Vibrio cholerae Represses Polysaccharide Synthesis To Promote Motility in Mucosa

The viscoelastic mucus layer of gastrointestinal tracts is a host defense barrier that a successful enteric pathogen, such asVibrio cholerae, must circumvent.V. cholerae, the causative agent of cholera, is able to penetrate the mucosa and colonize the epithelial surface of the small intestine. In this study, we found that mucin, the major component of mucus, promotedV. choleraemovement on semisolid medium and in liquid medium. A genome-wide screen revealed thatVibriopolysaccharide (VPS) production was inversely correlated with mucin-enhanced motility. Mucin adhesion assays indicated that VPS bound to mucin. Moreover, we found thatvpsexpression was reduced upon exposure to mucin. In an infant mouse colonization model, mutants that overexpressed VPS colonized less effectively than wild-type strains in more distal intestinal regions. These results suggest thatV. choleraeis able to sense mucosal signals and modulatevpsexpression accordingly so as to promote fast motion in mucus, thus allowing for rapid spread throughout the intestines.

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Vibrio cholerae Virulence Activator ToxR Regulates Manganese Transport and Resistance to Reactive Oxygen Species

Infection and Immunity ◽

10.1128/iai.00944-19 ◽

2019 ◽

Vol 88 (3) ◽

Author(s):

Hang-hang Jiang ◽

Yitian Zhou ◽

Ming Liu ◽

Jessie Larios-Valencia ◽

Zachariah Lee ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Vibrio Cholerae ◽

Reactive Oxygen ◽

Oxygen Species ◽

Content Type ◽

Genome Wide ◽

A Genome ◽

Its Gene ◽

Virulence Regulator ◽

Manganese Transport

ABSTRACT Like many other pathogens, Vibrio cholerae, the causative agent of cholera, can modulate its gene expression to combat stresses encountered in both aquatic and host environments, including stress posed by reactive oxygen species (ROS). We previously reported that the virulence activator AphB in V. cholerae is involved in ROS resistance. In this study, we found that another key virulence regulator, ToxR, was important for V. cholerae resistance to hydrogen peroxide. Through a genome-wide transposon screen, we discovered that a deletion in mneA, which encodes a manganese exporter, restored ROS resistance of the toxR mutant. We then showed that ToxR did not affect mneA transcription but that the ToxR-regulated major porin OmpU was critical for ROS resistance. The addition of manganese in culture medium restored ROS resistance in both the toxR and ompU mutants. Furthermore, elemental analysis indicated that the intracellular concentration of manganese in both the toxR and ompU mutants was reduced. This may result in intracellular ROS accumulation in these mutants. Our data suggest that ToxR plays an important role in the resistance to reactive oxygen species through the regulation of manganese transport.

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Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽

10.1099/mic.0.001098 ◽

2021 ◽

Vol 167 (10) ◽

Author(s):

Mengting Shi ◽

Yue Zheng ◽

Xianghong Wang ◽

Zhengjia Wang ◽

Menghua Yang

Keyword(s):

Mouse Model ◽

Vibrio Cholerae ◽

Biofilm Formation ◽

Type Species ◽

Wild Type ◽

Expression Library ◽

Content Type ◽

Genomic Expression ◽

Infant Mouse ◽

Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

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Host Genetic Factors Associated with Vaginal Microbiome Composition in Kenyan Women

mSystems ◽

10.1128/msystems.00502-20 ◽

2020 ◽

Vol 5 (4) ◽

Cited By ~ 2

Author(s):

Supriya D. Mehta ◽

Drew R. Nannini ◽

Fredrick Otieno ◽

Stefan J. Green ◽

Walter Agingu ◽

...

Keyword(s):

