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bioRxiv2017

The spent culture supernatant of Pseudomonas syringae contains azelaic acid

Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. A metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid azelaic acid which for several reasons was decided to further study. Firstly azelaic acid, which is a straight chained nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as mobile signal that primes systemic defenses. Secondly its structure, which is associated with fatty acid biosynthesis, is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). Analytical and structural studies by NMR spectroscopy confirmed that in the PSA spent supernatant azelaic acid was present. Quantification studies further revealed that 20 {micro}g/L of azelaic acid was present and was also found in spent supernatants of several other P. syringae pathovars. An RNAseq transcriptome study however did not reveal whether azelaic acid behaved as a QS molecule. This is the first report of the possible natural biosynthesis of azelaic acid by bacteria.

microbiology↗
bioRxiv2017

A proposed chaperone of the bacterial type VI secretion system functions to constrain a self-identity protein

The opportunistic bacterial uropathogen Proteus mirabilis can communicate identity through the export of the self-identity protein, IdsD, via the type VI secretion (T6S) system. Expression of the ids genes provides a fitness advantage during polymicrobial infections in a mouse infection model. Here we provide an answer to the unresolved question of how the activity of a T6S substrate, such as IdsD, is regulated before export. We demonstrate that IdsD is found in clusters that form independently of the T6S machinery and activity. We show that the protein IdsC, which is a member of the proposed DUF4123 chaperone family, is essential for the stability of these clusters as well as the IdsD protein itself. And we provide evidence that amino acid disruptions in IdsC are sufficient to disrupt IdsD export but not IdsD localization into stable subcellular clusters, strongly supporting that IdsC functions in at least two different ways: IdsD stabilization and IdsD export. We propose that IdsC, and likely other DUF4123-containing proteins, function to regulate T6S substrates before export by both stabilizing the protein and mediating export at the T6S machinery.

microbiology↗
bioRxiv2017

Conductive particles enable syntrophic acetate oxidation between Geobacter and Methanosarcina from coastal sediments

Coastal sediments are rich in conductive minerals, which could impact microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure and their survival depends on successful partnership. Here we demonstrate that conductive minerals facilitate a SAO partnership between Geobacter and Methanosarcina from the coastal sediments of the Bothnian Bay, Baltic Sea. Bothnian methanogenic sediments showed a high apparent isotopic fractionation (c 1.07) characteristic of CO2-reductive methanogenesis. The native community was represented by electrogens such as Geobacter and methanogens like Methanosarcina. Upon the addition of conductive particles (activated carbon and magnetite) methanogenesis from acetate increased four fold. Geobacter (96% related to G. psychrophilus) and Methanosarcina (99% related to M. subterranea) dominated the conductive particle-spiked SAO communities. Using NanoSIMS we demonstrated that during SAO, Geobacter incorporated 82% of the labeled acetate as compared to only 18% by Methanosarcina. At the same time Geobacter converted 27% of the 13C-acetate to 13CO2 as detected by IRMS. Indigenous soluble shuttles were not involved in SAO, since spiking fresh cultures with spent-media filtrate had no effect on methanogenic rates. Our results demonstrate that Geobacter oxidizes acetate to CO2 while transferring electrons extracellularly via conductive particles to Methanosarcina, which utilizes them for CO2 reduction to methane. In natural environments, mediation of SAO by conductive particles between electrogens and methanogens could impact the iron and methane cycles.\n\nSignificanceAcetate oxidizing bacteria are known to thrive in mutualistic consortia in which H2 or formate is shuttled to a methane-producing Archaea partner. Here we discovered that they could instead transfer electrons via conductive minerals. Mineral-SAO (syntrophic acetate oxidation) could be a vital pathway for CO2-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

microbiology↗
bioRxiv2017

A novel multi SNP based method for identifying subspecies and associated lineages and sub-lineages of the Mycobacterium tuberculosis complex by whole genome sequencing

The clinical phenotype of zoonotic tuberculosis, its contribution to the global burden of disease and prevalence are poorly understood and probably underestimated. This is partly because currently available laboratory and in silico tools have not been calibrated to accurately identify all subspecies of the Mycobacterium tuberculosis complex (Mtbc). We here present the first such tool, SNPs to Identify TB ( SNP-IT). Applying SNP-IT to a collection of clinical genomes from a UK reference laboratory, we demonstrate an unexpectedly high number of M. orygis isolates. These are seen at a similar rate to M. bovis which attracts much health protection resource and yet M. orygis cases have not been previously described in the UK. From an international perspective it is possible that M. orygis is an underestimated zoonosis. As whole genome sequencing is increasingly integrated into the clinical setting, accurate subspecies identification with SNP-IT will allow the clinical phenotype, host range and transmission mechanisms of subspecies of the Mtbc to be studied in greater detail.

