Biology subjects

Waldor, M. K.

Publications and source records attributed to Waldor, M. K..

53 records · Page 3Linked to original sources

bioRxiv2020

Genetic analyses link the conserved inner membrane protein CvpA to the σEextracytoplasmic stress response

The function of cvpA, a bacterial gene predicted to encode an inner membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpAs genetic interactions and identify common pathways that rescue the sensitivity of a {Delta}cvpA EHEC mutant to DOC. Collectively, these screens led to the hypothesis that the {Delta}cvpA mutant is impaired in its capacity to activate the {sigma}E-mediated stress response. This idea was supported by showing that mutations that activate {sigma}E, either indirectly or through its direct overexpression, can restore the {Delta}cvpA mutants resistance to DOC. Analysis of the distribution of CvpA homologs revealed that this inner membrane protein is conserved across bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA may function in triggering activation of the {sigma}E stress response pathway in response to DOC as well as additional stimuli. ImportanceSeveral enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require cvpA to robustly colonize the intestine. This inner membrane is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate, but its function is unknown. Genetic analyses carried out here suggest that cvpA is required to trigger the {sigma}E stress response pathway in response to deoxycholate. Since CvpA is conserved across diverse bacterial phyla, we propose that this inner membrane protein is important for activation of this stress response pathway in response to diverse perturbations of the cell envelope.

microbiology↗

bioRxiv2020

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into Ogawa and Inaba serotypes. The O-antigen is V. choleraes immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogenous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germ-free mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single and mixed strain infection assays, we found that all three HaitiV variants - InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa) - were immunogenic and protective, suggesting the impact of O1 serotype variation on OCV function may be minimal. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

microbiology↗

bioRxiv2020

Shiga toxin remodels the intestinal epithelial transcriptional response to Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that causes diarrheal disease and the potentially lethal hemolytic uremic syndrome. We used an infant rabbit model of EHEC infection that recapitulates many aspects of human intestinal disease to comprehensively assess colonic transcriptional responses to this pathogen. Cellular compartment-specific RNA-sequencing of intestinal tissue from animals infected with EHEC strains containing or lacking Shiga toxins (Stx) revealed that EHEC infection elicits a robust response that is dramatically shaped by Stx, particularly in epithelial cells. Many of the differences in the transcriptional responses elicited by these strains were in genes involved in immune signaling pathways, such as IL23A, and coagulation, including F3, the gene encoding Tissue Factor. RNA FISH confirmed that these elevated transcripts were found almost exclusively in epithelial cells. Collectively, these findings suggest that within the intestine, Stx primarily targets epithelial cells, and that the potent Stx-mediated modulation of innate immune signaling skews the host response to EHEC towards type 3 immunity. Significance StatementEnterohemorrhagic Escherichia coli (EHEC) is a potentially lethal foodborne pathogen. During infection, EHEC releases a potent toxin, Shiga toxin (Stx), into the intestine, but there is limited knowledge of how this toxin shapes the host response to infection. We used an infant rabbit model of infection that closely mimics human disease to profile intestinal transcriptomic responses to EHEC infection. Comparisons of the transcriptional responses to infection by strains containing or lacking Stx revealed that this toxin markedly remodels how the epithelial cell compartment responds to infection. Our findings suggest that Stx biases the intestinal innate immune response to EHEC and provide insight into the complex host-pathogen dialogue that underlies disease.

microbiology↗

bioRxiv2020

Transient intestinal colonization by a live-attenuated oral cholera vaccine induces protective immune responses in streptomycin-treated mice

Current mouse models for evaluating the efficacy of live oral cholera vaccines (OCVs) have important limitations. Conventionally raised adult mice are resistant to intestinal colonization by Vibrio cholerae, but germ-free mice can be colonized and have been used to study OCV immunogenicity. However, germ free animals have impaired immune systems and intestinal physiology; also, live OCVs colonize germ free mice for many months, which does not mimic the clearance kinetics of live OCVs in humans. Here, we leverage antibiotic-treated, conventionally raised adult mice to study the effects of transient intestinal colonization by a live OCV V. cholerae strain. In a single dose vaccination regimen, we found that HaitiV, a live-attenuated OCV candidate, was cleared by streptomycin treated adult mice within a week after oral inoculation. This transient colonization elicited far stronger adaptive immune correlates of protection against cholera than did inactivated whole-cell HaitiV. Infant mice from HaitiV vaccinated dams were also significantly protected from choleric disease than pups from inactivated-HaitiV dams. Our findings establish the benefits of antibiotic treated mice for live OCV studies as well as its limitations and underscore the immunogenicity of HaitiV. ImportanceOral cholera vaccines (OCVs) are being deployed to combat cholera but current killed OCVs require multiple doses and show little efficacy in young children. Live OCVs have the potential to overcome these limitations but small animal models for testing OCVs have shortcomings. We used an antibiotic treatment protocol for conventional adult mice to study the effects of short-term colonization by a single dose of HaitiV, a live OCV candidate. Vaccinated mice developed vibriocidal antibodies against V. cholerae and delivered pups that were resistant to cholera, whereas mice vaccinated with inactivated HaitiV did not. These findings demonstrate HaitiVs immunogenicity and suggest that this antibiotic treatment protocol will be useful for evaluating the efficacy of live OCVs.

