispensable for virus replication in vitro. Three proteomic screens of these mutants were performed, with each mutant represented in at least duplicate. From these data we have defined the genetic loci responsible for targeting >250 host proteins. Bioinformatic enrichment analysis on the targets of each mutant virus enabled attribution of novel functions to blocks of uncharacterised genes. Our approach was validated from analysis of the US1-11 genetic locus, which confirmed that the major function of US1-11 genes is the regulation of MHC class I molecules and other cell surface receptors. The data also suggests that the major functions of the poorly characterised blocks RL1-6 and US29-34A are the regulation of secreted proteins and the regulation of a family of cell surface adhesion molecules respectively. Overall this approach can be used to gain global insights into HCMV gene function, the study of which has previously been only been possible on a single gene basis.

Herpesviruses are large and complex DNA viruses that are composed of an icosahedral capsid, a proteinaceous layer termed the tegument, and a glycoprotein rich lipid envelope. One important area of host-pathogen interaction that is still poorly understood is the extensive change to intracellular organelles and cellular morphology that occur within the infected cell during active virus replication. In order to characterise the spatiotemporal dynamics of host cell remodelling caused by herpesvirus infection, we use novel multiparametric fluorescence microscopy methods compatible with live-cell imaging. In addition, we apply expansion microscopy to map 3D rearrangement in great detail. The remodelling of the host cell is correlated to the stage of virus replication which can highly vary between individual cells. Therefore, we have constructed a recombinant reporter virus that expresses eYFP-tagged ICP0, a multifunctional immediate early tegument protein, as well as mCherry-tagged glycoprotein C (gC), a late protein that is a major component of the viral envelope. The sequential expression of these two viral proteins provides us with an intrinsic time stamp for the stage of virus infection in each cell. With this fluorescent reporter virus, we are able to describe the remodeling of- the three-dimensional architecture of microtubules and the actin network,- compartments of the secretory and endocytic pathways which are intimately linked to viral envelope protein synthesis, maturation and transport,- and key antiviral and inflammatory signalling platforms (mitochondria and peroxisomes).

The predominance of specific bacteria within the Crohn’s disease intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Here we show that the intestinal short chain fatty acid propionic acid stimulates increased ethanolamine degradation by one such Crohn’s disease associated pathogen, adherent-invasive Escherichia coli (AIEC). This degradation occurs within bacterial microcompartments that are subsequently excreted in outer membrane vesicles. Additionally ethanolamine, added extracellularly at concentrations comparable to those in the human intestine, is accessible to intracellular AIEC and stimulates significant increases in growth within macrophages. Finally, expression of the operon for ethanolamine degradation (eut) is increased in children with active Crohn’s disease compared to healthy controls. After clinical remission was seen with exclusive enteral nutrition treatment, Crohn’s disease patient’s exhibit significantly reduced eutexpression. Our data indicates a role for ethanolamine metabolism in facilitating AIEC colonization of the Crohn’s disease intestine and warrants further study of its potential use as an indicator of inflammatory status in Crohn’s disease.

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infection for animals and plants. Our investigation is focussed on a current and growing concern: the distribution and formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures around 30 to 40 °C degrees rather than at higher temperatures above 60 °C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa into the environment. P. aeruginosa is one of the predominant bacteria found in washing machines and is highly resistant to many antibiotics. Little is known about environmental microniches present in biofilms. In this work, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilm formation. Here, we use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to variation in pH. Confocal laser scanning microscopy revealed that the nanosensors can penetrate biofilms within minutes and interact with the biofilm structure. Different washing detergents were tested resulting in altered biofilm formation and killing abilities. Using time lapse imaging, pH changes were tracked in real time at a microcolony and single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms, underpinning the development of more effective antimicrobials to limit the emergence of AMR.

Lyme disease (LD) is a multisystem infection caused by tick-borne spirochaetes of the Borrelia burgdorferii sensu lato group. UK and US laboratory diagnosis of LD involves the two-tier serological approach. The negative predictive value of the test has been challenged, particularly in early stage LD. There is considerable interest, therefore, in the development of improved diagnostic tests. The main aim of the project is to identify new markers that could form the basis for improved tests. A mass spectrometry biomarker discovery study was undertaken on LD positive and negative residual diagnostic samples from UK LD testing by Public Health England and a cohort of patient samples from collaboration with a research group in the Czech Republic. A ‘related-disease control group’ including serum samples from syphilis, leptospirosis and chronic fatigue syndrome was also included. Several proteins were found at a significantly higher or lower in abundance in the ld-positive patients compared with ld-negative. Of particular interest was Lipocalin-2 (LCN2), a protein involved in immunity. LCN2 has previously been found in increased abundance in mice exposed to B. burgdorferi. Further analysis of LD samples using Illumina RNA sequencing revealed further markers of interest. Transcriptomic analysis including Ingenuity Pathway Analysis (IPA) gave insights into the host response to LD infection. Proteins of interest from proteomic and transcriptomic analysis were taken forward for further analysis by WB or ELISA in a larger sample set.

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.

Cryptococcus neoformans is an opportunistic fungal infection that causes cryptococcal meningitis in immunocompromised individuals. Macrophages play a critical role in determining the outcome of infection, and can either phagocytose and kill the cryptococcal cells, or disseminate infection. While it is known that macrophages impact the progression of cryptococcal disease, it is not known how the macrophage intracellular niche contributes to complex infection outcomes. Clinical and experimental studies have identified potential genetic differences, in both host and pathogen, but statistical robustness has been difficult to achieve due the large variability in the outcome of infections. Therefore, in attempt to quantitatively explain this variability, we have used a zebrafish model of cryptococcal infection where we can directly relate the initial level of fungal infection with final infection outcome. We find that at low levels of initial fungal burden the outcome of infection is stochastic, while over high ranges of initial infection the outcome is linearly related to the initial burden, but with a further stochastic component that contributes to increased variability. Using these experimental data and data from clinical trials we have generated a computational simulation of in vivo cryptococcal infections which allows us to consider different infection variables and how they alter the progression of cryptococcosis. By combining such computational simulations with our experimental models we demonstrate that the macrophage intracellular niche determines the unknown stochastic component, independent of fungal burden. Using this knowledge, we can better identify the molecular, population genetic, and clinical parameters associated with the outcome of cryptococcosis.