microbiology↗
bioRxiv2017

YlmD and YlmE are required for correct sporulation-specific cell division in Streptomyces coelicolor A3(2)

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

microbiology↗
bioRxiv2017

Receptor guanylyl cyclase C confers protection during oral Salmonella Typhimurium infection by attenuating ileal damage and enhancing cytokine and antimicrobial peptide production

Non-typhoidal Salmonella disease contributes towards significant morbidity and mortality across the world. Host factors including IFN-{gamma}, TNF- and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we have investigated the roles of receptor guanylyl cyclase C (GC-C) that is predominantly expressed in the intestine, and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C (Gucy2c-/-) displayed accelerated mortality following infection via the oral route, in spite of possessing comparative systemic Salmonella infection burden. Survival following intra-peritoneal infection remained similar, indicating that GC-C offered protection via a gut-mediated response. Serum cortisol was higher in Gucy2c-/- mice, in comparison to wild type (Gucy2c+/+) mice, and an increase in infection-induced thymic atrophy, with loss in immature CD4+CD8+ double positive thymocytes, was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting and distortion of villus architecture, was seen in Gucy2c-/- mice, concomitant with a larger number of ileal tissue-associated bacteria. Transcription of key mediators in Salmonella-induced inflammation (IL-22/Reg3{beta}) were altered in Gucy2c-/- mice in comparison to Gucy2c+/+ mice. A reduction in fecal Lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c-/- mice. Gucy2c-/- mice cohoused with wild type mice continued to show reduced Lactobacilli and increased susceptibility to infection. Our study therefore suggests that receptor GC-C confers a survival advantage during gut-mediated S. Typhimurium pathogenesis, presumably by regulating Salmonella-effector mechanisms and maintaining a beneficial microbiome.

microbiology↗
bioRxiv2017

Divalent metal cations potentiate the predatory capacity of amoeba for Cryptococcus neoformans

Among the best studied interaction between soil phagocytic predators and a human pathogenic fungus is that of Acanthamoeba castellanii and Cryptococcus neoformans. The experimental conditions used in amoeba-fungal confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg2+ and Ca2+ in PBS, C. neoformans was consistently killed when incubated with A. castellanii. A. castellanii survived better in the presence of Mg2+ and Ca2+, even when incubated with C. neoformans. In the absence of Mg2+ and Ca2+, C. neoformans survived when incubated with A. castellanii, and the percentage of dead amoeba was higher than when incubated without yeast cells. These results show that the presence of Mg2+ and Ca2+ can make a decisive contribution toward tilting the outcome of the interaction in favor of amoeba. Of the two metals Mg2+ had a stronger effect than Ca2+. Cations enhanced A. castellanii activity against C. neoformans through enhanced phagocytosis, which is the major mechanism for amoeba to kill fungal cells. We found no evidence that amoeba uses extracellular killing mechanisms in their interactions with C. neoformans. In summary, the presence of Mg2+ and Ca2+ enhanced cell adhesion on surface and motility of amoeba, thus increasing the chance for contact of C. neoformans and the frequency of phagocytosis. Our findings imply that divalent cation concentration in soils could be an important variable for whether amoeba can control C. neoformans in the environment.\n\nImportanceGrazing of soil organisms by phagocytic predators such as amoeba is thought to select for traits that allow some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoeba and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the interaction between amoeba and the pathogenic fungus C. neoformans is influenced by the presence of magnesium and calcium in the assay, which potentiate amoeba. The results may also have practical applications since enriching soils with divalent cations may reduce C. neoformans numbers in contaminated soils.

microbiology↗
bioRxiv2017

Kombucha as a model system for multispecies microbial cooperation: theoretical promise, methodological challenges and new solutions ‘in solution’