microbiology↗

bioRxiv2020

A temperature-dependent translational switch controls biofilm development in Vibrio cholerae

The gastrointestinal pathogen Vibrio cholerae frequently forms biofilms during its life cycle. Biofilm formation is vital for protection against environmental stresses and is thought to facilitate intestinal colonization. Adaptation to temperature is crucial for V. cholerae survival, as the pathogen is exposed to seasonal temperature variations in the aquatic environment, and temperature fluctuations during host-environment transitions. Here, we show that V. cholerae strains naturally lacking the master biofilm transcriptional regulator HapR are unable to develop colony rugosity at low temperatures. We find that BipA, a ribosome-associated GTPase, accounts for this temperature-dependent control of biofilm formation by repressing translation of the primary biofilm transcriptional activators VpsR and VpsT at low temperatures. In vitro studies demonstrate that low temperatures influence BipA structural conformation and decrease its sensitivity to proteolysis. Proteomic analyses reveal that BipA exerts temperature-dependent control over >200 proteins in V. cholerae involved in a multitude of cell processes, including biofilm assembly. Our study reveals a remarkable new facet of the complex V. cholerae biofilm regulatory cascade and suggests that combined transcriptional-translational control could be a common mechanism by which bacteria adapt to environmental flux.

microbiology↗

bioRxiv2020

Functional remodeling of lysosomes by type I interferon modifies host defense

Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I interferon (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including Ifitm3, Slc15a3, and Cnp contribute to lysosome acidification. IFN-I-dependent lysosome acidification stimulated intracellular Salmonella virulence gene expression, leading to rupture of the Salmonella-containing vacuole and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium, where Salmonella initiates infection. Our findings explain how an intracellular bacterial pathogen co-opts epithelial IFN-I signaling. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.

cell biology↗

bioRxiv2019

An oral inoculation infant rabbit model for Shigella infection

Shigella species cause diarrheal disease globally. Shigellosis is typically characterized by bloody stools and colitis with mucosal damage and is the leading bacterial cause of diarrheal death worldwide. Following oral ingestion, the pathogen invades and replicates within the colonic epithelium through mechanisms that rely on its type III secretion system (T3SS). Currently, oral infection-based small animal models to study the pathogenesis of shigellosis are lacking. Here, we found that oro-gastric inoculation of infant rabbits with S. flexneri resulted in diarrhea and colonic pathology resembling that found in human shigellosis. Fasting animals prior to S. flexneri inoculation increased the frequency of disease. The pathogen colonized the colon, where both luminal and intraepithelial foci were observed. The intraepithelial foci likely arise through S. flexneri spreading from cell-to-cell. Robust S. flexneri intestinal colonization, invasion of the colonic epithelium, and epithelial sloughing all required the T3SS as well as IcsA, a factor required for bacterial spreading and adhesion in vitro. Expression of the proinflammatory chemokine IL-8, detected with in situ mRNA labeling, was higher in animals infected with wild-type S. flexneri versus mutant strains deficient in icsA or T3SS, suggesting that epithelial invasion promotes expression of this chemokine. Collectively, our findings suggest that oral infection of infant rabbits offers a useful experimental model for studies of the pathogenesis of shigellosis and for testing of new therapeutics. ImportanceShigella species are the leading bacterial cause of diarrheal death globally. The pathogen causes bacillary dysentery, a bloody diarrheal disease characterized by damage to the colonic mucosa and is usually spread through the fecal-oral route. Small animal models of shigellosis that rely on the oral route of infection are lacking. Here, we found that oro-gastric inoculation of infant rabbits with S. flexneri led to a diarrheal disease and colonic pathology reminiscent of human shigellosis. Diarrhea, intestinal colonization and pathology in this model were dependent on the S. flexneri type III secretion system and IcsA, canonical Shigella virulence factors. Thus, oral infection of infant rabbits offers a feasible model to study the pathogenesis of shigellosis and to develop and test new therapeutics.

