scholarly journals The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity

2012 ◽  
Vol 80 (5) ◽  
pp. 1794-1802 ◽  
Author(s):  
Benjamin Mudrak ◽  
Rita Tamayo
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Phosphate Transport ◽  
Uptake System ◽  
Content Type ◽  
Infant Mouse ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Infectious Form

ABSTRACTVibrio choleraeis the causative agent of the deadly diarrheal disease cholera. As part of its life cycle,V. choleraepersists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derivedV. choleraecells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity inV. cholerae, and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contributions to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in thepst2locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of twopst2components resulted in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm compared to shaking culture. Based on these data, we propose thatV. choleraecells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity inV. choleraewill improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host.

scholarly journals Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae

2015 ◽  
Vol 84 (2) ◽  
pp. 511-523 ◽  
Author(s):  
Eric D. Peng ◽  
Elizabeth E. Wyckoff ◽  
Alexandra R. Mey ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Iron Acquisition ◽  
Growth Conditions ◽  
Growth Defect ◽  
Null Mutant ◽  
Content Type ◽  
Infant Mouse ◽  
Iron Transporter

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number ofV. choleraeiron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter inV. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron sourcein vitro, we demonstrate that heme is not available toV. choleraein the infant mouse intestine.

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

Microbiology ◽  
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.

Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015–2016

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Mark Philip Bugayong ◽  
Hidemasa Izumiya ◽  
Josie M. Bilar ◽  
Masatomo Morita ◽  
Eiji Arakawa ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Type Species ◽  
Variable Number Tandem Repeat ◽  
The Philippines ◽  
Large Chromosome ◽  
Content Type ◽  
Link Type ◽  
Mlva Type ◽  
El Tor

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

scholarly journals Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

2013 ◽  
Vol 81 (9) ◽  
pp. 3068-3076 ◽  
Author(s):  
Carolyn R. Morris ◽  
Christen L. Grassel ◽  
Julia C. Redman ◽  
Jason W. Sahl ◽  
Eileen M. Barry ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Intracellular Growth ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

scholarly journals Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

2012 ◽  
Vol 80 (8) ◽  
pp. 2905-2913 ◽  
Author(s):  
Jessica Queen ◽  
Karla J. Fullner Satchell
Keyword(s):  
Immune Response ◽  
Vibrio Cholerae ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Infection Model ◽  
Proinflammatory Response ◽  
Necrosis Factor Alpha ◽  
Live Attenuated Vaccine ◽  
Content Type

ABSTRACTCholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection withVibrio choleraeand a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1V. choleraeinfection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1β and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.

scholarly journals Type III Secretion Is Essential for the Rapidly Fatal Diarrheal Disease Caused by Non-O1, Non-O139 Vibrio cholerae

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Ok S. Shin ◽  
Vincent C. Tam ◽  
Masato Suzuki ◽  
Jennifer M. Ritchie ◽  
Roderick T. Bronson ◽  
...  
Keyword(s):  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Intestinal Epithelium ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iii ◽  
Severe Diarrhea ◽  
Toxin Coregulated Pilus