Racial Differences ◽

Genome Wide Association Study ◽

Vaginal Microbiome ◽

Content Type ◽

Microbiome Composition ◽

Genome Wide ◽

A Genome ◽

Kenyan Women ◽

Host Genetic ◽

Women Of African Descent

ABSTRACT Bacterial vaginosis (BV) affects 20% of women worldwide and is associated with adverse reproductive health outcomes and increased risk for HIV. Typically, BV represents a shift in the vaginal microbiome from one that is dominated by Lactobacillus to one that is diverse. Persistent racial differences in BV and diverse vaginal microbiome composition overlap with racial disparities in risks for HIV and sexually transmitted infection, especially among women of African descent. Risk factors for BV and nonoptimal vaginal microbiome include sexual practices, yet racial differences persist when adjusted for behavioral factors, suggesting a host genetic component. Here, we perform a genome-wide association study on vaginal microbiome traits in Kenyan women. Linear regression and logistic regression were performed, adjusting for age and principal components of genetic ancestry, to evaluate the association between Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis, Shannon diversity index, and community state type (CST) with host genetic single nucleotide polymorphisms (SNPs). We identified novel genomic loci associated with the vaginal microbiome traits, though no SNP reached genome-wide significance. During pathway enrichment analysis, Toll-like receptors (TLRs), cytokine production, and other components of innate immune response were associated with L. crispatus, L. iners, and CST. Multiple previously reported genomic loci were replicated, including IL-8 (Shannon, CST), TIRAP (L. iners, Shannon), TLR2 (Shannon, CST), MBL2 (L. iners, G. vaginalis, CST), and MYD88 (L. iners, Shannon). These genetic associations suggest a role for the innate immune system and cell signaling in vaginal microbiome composition and susceptibility to nonoptimal vaginal microbiome. IMPORTANCE Globally, bacterial vaginosis (BV) is a common condition in women. BV is associated with poorer reproductive health outcomes and HIV risk. Typically, BV represents a shift in the vaginal microbiome from one that is dominated by Lactobacillus to one that is diverse. Despite many women having similar exposures, the prevalence of BV and nonoptimal vaginal microbiome is increased for women of African descent, suggesting a possible role for host genetics. We conducted a genome-wide association study of important vaginal microbiome traits in Kenyan women. We identified novel genetic loci and biological pathways related to mucosal immunity, cell signaling, and infection that were associated with vaginal microbiome traits; we replicated previously reported loci associated with mucosal immune response. These results provide insight into potential host genetic influences on vaginal microbiome composition and can guide larger longitudinal studies, with genetic and functional comparison across microbiome sites within individuals and across populations.

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Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death

mBio ◽

10.1128/mbio.02250-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e02250-20

Author(s):

Kathrin Stelzner ◽

Ann-Cathrin Winkler ◽

Chunguang Liang ◽

Aziza Boyny ◽

Carsten P. Ade ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Death ◽

Host Cell ◽

Intracellular Bacterium ◽

Tissue Destruction ◽

Content Type ◽

Host Cell Death ◽

Genome Wide ◽

A Genome ◽

Spread Of Infection

ABSTRACTThe opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

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Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

Infection and Immunity ◽

10.1128/iai.03050-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 1778-1788 ◽

Cited By ~ 41

Author(s):

Eveline M. Weerdenburg ◽

Abdallah M. Abdallah ◽

Farania Rangkuti ◽

Moataz Abd El Ghany ◽

Thomas D. Otto ◽

...

Keyword(s):

Insertion Site ◽

Mycobacterium Marinum ◽

Host Cells ◽

Intracellular Pathogens ◽

Virulence Determinants ◽

Phagocytic Cells ◽

General Hypothesis ◽

Content Type ◽

Genome Wide ◽

A Genome

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogenMycobacterium marinumwhether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show thatM. marinumcontains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore,M. marinumcontains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.

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A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression

Infection and Immunity ◽

10.1128/iai.00411-15 ◽

2015 ◽

Vol 83 (9) ◽

pp. 3381-3395 ◽

Cited By ~ 16

Author(s):

Qiyao Wang ◽

Yves A. Millet ◽

Michael C. Chao ◽

Jumpei Sasabe ◽

Brigid M. Davis ◽

...

Keyword(s):

Gene Expression ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Insertion Site ◽

Virulence Gene ◽

Loss Of Function ◽

Phosphotransferase System ◽

Content Type ◽

Virulence Gene Expression ◽

A Genome

Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression ofVibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression oftcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor. Besides identifying most of the genes known to modulatetcpAexpression, the screen yieldedptsIandptsH, which encode the enzyme I (EI) and Hpr components of theV. choleraephosphoenolpyruvate phosphotransferase system (PTS). In addition to reduced expression of TcpA, strains lacking EI, Hpr, or the associated EIIAGlcprotein produced less cholera toxin (CT) and had a diminished capacity to colonize the infant mouse intestine. The PTS modulates virulence gene expression by regulating expression oftcpPHandaphAB, which themselves control expression oftoxT, the central activator of virulence gene expression. One mechanism by which PTS promotes virulence gene expression appears to be by modulating the amounts of intracellular cyclic AMP (cAMP). Our findings reveal that theV. choleraePTS is an additional modulator of the ToxT regulon and demonstrate the potency of loss-of-function TIS sequencing screens for defining regulatory networks.

