- Volume 1, Issue 1A, 2019

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.