Kombucha is a sweetened tea fermented by bacteria and yeast into a carbonated, acidic drink, producing a surface biofilm pellicle (colloquially called a SCOBY) during the process. Typically, liquid and a biofilm pellicle from a previously fermented culture is used as a starter for new cultures; however, there is no standard protocol for growing kombucha in the laboratory. In order to establish a standard protocol with low variability between replicates, we tested whether we could begin a kombucha culture with only well-mixed liquid stock. We found that viable kombucha cultures can be grown from low percentages of initial inoculum stock liquid, that new pellicles can form from liquid alone (with no starter pellicle), and that the variation in the pellicle characteristics is lower when only a liquid starter is used (p = 0.0004). We also found that blending the pellicle before including it significantly reduces the variation among replicates, though the final pellicle was abnormal. We conclude that growing kombucha from only liquid stock is viable and provides a greater degree of experimental control and reproducibility compared to alternatives. Standardizing methodologies for studying kombucha in the lab can facilitate the use of this system for exploring questions about the evolutionary, ecological and cooperative/competitive dynamics within this multi-species system including resource transfers, functional dependence, genetic divergence, collective defense, and ecological succession. A better understanding of kombucha and other fermented foods may eventually allow us to leverage their pathogen inhibitory properties to develop novel antibiotics and bacteriocins.

microbiology↗
bioRxiv2017

Towards sustainable bioplastic production in resource limited environments using the photoferroautotrophic and photoelectroautotrophic bacterium Rhodopseudomonas palustris TIE-1

Bioplastics are an attractive alternative to petroleum-derived plastics because of the harmful environmental effects of conventional plastics and the impending fossil fuel crisis. Polyhydroxybutyrate (PHB) is a well-known bioplastic that is produced by several microbes using organic carbon sources. Autotrophic (using carbon dioxide or CO2) PHB production is reported for only a few organisms. Sustainable PHB bioproduction using other autotrophic microbes needs to be explored. Rhodopseudomonas palustris, a metabolically versatile purple non-sulfur bacterium (PNSB) is known to produce PHBs under photoheterotrophic conditions. Rhodopseudomonas palustris strain TIE-1 demonstrates extended metabolic versatility by using electron sources such as ferrous iron and poised electrodes for photoautotrophy. Here we report the ability of TIE-1 to produce PHB under photoferroautotrophic (light - energy source, ferrous iron - electron source and CO2 - carbon source) and photoelectroautotrophic (light - energy source, poised electrodes - electron source and CO2 - carbon source) growth conditions. PHB accumulation was observed both under nitrogen (N2) fixing and non-N2 fixing conditions. For comparison, we determined PHB production under chemoheterotrophic, photoheterotrophic and photoautotrophic conditions using hydrogen as the electron donor. Photoferroautotrophic and photoelectroautotrophic PHB production was on par with that observed from organic carbon substrates such as butyrate. PHB production increased during N2 fixation under photoheterotrophic conditions but not during photoautotrophic growth. Electron microscopy confirmed that TIE-1 cells accumulate PHBs internally under the conditions that showed highest production. However, gene expression analysis suggests that PHB cycle genes are not differentially regulated despite observable changes in biopolymer production.

microbiology↗
bioRxiv2017

Improving microbial electrosynthesis of polyhydroxybutyrate (PHB) from CO2 by Rhodopseudomonas palustris TIE-1 using an immobilized iron complex modified cathode

Microbial electrosynthesis (MES) is a promising bioelectrochemical approach to produce biochemicals. A previous study showed that Rhodopseudomonas palustris TIE-1 can directly use poised electrodes as electron donors for photoautotrophic growth at cathodic potentials that avoid electrolytic H2 production (photoelectroautotrophy). To make TIE-1 an effective biocatalyst for MES, we need to improve its electron uptake ability and growth under photoelectroautotrophic conditions. Because TIE-1 interacts with various forms of iron while using it as a source of electrons for photoautotrophy (photoferrotrophy), we tested the ability of iron-based redox mediators to enhance direct electron uptake. Our data show that soluble iron cannot act as a redox mediator for electron uptake by TIE-1 from a cathode poised at +100mV vs. Standard Hydrogen electrode. We then tested whether an immobilized iron-based redox mediator Prussian Blue (PB) can enhance electron uptake by TIE-1. Chronoamperometry indicates that cathodic current uptake by TIE-1 increased from 1.47 {+/-} 0.04 to 5.6 {+/-} 0.09 {micro}A/cm2 (3.8 times) and the production of the bioplastic, polyhydroxybutyrate (PHB) improved from 13.5 {+/-} 0.2 g/L to 18.8 {+/-} 0.5 g/L (1.4 times) on electrodes coated with PB. Overall, our data show that immobilized PB can increase direct electron uptake by TIE-1 and enhances PHB production.