Apicomplexans are human and animal protozoan pathogens responsible for diseases including malaria, cryptosporidiosis and toxoplasmosis. As obligate intracellular parasites they are highly organised cells with numerous novel and specialised sub-compartments that form the basis of their invasion biology, host defence evasion, and novel metabolic traits. However, our understanding of these cells is highly constrained by our limited knowledge of the locations and functions of most of the cell’s proteome. Even in the best-studied apicomplexans (Plasmodium spp. and Toxoplasma gondii) only a small fraction of proteins’ locations have been experimentally determined, with most assignments based on predictions from orthologues in distant relatives. Moreover, many parasite proteins are annotated as ‘hypotheticals’, for example 4113 of 8121 Toxoplasma proteins, and many are unique to parasites stymying even predictions of location or function by comparative biology. To address this deficit in our basic understanding of the compositional organisation of the apicomplexan cell, we have used a spatial proteomics method called hyper LOPIT to simultaneously capture the steady-state subcellular association of thousands of proteins in the apicomplexan Toxoplasma. These protein atlases reveal: extensive protein association networks throughout the cell providing testable hypotheses of their function; conservation and novelty of compartment proteomes between apicomplexans; differential selective pressures across the different cell compartments; and clear instances of protein relocation from one organelle to a different one during apicomplexan speciation. This new, global view of the organisation of the apicomplexan cell proteome provides a much more complete framework for understanding the mechanisms of function and biology of these cells.

Mechanosensitive channels are required for bacteria cells to survive hypoosmotic shock (transition from high to low osmolarity environment). YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. Previous studies have suggested that S. Typhimurium YnaI may be implicated in host colonization during infection of farmed animals. Disruption of ynaI impaired intestinal colonization in pigs, cattle and chicken. To investigate S. Typhimurium YnaI structure and function, the S.Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the S. Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. To further understand the role of YnaI in S. Typhimurium pathogenesis, S. Typhimurium ynaI was deleted and the ability of mutant to survive and replicate in host cells was investigated. The S. Typhimurium YnaI channel has unique characteristics when expressed in E. coli: S. Typhimurium YnaI channel conferred almost complete protection against 0.3 M NaCl hypoosmotic shock when overexpressed, but interestingly, high level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media. Deletion of ynaI from S. Typhimurium led to increased internalization in macrophages and epithelial cells. Data derived from this study reveals novel characteristics of S. Typhimurium YnaI which may provide insights into other functions of the S. Typhimurium YnaI channel.

The biocide triclosan is used extensively in household and hospital settings, resulting in chronic exposure to the biocide in individuals that use triclosan-containing products. Triclosan is thought to induce antibiotic tolerance and alter biofilm formation, although the underlying mechanisms causing these changes are yet to be elucidated. If true, the widely used biocide may contribute to antibiotic treatment failures, and therefore requires investigation. To determine how triclosan induces antibiotic tolerance, Staphylococcus aureus was pre-treated with triclosan prior to treatment with the clinically relevant antibiotics ciprofloxacin, rifampicin, and vancomycin. Planktonic S. aureus cultures pre-treated with triclosan had 1000 fold higher viable counts compared to non triclosan pre-treated cultures. Inspection of biofilms by live/dead staining found that triclosan pre-treatment protected S. aureus biofilms from treatment with otherwise lethal doses of ciprofloxacin, rifampicin, or vancomycin. Biofilms of mutant strains with a defective stringent response were not protected from antibiotic treatment, even in the presence of triclosan. Interestingly, stringent response mutants still exhibited triclosan-induced antibiotic tolerance in planktonic culture, but mutants with a defective agr quorum sensing system did not. Confocal laser scanning microscopy revealed that incubation of S. aureus with triclosan altered biofilm structure, resulting in increased proportions of polysaccharide in the biofilm matrix that could potentially mediate protection against antibiotics. Neither the stringent response mutants nor agr mutants influenced triclosan-induced biofilm changes, suggesting another, currently uncharacterised response. We suggest that triclosan triggers multiple global regulatory systems in S. aureus, subsequently inducing tolerance to multiple antibiotic classes and altering biofilm structure.

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.

We have designed a new Gram-negative antimicrobial- it is an oligonucleotide Transcription Factor Decoy (TFD) that binds to and inhibits bacterial transcription factors controlling genes essential for growth and pathogenicity. The TFD is highly effective in vitro and in vivo, tested in Galleria mellonella survival models and in a mouse model of intra-abdominal infection. TFDs are formulated as nanoparticles, composed of a proprietary lipidic molecule (CM2), that binds to essential prokaryotic phospholipids, such as Cardiolipin, to deliver the oligonucleotide across the bacterial membrane. Studies with models of bacterial membranes showed that translocation was dependent on the presence of Cardiolipin but occurred in the absence of ATP or a pH gradient. Flow Cytometry studies found that the efficiency of TFD delivery to bacterial cells was high, and Live/Dead staining confirmed that cells were not being lysed by the nanoparticles. Further investigation of the mechanism of delivery used a proteomics and metabolomics study of the response of E. coli to nanoparticulate delivery. A number of highly induced transcription factors were identified consistent with stress induced by disruption of respiratory centres bound by Cardiolipin within the membrane. A TFD was designed to inhibit one of the induced transcription factors. This TFD was shown to bind tightly to its cognate transcription factor, have a potent MIC in vitro and be efficacious in murine models of infection. Hence, the work demonstrates that TFDs can be rationally designed to create new antimicrobials to efficiently target bacterial transcription factors.

Burkholderia cepacia complex (Bcc) is a group of 22 closely related species of Gram-negative bacteria that cause chronic infections in people with cystic fibrosis (CF) that are rarely eradicated. The high-level antibiotic resistance and poorly understood mechanisms by which Bcc survive and persist during chronic infection mean that combatting these chronic infections is particularly challenging. We have previously found that a group of 20 low-oxygen-activated (lxa) encoded proteins were consistently upregulated in sequential Bcc isolates in two chronically infected CF patients over time of infection. Many of these proteins have not previously been studied in Bcc but the consistent upregulation over time of infection suggests that they play an important role during chronic infection. Two particular proteins of interest within the lxalocus that were consistently upregulated were a universal stress protein (USP) and a phospholipid binding protein (PBP). Single gene deletion mutants (Δusp and Δpbp) in the wildtype Bcc strain K56-2 (WT) both showed a 90 % reduction in attachment to CFBE41o- cells compared to WT. There was also a 5-fold reduction in the virulence of Δpbp in the acute infection model Galleria mellonella (P<0.005) and an increased sensitivity of Δusp to peroxide-induced oxidative stress (P<0.0001) and low pH (P<0.05) relative to WT. A reduction in both uptake and survival of Δusp in U937 macrophage-like cell line compared to WT, suggests the USP plays a role in the intra-macrophage survival of Bcc. Overall, these proteins, previously associated with low-oxygen conditions may play a considerable role in Bcc pathogenesis and its adaption during chronic infection.

Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, adept at colonising various ecological niches within the host. Colonisation of the nasopharynx, followed by asymptomatic carriage and non-inflammatory clearance is the predominant outcome of infection, but diverse disease manifestations including pneumonia, septicaemia and meningitis occur in a minority of individuals. Through experimental evolution of pneumococci in mouse disease models, we are investigating the genetic basis of the niche adaptations that enable pneumococci to switch from a commensal lifestyle in the nasopharynx, to a pathogenic phenotype in the lungs, brain or blood. Experimental evolution was carried out via serial passage of pneumococci separately through pneumonia and nasopharyngeal carriage mouse models, to generate lineages adapted to the lung and nasopharyngeal environments, respectively. Starting from a non-passaged (lab adapted) isolate, ten independently-evolved lineages of lung-adapted pneumococci have been generated, each having been passaged 20-times through a mouse pneumonia model. Pneumococci recovered from the infected lungs were used to inoculate further mice for the next passage round. We will present the results from whole genome sequencing and phenotypic analysis of lung-passaged bacterial isolates including growth characteristics, toxin production, adherence and invasion with lung epithelial cells. We will also describe how the acquired pneumococcal adaptations, which facilitate survival in the lung environment, can influence bacterial gene expression during exponential growth. These studies will provide insight into genetic changes associated with pneumococcal commensal to pathogen switch. Identifying such genetic determinants of virulence will be valuable for the development of vaccine candidates and targets for therapeutic intervention.

The Campylobacter lipooligosaccharides (LOS) can stimulate membrane-bound innate immune receptors in human macrophages. However, the association of Campylobacter LOS in the stimulation of cytosolic receptors or the inflammasome remains poorly characterised. Therefore, the aim of this study was to determine the role of Campylobacter LOS in the activation of NLRP3 inflammasome-dependent signalling in a human monocytic cell line. The induction of NLRP3 inflammasome-mediated IL-1β and Caspase-1 secretion in THP-1 supernatants was quantified using ELISA following co-culture of THP-1 cells with LOS extracts from wild type C. jejuni 11168, mutant C. jejuni 11 168 with reduced LOS and two wild type C. coli strains (RM1875 and 76339). Our results demonstrate that LOS purified from both C. jejuni and C. coli can induce Caspase-1 and IL-1β production in human macrophages. However, C. jejuni 11 168 mutant LOS with modified lipid A and lack of core oligosaccharides stimulated significantly reduced Caspase-1 and IL-1β. This result was also replicated in co-culture of live wild type and mutant C. jejuni with THP-1 cells. This study provides new insight into the interaction of Campylobacter with human macrophages and suggests that variation in LOS structure may alter NLRP3 inflammasome activation.

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via viral infection. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts have been considered as beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill species off starter cultures and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action in silico. Our deterministic model explains how killer Saccharomyces cerevisiae cells distress and consequently kill the sensitive members of the species, accounting for the K1, K2 and K28 toxin mode of action at high or low concentrations. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death, and allowed us to gain novel insight on these mechanisms. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately 3 times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.

The causative agent of TB, Mycobacterium tuberculosis (Mtb) is once again the world’s number one infectious killer. M. tuberculosis resides primarily within macrophages and metabolic reprogramming within this intracellular niche is a crucial determinant of virulence. We previously applied the metabolic modelling-based tool 13C-flux spectral analysis (13C-FSA) to show that intracellular M. tuberculosis co-metabolises multiple gluconeogenic and glycolytic carbon substrates by utilizing the reactions of the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (OAA) or anaplerotic (ANA) node. However, predicting the metabolic mode of operation required for intracellular survival is chellenging using a metabolic network as the ANA node consists of several apparently functionally redundant bidirectional reactions. Here we use multiple techniques including 13C isotopomer profiling, lipid analysis and fluorescent reporter strains to dissect the role of the ANA node. We show that this node has unexpected roles in the life cycle of M. tuberculosis including lipid biosynthesis, protection from known toxic intracellular carbon sources and redox regulation. Inhibiting enzymes at this node with novel therapeutic compounds restricts the growth of M. tuberculosis and limits the ability of this formidable pathogen to survive within the human host cell identifying the ANA node as a potential druggable pathway for controlling TB.

Heterogeneity in bacterial populations can manifest in various ways, such as resistant cells, which can be observed in harsh environments after the use of antibiotics. Many studies have looked at the evolution of resistance and the effect of inhibitory and sub-inhibitory concentrations of antibiotics by batch culture measurements without considering the heterogeneity of bacterial populations. But antibiotic susceptibility and fitness costs of resistance mutations or plasmids are affected by the growth rate and physiology of individual cells. Single-cell analysis in microfluidic systems has opened up new possibilities enabling us to investigate the various putative mechanisms behind the persistence phenomenon required direct observation under the microscope. In this study, we use a gradient mixer and a novel micro-chemostat, to create concentration gradients of growth substrates and/or antibiotics to study the effect of nutrient and antibiotic concentration on individual cells growing under constant and defined conditions in cell-sized channels. The single-cell elongation, morphology and growth rate of ribosome-targeting antibiotics resistant E. coli was tracked by combining the microfluidics, microscope phase contrast imaging and fluorescent tag in high throughput mode. A mechanistic cellular model was used to describe the reaction between antibiotics and ribosome and the resulting effects on bacterial growth; then we linked the intracellular chemical-reaction kinetics processes to the population level and predicted the behaviour of population responses. Our approach has enabled the investigation of single-cell individuality and predictions of population dynamics under different environment.

The bacterial order actinomycetales are responsible for the production of 65–70 % of microbially produced specialised metabolites with diverse biological activities, with some actinomycetale strains containing over 30 Biosynthetic Gene Clusters encoding for these metabolites. However, only approximately 10 % of these genes are typically transcribed in a mono-culture setting. Furthermore, it has been observed that microbial interactions may induce these cryptic gene clusters providing an ecological advantage to the producer strains. To understand the chemical exchange between strains isolated from the marine environment, microbial interactions were assessed using 49 actinomycetale strains, two Pseudomonas and one Bacillus strain. In total, 72 tri-cultures (three strains) were analysed resulting in 29 strains that showed an altered phenotypes as a result of the interaction. These were then evaluated in a one-to-one culture (two strains) followed by bioactivity screening. Using this data, nine tri-cultures and 27 one-to-one cultures were evaluated using tandem Mass Spectometry, enabling chemically interesting interactions to be prioritized for Time of Flight Secondary Ionisation Mass Spectometry (ToF-SIMS) analysis. ToF-SIMS enables the spatial distribution of parent ions within a sample, in this case, two bacterial strains interacting in a Petri dish. The results that will be presented demonstrate that microbial interactions induce the production of metabolites and ToF-SIMS represents an exciting strategy to study bacterial chemical ecology.

