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bioRxiv2017

Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteriaare implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O(LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.

microbiology↗

bioRxiv2017

Assessing and characterising the repertoire of constitutive promoter elements in soil metagenomic libraries in Escherichia coli

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we have developed a novel framework for prospecting, characterising and estimating the accessibility of promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Using the expression profiling of fluorescent clones from two independent libraries from soil samples, we directly analysed the regulatory dynamics of novel promoter elements, addressing the relationship between the \"metaconstitutome\" of a bacterial community and its environmental context. Through the construction and screening of plasmid-based metagenomic libraries followed by in silico analyses, we were able to provide both (i) a consensus exogenous promoter elements recognizable by Escherichia coli and (ii) an estimation of the accessible promoter sequences in a metagenomic library, which was close to 1% of the whole set of available promoters. The results presented here should provide new directions for the exploration through functional metagenomics of novel regulatory sequences in bacteria, which could expand the Synthetic Biology toolbox for novel biotechnological and biomedical applications.

microbiology↗

bioRxiv2017

Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase

Catalyzing the key step for anaerobic methane production and oxidation, methyl-coenzyme M reductase or Mcr plays a key role in the global methane cycle. The McrA subunit possesses up to five post-translational modifications (PTM) at its active site. Bioinformatic analyses had previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To examine its role, MMP1554, the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the specific loss of the 5-(S)-methylarginine PTM of residue 275 in the McrA subunit and a 40~60 % reduction in the maximal rates of methane formation by whole cells. Methylation was restored by complementations with the wild-type gene. However, the rates of methane formation of the complemented strains were not always restored to the wild type level. This study demonstrates the importance of Mmp10 and the methyl-Arg PTM on Mcr activity.

microbiology↗

bioRxiv2017

Differential and defective expression of Koala Retrovirus reveal complexity of host and virus evolution

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus free or to have only exogenous variants of KoRV with low rates of KoRV induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV induced disease such as lymphoma. This paper uses a combination of sequencing technologies, Illumina RNA sequencing of "southern" (south Australian) and "northern" (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of "southern" (South Australian and Victorian animals) to retrieve full length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication competent KoRV, are not in fact KoRV free but harbour defective, presumably endogenous, "RecKoRV" variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV induced disease.

microbiology↗

bioRxiv2017

Clostridium difficile differentially alters the structure and metabolism of distinct cecal microbiomes to promote persistent colonization during infection

Susceptibility to Clostridium difficile infection is primarily associated with previous exposure to antibiotics, which compromise the structure and function of the gut bacterial community. Specific antibiotic classes correlate more strongly with recurrent or persistent C. difficile infection. As such, we utilized a mouse model of infection to explore the effect of distinct antibiotic classes on the impact that infection has on community-level transcription and metabolic signatures shortly following pathogen colonization and how those changes may associate with persistence of C. difficile. Untargeted metabolomic analysis revealed that C. difficile infection had significantly larger impacts on the metabolic environment across cefoperazone and streptomycin-pretreated mice, which become persistently colonized compared to clindamycin-pretreated mice where infection quickly became undetectable. Through metagenome-enabled metatranscriptomics we observed that transcripts for genes associated with carbon and energy acquisition were greatly reduced in infected animals, suggesting those niches were instead occupied by C. difficile. Furthermore, the largest changes in transcription were seen in the least abundant species indicating that C. difficile may \"attack the loser\" in gut environments where sustained infection occurs more readily. Overall, our results suggest that C. difficile is able to restructure the nutrient-niche landscape in the gut to promote persistent infection.

microbiology↗

bioRxiv2017

Associations between sexual habits, menstrual hygiene practices, demographics and the vaginal microbiome as revealed by Bayesian network analysis