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The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00741-15 ◽

2016 ◽

Vol 198 (6) ◽

pp. 973-985 ◽

Cited By ~ 21

Author(s):

Andrew Rogers ◽

Loni Townsley ◽

Ana L. Gallego-Hernandez ◽

Sinem Beyhan ◽

Laura Kwuan ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Secretion System ◽

Lon Protease ◽

Human Pathogen ◽

Type Vi Secretion System ◽

Cyclic Diguanylate ◽

Content Type ◽

Infant Mouse ◽

Type Vi Secretion

ABSTRACTThe presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogenVibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in alonAmutant compared to the wild type. We further demonstrated that alonAmutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence ofV. cholerae.IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogenV. choleraeis in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding ofV. choleraephysiology.

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Production of the antimicrobial compound tetrabromopyrrole and the Pseudomonas quinolone system precursor, 2-heptyl-4-quinolone, by a novel marine species Pseudoalteromonas galatheae sp. nov.

Scientific Reports ◽

10.1038/s41598-020-78439-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Sara Skøtt Paulsen ◽

Thomas Isbrandt ◽

Markus Kirkegaard ◽

Yannick Buijs ◽

Mikael Lenz Strube ◽

...

Keyword(s):

Gene Clusters ◽

Marine Species ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

Halogenated Compound ◽

Genome Wide ◽

A Genome ◽

Rapid Spread ◽

Bioassay Guided Fractionation ◽

Type Strains

AbstractNovel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498T = NCIMB 15250T = LMG 31599T).

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Phylodynamic Analysis of Clinical and Environmental Vibrio cholerae Isolates from Haiti Reveals Diversification Driven by Positive Selection

mBio ◽

10.1128/mbio.01824-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 31

Author(s):

Taj Azarian ◽

Afsar Ali ◽

Judith A. Johnson ◽

David Mohr ◽

Mattia Prosperi ◽

...

Keyword(s):

Positive Selection ◽

Vibrio Cholerae ◽

Short Time Scale ◽

Single Nucleotide ◽

Content Type ◽

Nonsynonymous Substitutions ◽

Genome Wide ◽

Close Phylogenetic Relationship ◽

Phylodynamic Analysis ◽

Over Time

ABSTRACTPhylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates ofVibrio choleraefrom Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenicV. choleraeO1 isolates, four non-O1/O139 isolates, and a novel nontoxigenicV. choleraeO1 isolate with the classicaltcpAgene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenicV. choleraeO1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of the phylogeny showed a steady accumulation of synonymous substitutions and a progressive increase of nonsynonymous substitutions over time, suggesting diversification likely was driven by positive selection. Short-term accumulation of nonsynonymous substitutions driven by selection may have significant implications for virulence, transmission dynamics, and even vaccine efficacy.IMPORTANCECholera, a dehydrating diarrheal disease caused by toxigenic strains of the bacteriumVibrio cholerae, emerged in 2010 in Haiti, a country where there were no available records on cholera over the past 100 years. While devastating in terms of morbidity and mortality, the outbreak provided a unique opportunity to study the evolutionary dynamics ofV. choleraeand its environmental presence. The present study expands on previous work and provides an in-depth phylodynamic analysis inferred from genome-wide single nucleotide polymorphisms of clinical and environmental strains from dispersed geographic settings in Haiti over a 2-year period. Our results indicate that even during such a short time scale,V. choleraein Haiti has undergone evolution and diversification driven by positive selection, which may have implications for understanding the global clinical and epidemiological patterns of the disease. Furthermore, the continued presence of the epidemic strain in Haitian aquatic environments has implications for transmission.

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Vibrio cholerae Type VI Activity Alters Motility Behavior in Mucin

Journal of Bacteriology ◽

10.1128/jb.00261-20 ◽

2020 ◽

Vol 202 (24) ◽

Author(s):

Abby Frederick ◽

Yuhsun Huang ◽

Meng Pu ◽

Dean A. Rowe-Magnus

Keyword(s):

Small Intestine ◽

Vibrio Cholerae ◽

Swimming Speed ◽

Cell Tracking ◽

Structural Genes ◽

Wild Type ◽

Epithelial Surface ◽

Near Surface ◽

Content Type ◽

Distal Small Intestine

ABSTRACT Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS−). In the absence of mucin, wild-type and T6SS− cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS− cells to a lesser extent, and many continued to flick and produce RRF-like traces. ΔcheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucin-induced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface. IMPORTANCE V. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface.

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