Seaweeds are of huge interest in the food, pharmaceutical and agricultural industries due to their high nutritional content and the prevalence of useful bioactive compounds. Current extraction methods of macroalgal-derived metabolites are however problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular as they allow the development of more efficient and eco-friendly processes. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25°C and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina Miseq sequencing and culture-dependent approaches, including the novel iChip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay with a substantial decrease in the relative abundances of certain phyla including Planctomycetes and Verrucomicrobia observed during the decay period. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species from the phylum Bacteroidetes together with a number of Vibrio species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculous and were shown to be equally efficient as commercial enzymes in their extraction efficiencies.

Many microbes colonise the gut establishing interactions with their host and their nutritional environment. Studying genetics and metabolism brought about the drive and potential to engineer communities to promote health and improve industrial processes. However, structuring artificial communities in predictable ways is underdeveloped. We studied Escherichia coli’s genetic targets and physiological mechanisms during gut colonisation and adaptation and how metabolic environment/microbiota complexity shape these processes. We introduced a tractable E. coli K-12 in mice Germ-free or with polymicrobial communities. Whole Genome Sequencing identified potential adaptive targets. Here, we established phenotypic assays as well characterising effects of key mutations and metabolomics was performed with 1H-NMR of intestinal contents. Genes for sugar alcohol metabolism (gat) was the only target common to both mouse models, evidencing specificity. Facing complex microbiota E. coli targeted use of sugar alcohols (srlR, kdgR) and anaerobic respiration (dcuB, focA) [1] whereas alone, we observed instead mutations pointing to increased ability for amino acid use (lrp, dtpB, alaA). Mutations selected correlated dinamically with metabolomics: our results fit the model whereby other microbiota members scavenge oxygen and breakdown complex sugars, limiting E. coli to anaerobically respire simple by-product carbon sources. In the opposing scenario (functional absence) improved amino acid use are favoured colonisation factors. Through experimental evolution we gained insight on shaping E. coli’s metabolic traits through genetic engineering to colonise specific host environments. This work also highlights the versatility of E. coli as potential biotic sensor. [1] Barroso-Batista, J. et al. The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps, 2014.

In the blood stream, arginine is an essential amino acid that is required by phagocytes to synthesize iNOS. Previously we showed that the fungus Candida albicans induces host arginase production that diverts arginine from the pathway that leads to the production of nitrite oxide. We therefore investigated whether C. albicans arginase activity also contributed to the protection of the fungus by competing for arginine during infections. Three C. albicans genes had been annotated as putative arginase encoding genes. Heterologous expression of these genes suggested all three had some arginase activity and one gene product (Car1) encoded a bone fide arginase that was required for growth on arginine. However, single and double mutations in the two other genes (AGM1 and GBU1) did not affect growth on arginine as a single nitrogen source and were found instead to encode agmatinase and guanidinobutyrase respectively that participate in two other pathways related to arginine metabolism. This family of three enzymes therefore exhibits mixed biochemical activities and collectively participate in the catabolism of exogenous and endogenous sources of arginine. Virulence of the triple mutant lacking all three genes was reduced in a Galleria infection model, but single or double mutants were fully virulent. None of the single or multiple mutants affected host NO production suggesting they do not influence the oxidative burst of phagocytes. In addition, CAR1 expression was required for hyphal growth. This family of enzymes therefore represent a novel enzyme set that is essential for growth in vivo and indirectly for fungal virulence.

The airways of cystic fibrosis (CF) patients provide a rich and unique environmental niche, prone to lifelong chronic infection by a diverse and dynamic polymicrobial community. Such dense microbial ecosystems have a network of interspecies communication between each member of the community, serving to modulate virulence, impact metabolism and contribute towards antimicrobial resistance (AMR). Currently no models exist which enable the long-term culture of a true polymicrobial community. Most existing animal models are only suitable for short term infection studies, often utilising relatively healthy hosts and which use axenically cultured clonal strains, providing little parallel to the complex biochemical interactions occurring within chronic CF infections. Here we describe a simple in vitro model utilising artificial sputum medium to allow the successful coculture of major CF-associated pathogens and begin to recapitulate and maintain the CF microbiome within a relatively steady-state. An in vitro model confers several advantages for studying widespread community changes and pathogenic interactions. Perhaps most importantly, in vitro models can be easily perturbed through the addition of antibiotics or introduction of new species/strain variants, allowing the impact of external stressors upon the emergence and changes in lifestyles of key pathogens to be effectively studied. A simple, robust and physiologically relevant CF model could be applied to address any number of fundamental biological questions surrounding interspecies interactions occurring within polymicrobial infections.

Candida glabrata currently accounts for 25 % of all fungal cases in UK hospitals, second only to C. albicans. This number is expected to rise given the intrinsic anti-fungal resistance of this species and the difficulty in treating it. In an effort to identify novel-anti fungal targets in C. glabrata, we used comparative genomics within Saccharomycotina yeast to predict which genes are under positive selection in this species specifically. Such genes are predicted to have influenced the adaptation of C. glabrata from a free-living microbe to a human pathogen, potentially due to functional shift(s) of the proteins they encode. Our analysis predicts that histone acetylation pathways are under positive selection in C. glabrata. Thereforewe hypothesised that we could use histone deacetylase inhibitors (HDACi) to interfere with histone acetylation levels and impact C. glabrata virulence. By treating C. glabrata withbroad spectrum HDACis we show it has a reduced capacity to form biofilms, it is less well adapted to high salt conditions typically found within a human host, and most importantly, it reverts to a more anti-fungal sensitive state. RNAseq analysis indicates that HDACi treatment interferes with the C. glabrata transcriptional response to anti-fungal treatment, rendering it incapable of combating against these drugs. Furthermore, using an in vivo worm model of candidiasis, we show that HDACi treatment in conjunction with the anti-fungal fluconazole, can increase the survival rate of individuals with C. glabrata infections. Taken together our data suggest that the health threat posed by C. glabrata might be addressed by repurposing HDACi to treat this infection.

Dinoflagellate algae are ecologically and environmentally important, as symbionts of corals and many other aquatic organisms, and the causative agents of red tides. However, attempts over the last twenty years to establish genetic manipulation systems for dinoflagellates have met with little success. We have exploited the unusual chloroplast genome of dinoflagellates to establish a system for transformation of this organelle. The chloroplast genome of peridinin-containing (the ancestral state) dinoflagellates is highly reduced and composed of a number of small, plasmid-like molecules, referred to as ‘minicircles’. We have constructed shuttle vectors that are fusions of minicircles and Escherichia coli plasmids and carry selectable markers. We used biolistic transformation to introduce these into the model dinoflagellate Amphidinium carterae. We found that the plasmids confer the expected phenotype on the dinoflagellate cells, and we can detect the plasmid DNA and associated transcripts following selection, indicating successful transformation. This opens up the possibility of studying many aspects of dinoflagellate chloroplast biology, including the maintenance and expression of the minicircles, and the role of the chloroplast in phenomena such as coral bleaching.