The vaginal microbiome plays an influential role in several disease states in reproductive age women, including bacterial vaginosis (BV). While demographic characteristics are associated with differences in vaginal microbiome community structure, little is known about the influence of sexual and hygiene habits. Furthermore, associations between the vaginal microbiome and risk symptoms of bacterial vaginosis have not been fully elucidated. Using Bayesian network (BN) analysis of 16S rRNA gene sequence results, demographic and extensive questionnaire data, we describe both novel and previously documented associations between habits of women and their vaginal microbiome. The BN analysis approach shows promise in uncovering complex associations between disparate data types. Our findings based on this approach support published associations between specific microbiome members (e.g., Eggerthella, Gardnerella, Dialister, Sneathia and Ruminococcaceae), the Nugent score (a BV diagnostic) and vaginal pH (a risk symptom of BV). Additionally, we found that several microbiome members were directly connected to other risk symptoms of BV (such as vaginal discharge, odor, itch, irritation, and yeast infection) including L. jensenii, Corynebacteria, and Proteobacteria. No direct connections were found between the Nugent Score and risk symptoms of BV other than pH, indicating that the Nugent Score may not be the most useful criteria for assessment of clinical BV. We also found that demographics (i.e., age, ethnicity, previous pregnancy) were associated with the presence/absence of specific vaginal microbes. The resulting BN revealed several as-yet undocumented associations between birth control usage, menstrual hygiene practices and specific microbiome members. Many of these complex relationships were not identified using common analytical methods, i.e., ordination and PERMANOVA. While these associations require confirmatory follow-up study, our findings strongly suggest that future studies of the vaginal microbiome and vaginal pathologies should include detailed surveys of participants sanitary, sexual and birth control habits, as these can act as confounders in the relationship between the microbiome and disease. Although the BN approach is powerful in revealing complex associations within multidimensional datasets, the need in some cases to discretize the data for use in BN analysis can result in loss of information. Future research is required to alleviate such limitations in constructing BN networks. Large sample sizes are also required in order to allow for the incorporation of a large number of variables (nodes) into the BN, particularly when studying associations between metadata and the microbiome. We believe that this approach is of great value, complementing other methods, to further our understanding of complex associations characteristic of microbiome research.

microbiology↗

bioRxiv2017

Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially processed plant derived carbon

BackgroundThe transformation of plant photosynthate into soil organic carbon and its recycling to CO2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon.\n\nResultsWe conducted an experiment in which common wild oats (Avena fatua) were grown in a 13CO2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13C into their DNA. A 1.45 Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally-derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13C because they preyed upon these bacteria and/or directly from the labelled plant DNA. Isotopic labeling indicates that the population was replicating during the six-week period of plant growth. Interestingly, the genome is ~30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch and 1,3-{beta}-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate or lactate. The genome encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to make a variety of isoprenoids, including the plant hormone zeatin.\n\nConclusionsRhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

microbiology↗

bioRxiv2017

Lessons from simple marine models on the bacterial regulation of eukaryotic development

Molecular cues from environmental bacteria influence important developmental decisions in diverse marine eukaryotes. Yet, relatively little is understood about the mechanisms underlying these interactions, in part because marine ecosystems are dynamic and complex. With the help of simple model systems, including the choanoflagellate Salpingoeca rosetta, we have begun to uncover the bacterial cues that shape eukaryotic development in the ocean. Here, we review how diverse bacterial cues - from lipids to macromolecules - regulate development in marine eukaryotes. It is becoming clear that there are networks of chemical information circulating in the ocean, with both eukaryotes and bacteria acting as nodes; one eukaryote can precisely respond to cues from several diverse environmental bacteria, and a single environmental bacterium can regulate the development of different eukaryotes.\n\nHighlightsO_LICues from environmental bacteria influence the development of many marine eukaryotes\nC_LIO_LIThe molecular cues produced by environmental bacteria are structurally diverse\nC_LIO_LIEukaryotes can respond to many different environmental bacteria\nC_LIO_LISome environmental bacteria act as \"information hubs\" for diverse eukaryotes\nC_LIO_LIExperimentally tractable systems, like the choanoflagellate S. rosetta, promise to reveal molecular mechanisms underlying these interactions\nC_LI

microbiology↗

bioRxiv2017

Bacterium-triggered remodeling of chromatin identifies BasR, a novel regulator of fungal natural product biosynthesis

The eukaryotic epigenetic machinery is targeted by bacteria to reprogram the response of eukaryotes during their interaction with microorganisms. In line, we discovered that the bacterium Streptomyces rapamycinicus triggered increased chromatin acetylation and thus activation of the silent secondary metabolism ors gene cluster leading to the production of orsellinic acid in the fungus Aspergillus nidulans. Using this model we aim at understanding molecular mechanisms of communication between bacteria and eukaryotic microorganisms based on bacteria-triggered chromatin modification. By genome-wide ChIP-seq analysis of acetylated histone H3 (H3K9ac, H3K14ac) we uncovered the unique chromatin landscape in A. nidulans upon co-cultivation with S. rapamycinicus. Genome-wide acetylation of H3K9 correlated with increased gene expression, whereas H3K14 appears to function in transcriptional initiation by providing a docking side for regulatory proteins. In total, histones belonging to six secondary metabolism gene clusters showed higher acetylation during co-cultivation including the ors, aspercryptin, cichorine, sterigmatocystin, anthrone and 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde gene cluster with the emericellamide cluster being the only one with reduced acetylation and expression. Differentially acetylated histones were also detected in genes involved in amino acid and nitrogen metabolism, signaling, and genes encoding transcription factors. In conjunction with LC-MS/MS and MALDI-MS imaging, molecular analyses revealed the cross-pathway control and Myb-like transcription factor BasR as regulatory nodes for transduction of the bacterial signal in the fungus. The presence of basR in other fungal species allowed forecasting the inducibility of ors-like gene clusters by S. rapamycinicus in these fungi, and thus their effective interaction with activation of otherwise silent gene clusters.