microbiology↗
bioRxiv2017

The Distribution of Bacterial Doubling Times in the Wild

Generation time varies widely across organisms and is an important factor in the life cycle, life history and evolution of organisms. Although the doubling time (DT), has been estimated for many bacteria in the lab, it is nearly impossible to directly measure it in the natural environment. However, an estimate can be obtained by measuring the rate at which bacteria accumulate mutations per year in the wild and the rate at which they mutate per generation in the lab. If we assume the mutation rate per generation is the same in the wild and in the lab, and that all mutations in the wild are neutral, an assumption that we show is not very important, then an estimate of the DT can be obtained by dividing the latter by the former. We estimate the DT for four species of bacteria for which we have both an accumulation and a mutation rate estimate. We also infer the distribution of DTs across all bacteria from the distribution of the accumulation and mutation rates. Both analyses suggest that DTs for bacteria in the wild are substantially greater than those in the lab, that they vary by orders of magnitude between different species of bacteria and that a substantial fraction of bacteria double very slowly in the wild.

microbiology↗
bioRxiv2017

Streptococcus pyogenes infection and the human proteome with a special focus on the IgG-cleaving enzyme IdeS

Infectious diseases are characterized by a complex interplay between host and pathogen, but how these interactions impact the host proteome is unclear. Here we applied a novel mass spectrometry based proteomics strategy to investigate how the human proteome is transiently modified by the pathogen Streptococcus pyogenes, with a particular focus on bacterial cleavage of IgG in vivo. In invasive diseases, S. pyogenes evokes a massive host response in blood, whereas superficial diseases are characterized by a local leakage of several blood plasma proteins at the site of infection including IgG. S. pyogenes produces IdeS, a protease cleaving IgG in the lower hinge region and we find highly effective IdeS-cleavage of IgG in samples from local IgG poor microenvironments. The results show that IdeS contributes to the adaptation of S. pyogenes to its normal ecological niches. Additionally, the work identifies novel clinical opportunities for in vivo pathogen detection.

microbiology↗
bioRxiv2017

Isolation of a natural DNA virus of Drosophila melanogaster, and characterisation of host resistance and immune responses

Drosophila melanogaster has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in Drosophila have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of D. melanogaster have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of D. melanogaster. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males and movement and late fecundity in females. Using the Drosophila Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including Cdc42-interacting protein 4. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection, and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between D. melanogaster and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects.\n\nAuthor SummaryThe fruit fly Drosophila melanogaster is a useful model species to study host-virus interaction and innate immunity. However, few natural viruses of Drosophila have been available for experiments, and no natural DNA viruses of Drosophila melanogaster have been available at all. Although infecting flies with viruses from other insects has been useful to uncover general immune mechanisms, viruses that naturally infect wild flies could help us to learn more about the coevolutionary process, and more about the genes that underlie the host-virus interaction. Here we present an isolate of a DNA virus (named Kallithea Virus) that naturally infects the model species Drosophila melanogaster in the wild. We describe the basic biology of infection by this virus, finding that both male and females flies die from infection, but females are more tolerant of infection than males, while laying lay fewer eggs than uninfected females. We quantify genetic variation for virus resistance in the flies, and we use RNA sequencing to see which genes are expressed in male and female flies in response to infection. These results will form the basis for further research to understand how insects defend themselves against infection by DNA viruses, and how DNA viruses can overcome antiviral defence.

microbiology↗
bioRxiv2017

Calcimycocavitological Studies on Seashells from Beaches of North Goa, India

Calcimycocavitology deals with the study of hollowing out of spaces in hard calcareous seashells by the fungi called calcimycocavites. Endolithic fungi in the shells were first reported and named as the trace fossils in 1889 by Bornet and Flahault. Endolithic fungi bore inside the shell by the process of bioturbation by using organic acids (oxalic acid, citrate) and enzymes (proteases, dehydrogenases and oxidoreductases). Previous reports show the presence of tunnels formed by fungal hyphae in the shells. The present work reports preliminary results of calcimycocavitological studies on the seashells from beaches of north Goa, India. The calcareous sand samples were collected from Arambol, Ashvem, Morjim, Vagator, Anjuna, Baga and Miramar beaches by pool sampling method and were separated into different fractions by using standard sieves. Each fraction of sand was subjected to stereomicroscopic studies which revealed that the sand fraction between 150-250 m included irregular shell fragments showing positive colonization by calcimycocavites. Hydrochloric acid treatment was used to dissolve the shells and release calcimycocavites biomass which was stained with Congo Red and tentatively identified as distinct microfungal forms. SEM studies of the calcareous shell fragments revealed the microtunneling behavior of the fungi. Digital analysis of SEM images using Mountain premium 7.2 software revealed the fine topography of calcimycocavites hyphae along with unidentified presumptive biomineral encrustations. The ecological, biological and biogeochemical implications of the findings are presented with respect to possible role of calcimycocavites in Calcium and Carbon cycling by breakdown of the calcareous shells and release of inorganic and organic components in the ecosystem.