Thraustochytrids are abundant and ubiquitous osmoheterotrophic marine protists (labyrinthulomycetes, stramenopiles) thought to function ecologically as fungus-like decomposers. Some thraustochytrids have the ability to synthesize carotenoids, including carotenes (e.g. beta-carotene) and xanthophylls (e.g. astaxanthin), which is uncommon among heterotrophic eukaryotes. Carotenogenic thraustochytrids appear to have acquired carotenoid biosynthetic enzymes by horizontal gene transfer from bacteria. Heterotrophic production of carotenoids is typically associated with protection against oxidative stress, and in thraustochytrids may be particularly associated with protecting large amounts of essential omega-3 polyunsaturated fatty acids stored in lipid droplets. To gain better understanding of carotenoid function in thraustochytrids, and thus new insight into the ecophysiology of these organisms, we have produced mutants of the thraustochytrid Aurantiochytrium limacinum in which the trifunctional gene Aurli_150841, encoding the first three carotenogenesis-specific reactions (phytoene synthase, phytoene desaturase, lycopene cyclase), has been interrupted by double homologous recombination with a construct containing a zeocin resistance (BleoR, shble) expression cassette. As predicted, the Aurli_150841 knockout mutants lack the carotenoid pigmentation found in the wild-type. Complementation with the wild-type Aurli_150841 to confirm that this phenotype is due to the knockout is in progress. Differences between the wild-type and Aurli_150841 knockout mutants in features such as growth rate and biomass yield, lipid content, survival in stationary phase and response to oxidative stress are being evaluated under growth conditions that induce different amounts of carotenoid accumulation in the wild-type.

Naegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.

To address biological questions that cannot be answered by current model organisms, we need to develop genetic tools in the specific taxa that can provide the best answers. Such is the case of the origin of animals in which, genetic tools need to be developed among the closest unicellular relatives of animals. To fill this gap, we are developing genetic tools in Corallochytrium limacisporum, a close unicellular relative of animals that also has a fascinating biology. Corallochytrium is a marine free-living walled saprotroph that develops through a choenocyte. Moreover, because of the basal phylogenetic position of Corallochrytria together with Ichthyosporea, this lineage is especially informative to fill the void of information between yeast and metazoans. We have successfully developed transient and a stably transfection protocols by introducing the resistance gene to puromycin, allowing us to select individual transformants. Deep characterization of the established transformed lines has revealed important biological features of this organism such as the plasticity of its genome, the mode of plasmid integration, and some differences between the two known strains (Hawaii and India). Currently we are better characterizing these features and, in parallel, developing genome-editing technologies. Progress and the potential implications of our research will be presented and further discussed.

Chytridiomycota (Chytrids) are the most basal lineage within the true fungi, however they have largely remained in the dark in terms of their fundamental cell biology. In aquatic ecosystems, chytrids can dominate ‘dark matter’ surveys and are important saprotrophs of recalcitrant organic carbon. They therefore play an integral biogeochemical role in carbon cycling. Unlike ‘higher’ dikaryan fungi that feed via multicellular hyphae, chytrids are unicellular and develop an anucleate rhizoid system that acts as the trophic interface of the cell. Understanding the functions of the rhizoid has the potential to shed light on the trophic biology of ‘dark matter’ chytrids. We applied 3D and 4D live-cell confocal microscopy to morphometrically quantify rhizoid development in the model saprotrophic chytrid Rhizoclosmatium globosum under different nutrient treatments. Rhizoid branching was highest under carbon-rich conditions, whereas under carbon-starved treatments, rhizoids grew significantly longer and were less branched, in what we interpret to be a ‘search strategy’ for nutrient sources. F-actin and the cell wall were identified throughout the rhizoid system. Chemical inhibition of actin and cell wall glucan synthesis induced the development of hyperbranched paramorphs, suggesting that these components underpin rhizoid branching and organising cell polarity at the rhizoid tip. Previous studies have shown that inhibition of these components induces an identical phenotype in dikaryan fungi. These findings represent an important step in understanding the trophic biology of a biogeochemically important microbe and unveil striking similarities in cell development between early-diverging and ‘higher’ dikaryan fungi.

The human gut microbiota composition is linked to both health and disease, but knowledge of individual microbial species is needed to decipher their biological role. Despite extensive culturing and sequencing efforts, the complete bacterial repertoire of the human gut microbiota remains undefined. Here we identify 1952 uncultured candidate bacterial species by reconstructing 92 143 metagenome-assembled genomes from 11 850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281 % increase in phylogenetic diversity. Whilst the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by over 200 %. These candidate species encode hundreds of novel biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. We also highlight newly identified species overrepresented in patients with gastrointestinal diseases, suggesting an underappreciated role in human health and disease. Our work uncovers the uncultured gut bacterial diversity, providing unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.

Marine fungi are a major part of ‘microbial dark matter’, with most organisms known from sequence data and currently not in culture. Interest in marine fungi has substantially increased over the past decade, and studies using culture independent methods have indicated that fungal diversity in the oceans may be greater than previously estimates based on cultivation alone. There remains much to learn about the true diversity of marine fungi in the global oceans and the ecological roles that they could play. The Tara Oceans expedition has allowed for significant advancements in our understanding of the global diversity of planktonic microorganisms. Interrogation of the Tara Oceans 18S rRNA gene dataset for fungal sequences shows that fungi are found throughout the global oceans, appearing in all marine regions covered by the Tara Oceans expedition. In the survey, Ascomycota and Basidiomycota were common, while some locations had increased abundances of Chytridiomycota. Differences in community composition were observed between oceanic regions and, although clear signals were not apparent due to the nature of the sampling, there was some indication of community variation between upwelling, coastal, shelf and gyral provinces. Different size sampling fractions appeared to capture different portions of the pelagic fungal community. These findings highlight a number of ecological questions: How important are oceanic currents in determining fungal biogeography? What is the relationship between marine fungi and biogeochemical processes? What is actually there? By targeting these questions directly, we will be able to bring the dark matter of marine fungi into the light.