microbiology↗

bioRxiv2017

Detection of cytosolic Shigella flexneri via a C-terminal triple-arginine motif of GBP1 inhibits actin-based motility

Dynamin-like guanylate binding proteins (GBPs) are gamma interferon (IFN{gamma})-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique amongst the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS) mutants of S. flexneri co-localize with GBP1 less frequently than wildtype S. flexneri, suggesting that host recognition of O-antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C-terminus, promotes the co-recruitment of four additional GBP paralogs (GBP2, GBP3, GBP4 and GBP6). GBP1-decorated Shigella replicate but fail to form actin tails leading to their intracellular aggregation. Consequentially, wildtype but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular glue trap inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program S. flexneri has evolved an effective counter-defense to restrict GBP1 recruitment.\n\nImportanceSeveral pathogenic bacterial species evolved to invade, reside and replicate inside the cytosol of their host cells. One adaptation common to most cytosolic bacterial pathogens is the ability to co-opt the hosts actin polymerization machinery, in order to generate force for intracellular movement. This actin-based motility enables Gram-negative bacteria such as Shigella to propel themselves into neighboring cells thereby spreading from host cell to host cell without exiting the intracellular environment. Here, we show that the human protein GBP1 acts as a cytosolic glue trap capturing cytosolic Gram-negative bacteria through a unique protein motif and preventing disseminated infections in cell culture models. To escape from this GBP1-mediated host defense, Shigella employs a virulence factor that prevents or dislodges the association of GBP1 with cytosolic bacteria. Thus, therapeutic strategies to restore GBP1 binding to Shigella may lead to novel treatment options for shigellosis in the future.

microbiology↗

bioRxiv2017

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d{inverted exclamation}-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbCs calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.\n\nImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d{inverted exclamation}-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a \"cloud\" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

microbiology↗

bioRxiv2017

Membrane curvature and the Tol-Pal complex determine polar localization of the chemoreceptor Tar in E. coli

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years different mechanisms have been put forward to explain this polar localization; from stochastic clustering, membrane curvature driven localization, interactions with the Tol-Pal complex, to nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at cell poles. This finding also implies that the curvature of cell poles does not attract chemoreceptor complexes. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterised by strong membrane curvature. Chemoreceptors, like Tar, form trimer-of-dimers that bend the cell membrane due to a rigid tripod structure with an estimated curvature of approximately 37 nm. This curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favourable as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or cell poles. These findings favour a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.\n\nImportanceBacteria have exquisite mechanisms to sense and to adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli, and found that membrane curvature at cell division sites and interaction with the Tal-pol protein complex, localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

microbiology↗

bioRxiv2017

Gene network analysis identifies a central post-transcriptional regulator of cellular stress survival

Cells adapt to shifts in their environment by remodeling transcription. Measuring changes in transcription at the genome scale is now routine, but defining the functional significance of individual genes within large gene expression datasets remains a major challenge. We applied a network-based algorithm to interrogate publicly available transcription data to predict genes that serve major functional roles in Caulobacter crescentus stress survival. This approach identified GsrN, a conserved small RNA that is directly controlled by the general stress sigma factor, {sigma}{tau}, and functions as a potent post-transcriptional regulator of survival under multiple stress conditions. GsrN expression is both necessary and sufficient to protect cells from hydrogen peroxide, where it functions by base pairing with the leader of katG mRNA and promoting catalase/peroxidase expression. We conclude that GsrN convenes a post-transcriptional layer of gene expression that serves a central functional role in stress physiology.

microbiology↗

bioRxiv2017

Modeling the temporal dynamics of the gut microbial community in adults and infants