microbiology↗
bioRxiv2017

Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra of single bacterial colonies from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis colonies in under 30 minutes, which differ by a single genomic mutation, solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin. Next, we employed our IDBac technique to detect subtle intra-species differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we employed our method to simultaneously extract protein and specialized metabolite MS profiles from unidentified species of Lake Michigan sponge-associated bacteria cultivated on an agar plate. In just 3 hours, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 samples) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on inter- and intra-species differences in specialized metabolite production. To our knowledge, IDBac is the first attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow the distinction of closely related bacterial colonies.\n\nSignificanceMass spectrometry is a powerful technique that has been used to identify bacteria via protein content, and to assess bacterial function in an environment via analysis of specialized metabolites. However, until now these analyses have operated independently, and this has resulted in the inability to rapidly connect bacterial phylogenetic identity with patterns of specialized metabolism. To bridge this gap, we designed a MALDI-TOF mass spectrometry data acquisition and bioinformatics pipeline (IDBac) to discriminate both intact protein and specialized metabolite spectra directly from bacterial cells grown on agar. To our knowledge, this is the first technique that organizes bacteria into highly similar phylogenetic groups and allows for comparison of metabolic differences of hundreds of isolates in just a few hours.

microbiology↗
bioRxiv2017

MetQy: an R package to query metabolic functions of genes and genomes

SummaryWith the rapid accumulation of sequencing data from genomic and metagenomic studies, there is an acute need for better tools that facilitate their analyses against biological functions. To this end, we developed MetQy, an open-source R package designed for query-based analysis of functional units in [meta]genomes and/or sets of genes using the The Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Furthermore, MetQy contains visualization and analysis tools and facilitates KEGGs flat file manipulation. Thus, MetQy enables better understanding of metabolic capabilities of known genomes or user-specified [meta]genomes by using the available information and can help guide studies in microbial ecology, metabolic engineering and synthetic biology.\n\nAvailability and ImplementationThe MetQy R package is freely available and can be downloaded from our groups website (http://osslab.lifesci.warwick.ac.uk) or GitHub (https://github.com/OSS-Lab/MetQy).\n\nContactO.Soyer@warwick.ac.uk

microbiology↗
bioRxiv2017

Mannan molecular sub-structures control nanoscale glucan exposure in Candida

N-linked mannans (N-mannans) in the cell wall of Candida albicans are thought to mask {beta}-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Lateral cell wall exposures of glucan on Candida albicans are predominantly single receptor-ligand interaction sites and are restricted to nanoscale geometries. Candida species exhibit a range of basal glucan exposures and their mannans also vary in size and complexity at the molecular level. We used super resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and -(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and exhibited similar changes to nanoscopic glucan exposure geometry. We conclude that acid labile N-mannan controls glucan exposure geometry at the nanoscale. Furthermore, variations in glucan nanoexposure characteristics are clinically relevant and are likely to impact the nature of the pathogenic surface presented to innate immunocytes at dimensions relevant to receptor engagement, aggregation and signaling.

microbiology↗
bioRxiv2018

Microbiota metabolic product deoxycholic acid controls chicken necrotic enteritis

Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into NE pathogenesis, microbiome relationships, and host responses. Here we reported that the metabolic byproduct of microbial metabolism of bile acids to deoxycholic acid (DCA), at as low as 50 M, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenge strongly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and lamina propria immune cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into villi without significantly altering the bacterial luminal colonization. Molecular analysis showed that DCA reduced inflammatory mediators of Inf{gamma}, Litaf (Tnf), Il1{beta}, and Mmp9 mRNA accumulation in ileal tissue. Mechanically, C. perfringens induced elevated expression of inflammatory cytokines of Inf{gamma}, Litaf, and Ptgs2 (COX-2 gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin attenuated INF{gamma}- or TNFa-induced inflammatory response in the splenocytes. Consistently, chickens fed 0.12 g/kg aspirin diet resisted against NE-induced BW loss, ileal inflammation, and villus apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through curbing inflammatory response. These novel findings could serve as a stepping-stone for developing next generation antimicrobial alternatives against NE.

microbiology↗