microbiology↗

bioRxiv2019

Modulation of peptidoglycan synthesis by recycled cell wall tetrapeptides

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized by the cell, where they are recycled for synthesis of new PG. Although the PG recycling pathway is widely conserved, its components are not essential and its roles in cell wall homeostasis are not well-understood. Here, we identified LdcV, a Vibrio cholerae L,D-carboxypeptidase that cleaves the terminal D-Alanine from recycled murotetrapeptides. In the absence of ldcV, recycled tetrapeptides accumulated in the cytosol, leading to two toxic consequences for the cell wall. First, incorporation of tetrapeptide-containing PG precursors into the cell wall led to reduction in D,D-cross-linkage between stem peptides, diminishing PG integrity. Second, tetrapeptide accumulation led to a decrease in canonical UDP-pentapeptide precursors, reducing PG synthesis. Thus, LdcV and the recycling pathway promote optimal cell wall assembly and composition. Furthermore, Ldc substrate preference for murotetrapeptides containing canonical (D-Alanine) vs. non-canonical (D-Methionine) D-amino acids is conserved, suggesting that accumulation of tetrapeptide recycling intermediates may modulate PG homeostasis in environments enriched in non-canonical-muropeptides via substrate competition.

microbiology↗

bioRxiv2019

Surveying the landscape of tRNA modifications by combining tRNA sequencing and RNA mass spectrometry

Chemical modification of the nucleosides that comprise tRNAs are diverse1-3. Such modifications impact tRNA structure, stability, and mRNA decoding3,4. Although tRNA modifications are present in all kingdoms of life1, the structure, location, and extent of modifications have been systematically charted in very few organisms, in part because mapping modifications to tRNA sequences has been technically challenging. Here, we describe a new approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput tRNA-sequencing (tRNA-seq) data is coupled with chemical analysis and identification of tRNA modifications through RNA mass spectrometry (tRNA-SMS). As proof of concept, we applied this method to study tRNA modification profiles in two phylogenetically close bacteria, E. coli and Vibrio cholerae. Comparative tRNA-seq enabled prediction of several V. cholerae modifications that are absent from E. coli and showed the effects of various environmental conditions on V. cholerae tRNA modification profiles. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while another results from C-to-U RNA editing, a process not described before in bacteria. By combining comparative genomics with mass spectrometry, we identified a putative N- acetyltransferase required for acacp3U acetylation. These findings demonstrate the utility of the tRNA-SMS approach for rapid characterization of tRNA modification profiles and environmental control of tRNA modification. Moreover, our identification of a new modified nucleoside and RNA editing process suggests that there are many tRNA modifications awaiting discovery.

biochemistry↗

bioRxiv2019

A New Suite of Allelic Exchange Vectors for the Scarless Modification of Proteobacterial Genomes

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counter-selection escape, which prevents isolation of cells with the desired mutation, limits its utility. To circumvent this limitation, we engineered a series of \"pTOX\" allelic exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important Proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the blue amilCP or magenta tsPurple non-fluorescent chromoproteins facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri. Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 orthologous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC beta-lactamase activity and corresponding beta-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high level ampC derepression and beta-lactam resistance. These data suggest why beta-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens like E. cloacae. Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria.\n\nImportanceTargeted modification of bacterial genomes is critical for genetic analyses of microorganisms. Allelic exchange is a technique that relies on homologous recombination to substitute native loci for engineered sequences. However, current allelic exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic exchange vectors, pTOX, which were validated in several medically important Proteobacteria. They encode visible non-fluorescent chromoproteins that enable easy identification of colonies bearing integrated vector, and permit stringent selection for the second step of homologous recombination, yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam antibiotic resistance.

microbiology↗

bioRxiv2019

A Vibrio cholerae BolA-like protein is required for proper cell shape and cell envelope integrity

BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogens morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide cells that predominated in exponential phase {Delta}ibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae {Delta}ibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall acting antibiotics and bile, and was defective in intestinal colonization. {Delta}ibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutants morphological defects and sensitivity to envelope stressors. Transposon-insertion sequencing analysis of ibaGs genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.\n\nImportanceBolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

microbiology↗

bioRxiv2019

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and commensal E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHECs but not to K-12s growth in vitro, suggesting that gene acquisition during EHEC evolution has heightened the pathogens reliance on certain metabolic processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Numerous mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses encountered in vivo. cvpA, which is also required for intestinal growth of several other enteric pathogens, proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine. Author SummaryEnterohemorrhagic E. coli (EHEC) are important food-borne pathogens that infect the colon. We created a highly saturated EHEC transposon library and used transposon insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit model of intestinal colonization, and refined two analytic approaches to facilitate rigorous identification of new EHEC genes that promote fitness in vivo. Besides the known type III secretion system, more than 200 additional genes were found to contribute to EHEC survival and/or growth within the intestine. The requirement for some of these new in vivo fitness factors was confirmed, and their contributions to infection were investigated. This set of genes should be of considerable value for future studies elucidating the processes that enable the pathogen to proliferate in vivo and for design of new therapeutics.