Given the highly dynamic and complex nature of the human gut microbial community, the ability to identify and predict time-dependent compositional patterns of microbes is crucial to our understanding of the structure and function of this ecosystem. One factor that could affect such time-dependent patterns is microbial interactions, wherein community composition at a given time point affects the microbial composition at a later time point. However, the field has not yet settled on the degree of this effect. Specifically, it has been recently suggested that only a minority of the operational taxonomic units (OTUs) depend on the microbial composition in earlier times. To address the issue of identifying and predicting temporal microbial patterns we developed a new model, MTV-LMM (Microbial Temporal Variability Linear Mixed Model), a linear mixed model for the prediction of the microbial community temporal dynamics based on the community composition at previous time stamps. MTV-LMM can identify time-dependent microbes in time series datasets, which can then be used to analyze the trajectory of the microbiome over time. We evaluated the performance of MTV-LMM on three human microbiome time series datasets, and found that MTV-LMM significantly outperforms all existing methods for microbiome time series modeling. Particularly, we demonstrate that the effect of the microbial composition in previous time points on the abundance levels of an OTU at a later time point is underestimated by a factor of at least 10 when applying previous approaches. Using MTV-LMM, we demonstrate that a considerable proportion of the human gut microbiome, both in infants and adults, has a significant time-dependent component that can be predicted based on microbiome composition in earlier time points. This suggests that microbiome composition at a given time point is a major factor in defining future microbiome composition and that this phenomenon is considerably more common than previously reported for the human gut microbiome.

microbiology↗

bioRxiv2017

Low affinity membrane transporters can increase net substrate uptake rate by reducing efflux

Cells require membrane-located transporter proteins to import nutrients from the environment. Many organisms have several similar transporters for the same nutrient, which differ in their affinity. Typically, high affinity transporters are expressed when substrate is scarce and low affinity ones when substrate is more abundant. The benefit of using low affinity transporters when high affinity ones are available has so far remained unclear. Here, we investigate two hypotheses. First, it was previously hypothesized that a trade-off between the affinity and the maximal catalytic rate explains this phenomenon. We find some theoretical and experimental support for this hypothesis, but no conclusive evidence. Secondly, we propose a new hypothesis: for uptake by facilitated diffusion, at saturating extracellular substrate concentrations, lowering the affinity enhances the net uptake rate by reducing the substrate efflux rate. As a consequence, there exists an optimal, external substrate concentration dependent transporter affinity. An in silico analysis of glycolysis in Saccharomyces cerevisiae shows that using the low affinity HXT3 transporter instead of the high affinity HXT6 enhances the steady-state flux by 36%. We tried to test this hypothesis using yeast strains expressing a single glucose transporter that was modified to have either a high or a low affinity. Due to the intimate and reciprocal link between glucose perception and metabolism, direct experimental proof for this hypothesis remained inconclusive in our hands. Still, our theoretical results provide a novel reason for the presence of low affinity transport systems which might have more general implications for enzyme catalyzed conversions.

microbiology↗

bioRxiv2017

The ventral disc is a flexible microtubule organelle that depends on domed ultrastructure for functional attachment of Giardia lamblia

The parasite Giardia lamblia interacts with its host by directly attaching the lumen of the small intestine. Attachment is mediated by a cytoskeletal structure termed the ventral disc and proceeds in four distinct stages: skimming, seal formation, cell body contacts, and bare area contacts. The precise mechanism of disc-mediated attachment is unclear and attachment models rely heavily on whether or not the ventral disc is a dynamic structure. We sought to investigate the second stage of attachment in which a seal is formed beneath the ventral disc. Three-dimensional, live imaging of Giardia expressing specific ventral disc markers to the lateral crest, ventral groove, and disc body indicate dynamic movement in all of these regions. We observe seal formation by the lateral crest and determine that movement of the ventral groove region aids lateral crest seal formation. We also report the discovery of a new protein that is necessary for ventral disc formation and functional attachment (DAP_7268). Lastly, we observed that attachment largely depends on ventral disc ultrastructure as flattened discs display hindered attachment proficiency whether or not they retain the ability to form a seal. We propose a synthesized mechanism for attachment that includes flagellar hydrodynamic flow to help generate suction as well as disc conformational dynamics to aid in both hydrodynamic flow and the maintenance of negative pressure.

microbiology↗

bioRxiv2017

A Cell-Type-Specific Defect in Assembly of Influenza A Virus Particles in Primary Human Macrophages

