- 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.