microbiology↗

bioRxiv2019

Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiVs immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera. Author summaryVaccines for cholera are gaining acceptance as public health tools for prevention of cholera and curtailing the spread of outbreaks. However, current killed vaccines provide minimal protection in young children, who are especially susceptible to this diarrheal disease, and do not stimulate immunity against antigens that may only be expressed by live bacteria during infection. We recently developed HaitiV, an extensively engineered live-attenuated oral cholera vaccine candidate, derived from a clinical isolate from the Haiti cholera outbreak. Here, we found that the HaitiV induces immunological correlates of protection against cholera in germ free mice and leads to protection against disease in their offspring. Protection in this model was dependent on passively acquired factors in the milk of immunized dams and not transmission or colonization of HaitiV. Coupling the immunogenicity data presented here with our previous observation that HaitiV can protect from cholera prior to the induction of adaptive immunity, suggests that HaitiV may provide both rapid-onset short-term protection from disease while eliciting stable and long-lasting immunity against cholera.

microbiology↗

bioRxiv2018

Genetic determinants of penicillin tolerance in Vibrio cholerae

Many bacteria are resistant to killing (\"tolerant\") by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae. Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well-characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A Penicillin Binding Proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. TnSeq analyses revealed that LPS and cell wall biogenesis genes as well as the sigma E cell envelope stress response were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than Lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for post-antibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

microbiology↗

bioRxiv2018

CRISPR screen reveals that EHEC’s T3SS and Shiga toxin rely on shared host factors for infection

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.\n\nImportanceEnterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3), and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogens key virulence factors.

microbiology↗

bioRxiv2017

Deciphering Functional Redundancy in the Human Microbiome

Although the taxonomic composition of the human microbiome varies tremendously across individuals, its gene composition or functional capacity is highly conserved1-5---implying an ecological property known as functional redundancy. Such functional redundancy is thought to underlie the stability and resilience of the human microbiome6,7, but its origin is elusive. Here, we investigate the basis for functional redundancy in the human microbiome by analyzing its genomic content network --- a bipartite graph that links microbes to the genes in their genomes. We show that this network exhibits several topological features, such as highly nested structure and fat-tailed gene degree distribution, which favor high functional redundancy. To explain the origins of these topological features, we develop a simple genome evolution model that explicitly considers selection pressure, and the processes of gene gain and loss, and horizontal gene transfer. We find that moderate selection pressure and high horizontal gene transfer rate are necessary to generate genomic content networks with both highly nested structure and fat-tailed gene degree distribution, and consequently favor high functional redundancy. These findings provide insights into the relationships between structure and function in complex microbial communities. This work elucidates the potential ecological and evolutionary processes that create and maintain functional redundancy in the human microbiome and contribute to its resilience.

microbiology↗

bioRxiv2017

Mapping the ecological networks of microbial communities from steady-state data

Microbes form complex and dynamic ecosystems that play key roles in the health of the animals and plants with which they are associated. Such ecosystems are often represented by a directed, signed and weighted ecological network, where nodes represent microbial taxa and edges represent ecological interactions. Inferring the underlying ecological networks of microbial communities is a necessary step towards understanding their assembly rules and predicting their dynamical response to external stimuli. However, current methods for inferring such networks require assuming a particular population dynamics model, which is typically not known a priori. Moreover, those methods require fitting longitudinal abundance data, which is not readily available, and often does not contain the variation that is necessary for reliable inference. To overcome these limitations, here we develop a new method to map the ecological networks of microbial communities using steady-state data. Our method can qualitatively infer the inter-taxa interaction types or signs (positive, negative or neutral) without assuming any particular population dynamics model. Additionally, when the population dynamics is assumed to follow the classic Generalized Lotka-Volterra model, our method can quantitatively infer the inter-taxa interaction strengths and intrinsic growth rates. We systematically validate our method using simulated data, and then apply it to four experimental datasets of microbial communities. Our method offers a novel framework to infer microbial interactions and reconstruct ecological networks, and represents a key step towards reliable modeling of complex, real-world microbial communities, such as the human gut microbiota.

ecology↗