The primary target of Influenza A virus (IAV) is epithelial cells in the respiratory tract. In contrast to epithelial cells, productive infection of most IAV strains is either blocked or highly inefficient in macrophages. The exact nature of the defect in IAV replication in human macrophages remains unknown. In this study, we showed that primary human monocyte-derived macrophages (MDM) are inefficient in IAV release even when compared to a monocytic cell line differentiated to macrophage-like cells, despite comparable levels of expression of viral glycoproteins at the plasma membrane. Correlative fluorescence scanning electron microscopy revealed that formation of budding structures at the cell surface is inefficient in MDM even though clustering of a viral glycoprotein, hemagglutinin (HA), is observed, suggesting that IAV particle assembly is blocked in human MDM. Using an in situ proximity ligation assay, we further determined that association between HA and the viral ion channel protein M2 is defective at the plasma membrane of MDM. In contrast, HA and another glycoprotein neuraminidase (NA) associate with each other on the MDM surface efficiently. Notably, the defect in association between HA and M2 in MDM was reversed upon inhibition of actin polymerization by cytochalasin D. Altogether, these results suggest that HA-M2 association on the plasma membrane is a discrete step in the IAV assembly process, which is separable from the association between HA and NA and susceptible to suppression by actin cytoskeleton. Overall, our study revealed the presence of a cell-type-specific mechanism negatively regulating IAV assembly at the plasma membrane.\n\nImportanceIdentification of host cell determinants promoting or suppressing replication of many viruses has been aided by analyses of host cells that impose inherent blocks on viral replication. In this study, we show that primary human MDM are not permissive to IAV replication due to a defect at the virus particle formation step. This defect is specific to primary human macrophages, since a human monocytic cell line differentiated to macrophage-like cells supports IAV particle formation. We further identified association between two viral transmembrane proteins, HA and M2, on the cell surface as a discrete assembly step, which is defective in MDM. Defective HA-M2 association in MDM is rescued by disruption of the actin cytoskeleton, revealing a previously unknown, negative role for actin polymerization, which is generally thought to play positive roles in IAV assembly. Overall, our study uncovered a host-mediated restriction of association between viral transmembrane components during IAV assembly.

microbiology↗

bioRxiv2017

Metagenomics for chronic meningitis: clarifying interpretation and diagnosis

ImportanceIdentifying infectious causes of subacute and chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed.\n\nObjectiveTo present a case series of patients with diagnostically challenging subacute and chronic meningitis in whom metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF), supported by a statistical framework generated from mNGS sequencing of non-infectious patients and environmental controls, identified a pathogen.\n\nDesignCase series. Using mNGS data from the CSF of 94 non-infectious neuroinflammatory cases and 24 water and reagent controls, we developed and implemented a weighted scoring metric based on z-scores at the species and genus level for both nucleotide and protein databases to prioritize and rank mNGS results. We performed mNGS on total RNA extracted from CSF of patients with subacute or chronic meningitis and highlight seven cases representing a diverse array of pathogens.\n\nSettingA multi-center study of mNGS pathogen discovery in patients with suspected neuroinflammatory conditions.\n\nParticipantsPatients with diagnostically challenging subacute or chronic meningitis enrolled in a research study of mNGS performed on CSF.\n\nInterventionmNGS was performed on total RNA extracted from CSF (0.25-0.5 mL). A weighted z-score was used to filter out environmental contaminants and facilitate efficient data triage and analysis.\n\nMain Outcomes1) Pathogens identified by mNGS and 2) ability of a statistical model to prioritize, rank, and simplify mNGS results.\n\nResultsmNGS identified parasitic worms, fungi and viruses in seven subjects: Taenia solium (n=2), Cryptococcus neoformans, human immunodeficiency virus-1, Aspergillus oryzae, Histoplasma capsulatum, and Candida dubliniensis. Evaluating mNGS data with a weighted z-score based scoring algorithm effectively separated bona fide pathogen sequences from spurious environmental sequences.\n\nConclusions and RelevancemNGS of CSF identified a diversity of microbial pathogens in patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for one year, the first case of CNS vasculitis caused by Aspergillus oryzae, and the fourth reported case of Candida dubliniensis meningitis. Filtering metagenomic data with a scoring algorithm greatly clarified data interpretation and highlights the difficulties attributing biological significance to organisms that may be present in control samples used for metagenomic sequencing studies.\n\nKey PointsQuestion: How can metagenomic next-generation sequencing of cerebrospinal fluid be leveraged to aid in the diagnosis of patients with subacute or chronic meningitis?\n\nFindings: Metagenomic next-generation sequencing identified parasitic worms, fungi and viruses in a case series of seven subjects. A database of water-only and healthy patient controls enabled application of a z-score based scoring algorithm to effectively separate bona fide pathogen sequences from spurious environmental sequences.\n\nMeaning: Our scoring algorithm greatly simplified data interpretation in a series of patients with a wide range of challenging infectious causes of subacute or chronic meningitis identified by metagenomic next-generation sequencing.

microbiology↗