The development of new microbial growth and analytical techniques is becoming increasingly relevant in relation to ‘unculturable’ organisms. This may involve the modification of existing methods or the development of new, custom procedures. One important application of this is in the symbiotic bacteria of insects. Symbionts, due to adaptations to their host, are often difficult to culture in vitro. With the growing interest in the use of modified microbiomes to control vector-borne diseases, improved culture techniques that further the understanding of an insect’s microbiome are becoming increasingly important. The tsetse fly, genus Glossina, is the insect vector for Trypanosoma brucei. This parasite is responsible for human African trypanosomiasis (HAT), endemic in sub-Saharan Africa, as well as the wasting disease nagana in cattle. The tsetse’s secondary symbiont, Sodalis glossinidius, provides a unique potential target for reducing the spread of T. brucei. Here, we describe the use of metabolic modelling to design an entirely defined growth medium for S. glossinidius. This medium was used to verify predictions about carbon and nitrogen usage in the symbiont, including amino acid and vitamin auxotrophies. Furthermore, we discuss the use of multiobjective evolutionary algorithms combined with flux balance analysis to investigate computationally the evolution of symbioses, with S. glossinidius and its free-living relative S. praecaptivus as an exemplar. This work not only improves our understanding of the metabolic interactions within the tsetse microbiome, but serves also as a template for future investigations into symbiont evolution.

Many honey bee colonies suffer large losses due to colony collapse disorder. This phenomenon, which has dramatically increased in frequency since 2006, has led to widespread efforts in sequencing honey bee pathogens, including RNA viruses such as deformed wing virus. However, honey bees coexist with a number of other arthropods, whose viruses are less thoroughly characterised. Many viruses currently classified as honey bee pathogens may therefore have a much wider host range. In particular, ants, which like bees are members of the Hymenoptera order, often coexist with bees and the two groups have previously been shown to exchange viruses. Parasitism by Varroamites, known to act as effective vectors for a number of RNA viruses, is also almost ubiquitous amongst honey bees, but little is known about viruses endemic to mites. We have previously demonstrated that it is possible to detect and characterise viral RNA in publicly available RNA-seq datasets. There are over 3000 such datasets for diverse Hymenoptera and mite species. We have developed a computational pipeline to identify viral transcripts in these datasets. This pipeline performs quality control, removes low complexity reads and reads generated from host RNA and various known contaminants, assembles the remaining reads into transcripts and detects the presence of regions with homology to known RNA viruses. Viral fragments identified with this pipeline will be examined phylogenetically to identify novel pathogens, clarify host range and specificity, and characterise transmission patterns.

Mitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.

The twin-arginine translocation (Tat) machinery mediates the transport of folded proteins across the cytoplasmic membranes of prokaryotes and thylakoid membranes of chloroplasts. In Gram-negative bacteria three integral membrane components, TatA, TatB and TatC, are essential for generating an active complex. Most Gram-positive bacteria however, have a minimalist Tat complex formed solely from TatA and TatC subunits. Bacillus subtilis encodes two TatAC systems; TatAdCd and TatAyCy. These complexes operate in parallel but have differing substrate specificities. To date, little is known about how the B. subtilis TatAC-complexes assemble, however, the Escherichia coli TatABC complex is well studied and a substrate-triggered positional exchange of TatA and TatB has been identified as the first step in the assembly of an active complex. In this study, we aim to identify site specific interactions taking place between the B. subtilis Tat components and how these interactions differ whilst the complex is engaged in transport of a substrate. Molecular modelling was used to identify likely interaction interfaces between TatA and TatC, and site-directed mutagenesis was used to generate single cysteine variants of both proteins. Subsequently in vivo disulfide crosslinking was undertaken in both E. coli and B.subtilis, in the presence and absence of overproduced Tat substrates, to determine changes that occur between resting state and activated complexes. Overall, this work will enable us to outline a possible mechanism of assembly in TatAC-complexes of Gram-positive bacteria and identify whether the two distinct translocases assemble in a similar fashion.

Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developed in vitro and in vivo competition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.

Microbial production strains need to operate under sub- optimal growth conditions such as low pH, high osmolarity and thermal stress. The capacity to carry out industrial fermentations at higher temperatures reduces the risk of bacterial contamination and lowers cooling costs. We want to understand the basis of thermotolerance in the industrial yeast Kluyveromyces marxianus. As part of the EU-funded project, CHASSY, K. marxianus was grown in chemostat cultures under different stress conditions and a multi-omics analysis performed to study a range of stress responses, including elevated temperature (40 °C). Transcriptomes were generated from steady state cultures growing at identical growth rates under different stress conditions and gene set enrichment analysis (GSEA) performed. A range of functions were identified as being specifically expressed at higher temperatures and these are now being further investigated. One example is the temperature-specific expression of two putative hexose transport genes. Subsequent mutational inactivation using CRISPR and heterologous complementation established that at least one of these two genes is required for growth at (40 °C). We are now trying to determine the substrates for, and the precise function of, these genes. We also developed a ribosome profiling pipeline for K. marxianus and are using this to investigate the translational response to temperature stress. The combined study of both transcription and translation at steady state and as a culture responds to a temperature shift will give a comprehensive view of the basis of thermotolerance in K. marxianus and should identify strategies to exploit this in biotechnological processes.

Due to their intrinsic thermodynamic properties, RNA can misfold easily in cells. One way to mitigate RNA misfolding is through the actions of RNA chaperones, which bind and unwind structured RNA molecules and thereby offer opportunities for these misfolded species to refold properly. Such rescue activity has implications for the fitness effects of individual mutations-- at least mutations that compromise RNA folding or structure might be buffered by RNA chaperones. However, little is known about the rules governing such mutation buffering. Here, we describe how a model RNA chaperone, the DEAD-box RNA helicase CYT-19, affects the fitness effects of mutations in a model structured RNA, the Tetrahymenagroup I intron, whose self-splicing activity is dependent on its structure. We performed deep mutational scanning on the P1ex region of the intron which is critical for its self-splicing activity, and assayed differential splicing activity of all possible P1ex mutants in the presence and absence of CYT-19 to identify mutations that are buffered by RNA chaperone activity. I will discuss the properties of the chaperone-dependent and chaperone-independent mutation pools. Our results highlight that, to understand RNA robustness in vivo, we need to consider how mutational fitness effects are modulated by RNA chaperones and other trans-acting factors.

The development of optical microscopy has been traditionally driven by the needs of eukaryotic cell biology. Therefore, there are a number of unmet requirements for the application of advanced microscopy techniques to the field of microbiology. Conventional techniques, such as widefield epi-fluorescence and confocal laser scanning microscopy, are common-place in most laboratories, however these methods have trade-offs in terms of their attainable resolution and limited imaging volume. Here we present the application of several optical microscopy methods with the aim addressing the unmet needs of the field; increasing spatial resolution and sampling volume. We demonstrate the use of Interference Reflection Microscopy (IRM) for investigating the morphology and gliding motility of Myxococcus xanthus. This label-free technique provides super-resolution in the axial plane where changes in cell shape on the order of 100 nm can be detected in live cells. Using IRM we show novel insights into the gliding behaviour of these bacteria. We also present the application of the Mesolens to microbiology. The Mesolens is a large optical microscope with the unique combination of a low magnification and a high numerical aperture which results in an imaging volume >100 mm 3 with isotropic sub-cellular resolution. We demonstrate the use of the Mesolens to image live bacterial communities at multiple spatial scales simultaneously and offer new insights for bacterial community dynamics and biofilm architecture. Our work details novel applications of advanced microscopy to the field, and in doing so fills the technology gap which has previously restricted the study of complex microbial behaviours.

Chitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.

CRISPR-associated Rossmann fold (CARF) domain signalling underpins the modulation of CRISPR-Cas systems. Unlike the majority of known CARF domains associated with nuclease activity in CRISPR-Cas systems, the CARF domain of the transcriptional regulator RtcR modulates the opposite function by activating an RNA end sealing system. The Rtc RNA repair system in Escherichia coli consists ofthe universally conserved RNA cyclase RtcA and RNA ligase RtcB, and is known to be induced by antibiotics and oxidative stress. We aim to investigate the CARF domain mediated transcriptional regulation of the Rtc system in vivo and in vitro. A reporter based assay confirmed that the RtcR CARF domain has a negative regulatory effect on RtcR activity and subsequent induction of the rtcBA operon. Both predictive modelling and site-directed mutagenesis suggest the Rtc induction is not due to oxidation of cysteines present in the RtcR regulatory CARF domain. Interestingly, the enzymes RtcA and RtcB, products of the actions of RtcR as a transcription regulator, are required for RtcR to stimulate expression of the rtcBA operon and directly bind to the RtcR CARF domain as shown in vivo by two-hybrid and in vitro by gel filtration. Given that CARF domains are known to be activated by RNA binding, a range of RNA molecules linked to the Rtc system or CARF domains in general are currently being investigated as potential inducers in an in vitro transcription system. Together, our data indicate an expanded range for CARF domain signalling, including via protein-protein interactions.

Burkholderia cenocepacia (Bc) is an environmental opportunistic pathogen that causes persistent, often severe, lung infections in individuals with cystic fibrosis and other underlying diseases. Rho GTPases are central molecular switches that regulate cytoskeletal dynamics, trafficking, immune responses and cell proliferation in eukaryotic cells. Many microbes produce proteins that target Rho GTPase signalling. Bc employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho GTPases and causing actin cytoskeletal defects. Bc protein TecA is a non-VgrG T6SS effector that is responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces Pyrin inflammasome activation1. Our goal is to determine the detailed TecA secretion mechanism and the interacting partners inside the bacterial cytoplasm and inside the macrophages. In this study, we found by Co-IP/MS analysis that TecA interacts with the T6SS tube protein HcP, the membrane anchored TssM and with the elongation factor Tu. We also found that TecA is secreted in the absence of HcP and the secretion mechanism is discussed. 1. Aubert, D.F., X. Hao, J. Yang, X. Shi, W. Gao, L. Li, F. Bisaro, S. Chen, M.A. Valvano, and F. Shao. 2016. A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome. Cell Host and Microbe 19 : 664–674.

Microbial eukaryotes (parasites/protists) are widely distributed and are common inhabitants of the gastro-intestinal (GI) tract of humans and animals. Some species, including Giardia, Entamoeba and Cryptosporidium are associated with symptomatic gastro-intestinal illness. However, others, for example Blastocystis, have questionable pathogenicity as they can be found in symptomatic and asymptomatic individuals. The aim of this study is to investigate selected protists which present health concerns to humans or animals. To date, approximately 180 faecal samples from 33 mammalian species, four bird species and one reptile across two wildlife parks in the Southeast England have been collected. A combination of cell culturing techniques, microscopy and molecular biology have been carried out to positively identify different protists including Blastocystis, Cryptosporidium, Eimeria, Entamoeba, Giardia and Isospora. Preliminary data show over fifty percent of the animals are sequence positive for at least one species, with approximately thirty percent exhibiting co-habitation with two or more different species. This study provides one of the first thorough investigations into distribution and prevalence of GI tract protists in wildlife parks in the UK. As a result, it has enhanced our awareness regarding what may constitute a normal eukaryotic component of the gut microbiome, in addition to aiding conservation efforts by examining the impact captivity has on an animal’s microbiome and potential implications this may have on their release.

In recent years, multiple studies have examined the bacterial communities present in the gastric microbiota during the progression to gastric cancer (GC). Although Helicobacter pylori is the biggest risk factor for GC, the microbiota of GC comprises a decreased load of H. pylori and an enrichment of bacteria, such as Prevotella spp., Veillonella spp., Actinomyces spp. However, interactions between H. pylori and these cancer-associated bacteria is hugely understudied. Here, we have used hypoxic growth conditions (5 % O₂) to investigate polymicrobial interactions between cancer-associated bacteria and H. pylori in vitro. We found that whilst the co-culture of H. pylori with Prevotella spp. and Veillonella spp. had no effect on growth of either bacteria, Actinomyces oris completely inhibited the growth of H. pylori. Moreover, A. oris did not inhibit the growth of other Gram-negative pathogens such as Salmonella Typhimurium and E. coli, whilst there was a slight growth inhibition of Campylobacter jejuni. Furthermore, ultrafiltration of A. oris culture supernatants revealed that inhibition is mediated by a secreted factor larger than 5 kDa, which can be heat inactivated. Interestingly, Actinomyces viscosus can also specifically kill H. pylori suggesting that this inhibition could be conserved across the Actinomyces genus. We are currently identifying the inhibitory factor responsible for inhibiting H. pylori growth. Furthermore, we are investigating whether A. oris can clear gastric H. pylori infection in a mouse model of infection and the implications of this on gastric carcinogenesis. In conclusion, whilst data-rich microbiota studies continue to thrive, it is imperative that we understand the mechanisms underpinning changes to the gastric microbiota and whether these bacteria are drivers or ‘passengers’ of gastric carcinogenesis.

Tick borne diseases (TBDs) have significant impact on the health and welfare of domestic animals and humans. There is extremely little data on the prevalence of tick species, TBDs or their impact in Nigeria. Nigeria’s scenario is further worsened by lack of basic diagnostic facilities and treatment, compared with the average person’s income. The multiple climate zones and animal husbandry practices in Nigeria also make it difficult to extrapolate studies from one zone to the other five geopolitical zones. TBDs reported in Nigerian dogs include Anaplasma species. (A. platys A. omatnenne), Babesia species (B. rossi, B. canis and B. gibsoni), Theileria species. (T. equi, T. sable), Ehrlichia sp. (E. canis, E. ruminantum), Hepatozoon species (H. canis) and Candidatus Neoehrlichia mikurensis. The prevalence of zoonotic pathogens of dogs such as Borrelia sp., in humans in West Africa also indicates that these are likely to present in dogs in Nigeria. This main study aim is to identify ticks taken from dogs in Nigeria using morphological and molecular methods, determine which host species the ticks have fed on, and identify pathogens that they are carrying. It also aims to compare molecular and point of care diagnostics for TBDs in blood from Nigerian dogs. The overall objective is to provide robust data on which tick species and TBDs are present in Nigerian dogs and the potential zoonotic or epizootic risk.

Staphylococcus aureus subsp. anaerobius is a microaerophilic, catalase-negative bacterium responsible for abscess pathology (Morel’s disease) in small ruminants. We performed whole-genome sequencing to a collection of isolates taken in Europe and Africa over the last 30 years, and carried out an evolutionary genomic analysis to understand the molecular bases of its host adaptation and restricted metabolism. Phylodynamic analyses showed that S. anaerobius emerged from a Staphylococcus aureus progenitor about 1000 years ago (716–1184), with an evolutionary rate of ∼1.2 SNPs/year (approximately 10-fold slower than S. aureus clones), before differentiating into two distinct lineages separating African and European isolates. The S. anaerobius genome displays signatures of extreme adaptation to a highly specific niche, with 205 pseudogenes that together represent over 10 % of the genome and affect many metabolic and pathogenic pathways. In addition, S. anaerobius contains 87 highly similar insertion sequences (IS) located in intergenic regions. Our functional analysis suggests that the IS transcription could affect the expression of their flanking genes, using at least two complementary strategies: antisense RNA or modification of promoter regions. We propose that the IS-mediated control of gene expression underpins an orchestrated mechanism of host adaptation. Remarkably, we also identified 6 large genomic transversions (size range: 70–346 kb) flanked by IS, presumably the result of homologous recombination. In summary, S. anaerobius evolved from S. aureus undergoing restrictive host specialisation, which shaped its genome through widespread pseudogenisation, accumulation of IS that modulate gene expression, and large chromosomal rearrangements.

Mastitis is a disease of the mammary gland which affects most mammals and is one of the costliest ongoing challenges in modern dairy farming. The most significant challenges in mastitis management are the speed and accuracy of diagnosis and the use of antibiotics. Current mastitis diagnosis in veterinary practices in the United Kingdom utilises culture-based techniques. However, this approach has some limitations including; processing time, species selection biases and culture failure. This ongoing PhD project seeks to assess the utility of high-throughput sequencing technology in addressing the current challenges in mastitis diagnosis. Samples were obtained from cattle from farms in North Yorkshire, United Kingdom. Cattle were selected from monthly somatic cell count records as mastitis positive cases with a cell count between 250 000 and 550 000 cells ml−1 or control animals with a count below 200 000 cells. Mastitis positive cases were further split into bacteriology positive and negative groups. During sampling a cow-side somatic cell test (California Milk Test) was performed to confirm the monthly database readings. All farms sampled were conventional dairy farms with comparable management systems. Samples were collected by trained veterinarians following a pre-defined protocol designed to limit sample contamination. Microbial community diversity, richness and composition will be compared between sample groups to survey for variations which may explain why culture fails in culture negative subclinical cases. 131 sample animals have been assigned to study groups with samples collected, DNA extracted and prepared for sequencing on the Illumina MiSeq platform with results expected in January 2019.

Dendritic cells (DCs) are the sentinels of the immune system, responsible for recognising invading pathogens and priming the adaptive immune system for appropriate responses. Hence they are considered potential targets for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Little is known of the events of FMDV replication in bovine moDCs. Present work therefore sought to characterize FMDV and its immune complex (IC) replication in bovine moDCs in vitro. A chimeric heparin sulphate FMDV (O1M) was used in this study. Immuno-fluorescence microscopy (IFM) and quantitative RT-PCR was used to analyse viral replication at 0–6, 8, 16 and 24 hpi. Plaque assays were used to investigate the yields of live virus produced in moDCs at 0, 4, 8 and 24 hpi. FMDV and IC FMDV could infect moDC. In moDC infected with FMDV alone, or with immune-complexed (IC) FMDV, replication was observed by IFM between 2–4 and 1–16 hpi. In contrast, for both FMDV and FMDV IC infections RT-PCR analyses showed viral replication peaked at 4 hpi and then decreased between 8 to 24 hpi. Plaque assays using supernatants of the infected moDC showed no evidence of an increase in viral titre at 24 hpi. The detection of viral nsp (3AB and derivatives) suggests replication of FMDV persists for longer in moDCs when entry is mediated by IC. However, the lack of increase in virus yield suggests replication is abortive. One possible explanation for this difference could be that bovine moDCs are able to recognise non-immune complexed FMDV more rapidly.

Bovine ephemeral fever virus (BEFV) [Family Rhabdoviridae: genus Ephemerovirus] causes a transient febrile illness in cattle that results in economic losses and morbidity, with occasional mortality. Epidemics of the disease have been particularly costly in countries of the Middle East, including Israel. The virus is considered a vector-borne pathogen although the exact relationship with a number of blood-feeding arthropods has not been established. In order to improve developments in diagnostic detection, phylogeographic investigations and virus-vector relationships of BEFV we have derived the first genome of this virus from an isolate from Israel and used this to assess recent outbreaks of disease. We have also investigated the vector competence of BEFV with a number of target arthropod species. The complete genome sequence of BEFV (Israel strain 1) is 14 850 base pairs in length and shows over 95 % identity with the only other Middle Eastern genome for BEFV from Turkey. A wider phylogeny shows that these viruses form a clade, previously described as cluster II, which is clearly distinct from BEFV strains derived from China, Japan and Australia. However, there is a distinct separation of those viruses from Israel to others in the Middle East. Preliminary vector competence studies suggest that at least three species of mosquito that are potential transmission vectors of the virus are incapable of infection with, or transmission of, BEFV. Current studies are ongoing to assess other blood-feeding insect species as potential vectors.

Mycobacterium bovisis the causative agent of bovine tuberculosis (bTB), one of the most costly and persistent agricultural infectious diseases still widespread in England today. However, not all parts of England are created equally with respect to the incidence of bTB. In 2012 the Department for the Environment, Food and Rural Affairs (DEFRA) divided the country into three risk areas; the high, low and edge risk areas. The perpetuation of the bTB epidemic is often blamed on the known wildlife host of M. bovis, the European badger (Meles meles). Despite this, no large scale studies examining both the prevalence and transmission patterns of M. bovis in this species has been undertaken. Here we describe the results of a major survey of 600 road-kill badgers from 6 counties within the edge risk area of England. Mycobacterium like colonies were isolated from over 80 carcasses of which, 65 were confirmed to be tuberculosis complex positive by PCR. These isolates were spoligotyped, VNTR typed and then subsequently whole-genome sequenced. We describe a SNP based transmission analysis of the sequenced isolates that provides a higher degree of resolution between the badgers compared to the described molecular typing methods.