ABSTRACTCholera is a severe diarrheal disease typically caused by O1 serogroup strains ofVibrio cholerae. The pathogenicity of all pandemicV. choleraeO1 strains relies on two critical virulence factors: cholera toxin, a potent enterotoxin, and toxin coregulated pilus (TCP), an intestinal colonization factor. However, certain non-O1, non-O139V. choleraestrains, such as AM-19226, do not produce cholera toxin or TCP, yet they still cause severe diarrhea. The molecular basis for the pathogenicity of non-O1, non-O139V. choleraehas not been extensively characterized, but many of these strains encode related type III secretion systems (TTSSs). Here, we used infant rabbits to assess the contribution of the TTSS to non-O1, non-O139V. choleraepathogenicity. We found that all animals infected with wild-type AM-19226 developed severe diarrhea even more rapidly than rabbits infected withV. choleraeO1. UnlikeV. choleraeO1 strains, which do not damage the intestinal epithelium in rabbits or humans, AM-19226 caused marked disruptions of the epithelial surface in the rabbit small intestine. TTSS proved to be essential for AM-19226 virulence in infant rabbits; an AM-19226 derivative deficient for TTSS did not elicit diarrhea, colonize the intestine, or induce pathological changes in the intestine. Deletion of either one of the two previously identified or two newly identified AM-19226 TTSS effectors reduced but did not eliminate AM-19226 pathogenicity, suggesting that at least four effectors contribute to this strain’s virulence. In aggregate, our results suggest that the TTSS-dependent virulence in non-O1, non-O139V. choleraerepresents a new type of diarrheagenic mechanism.IMPORTANCECholera, which is caused byVibrio cholerae, is an important cause of diarrheal disease in many developing countries. The mechanisms of virulence of nonpandemic strains that can cause a diarrheal illness are poorly understood. AM-19226, like several other pathogenic, nonpandemicV. choleraestrains, carries genes that encode a type III secretion system (TTSS), but not cholera toxin (CT) or toxin coregulated pilus (TCP). In this study, we used infant rabbits to study AM-19226 virulence. Infant rabbits orally inoculated with this strain rapidly developed a fatal diarrheal disease, which was accompanied by marked disruptions of the intestinal epithelium. This strain’s TTSS proved essential for its pathogenicity, and there was no diarrhea, intestinal pathology, or colonization in rabbits infected with a TTSS mutant. The effector proteins translocated by the TTSS all appear to contribute to AM-19226 virulence. Thus, our study provides insight intoin vivomechanisms by which a novel TTSS contributes to diarrheal disease caused by nonpandemic strains ofV. cholerae.

scholarly journals Molecular Characterization of ISCR1-MediatedblaPER-1in a Non-O1, Non-O139 Vibrio cholerae Strain from China

2015 ◽  
Vol 59 (7) ◽  
pp. 4293-4295 ◽  
Author(s):  
Jun Wu ◽  
Lianyan Xie ◽  
Fangfang Zhang ◽  
Yuxing Ni ◽  
Jingyong Sun
Keyword(s):  
Vibrio Cholerae ◽  
Molecular Characterization ◽  
Cholerae Strain ◽  
Class 1 Integron ◽  
Content Type ◽  
Conserved Sequence ◽  
Circular Molecule ◽  
Class 1 ◽  
Insight Into

ABSTRACTWe report the detection of PER-1 extended-spectrum β-lactamase (ESBL) in a clinical non-O1, non-O139Vibrio choleraestrain from China. ISCR1-mediatedblaPER-1was embedded in a complex In4family class 1 integron belonging to the lineage of Tn1696on a conjugative IncA/C plasmid. A free 8.98-kb circular molecule present with the ISCR1-blaPER-1–truncated 3′-conserved sequence (CS) structure was detected in this isolate. These findings may provide insight into the mobilization ofblaPER-1.

scholarly journals Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Eduardo Soto ◽  
Norma Espinosa ◽  
Miguel Díaz-Guerrero ◽  
Meztlli O. Gaytán ◽  
José L. Puente ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Functional Characterization ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Content Type ◽  
Enterocyte Effacement

ABSTRACT The type III secretion system (T3SS) is a supramolecular machine used by many bacterial pathogens to translocate effector proteins directly into the eukaryotic host cell cytoplasm. Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrheal disease in underdeveloped countries. EPEC virulence relies on a T3SS encoded within a chromosomal pathogenicity island known as the locus of enterocyte effacement (LEE). In this work, we pursued the functional characterization of the LEE-encoded protein EscK (previously known as Orf4). We provide evidence indicating that EscK is crucial for efficient T3S and belongs to the SctK (OrgA/YscK/MxiK) protein family, whose members have been implicated in the formation of a sorting platform for secretion of T3S substrates. Bacterial fractionation studies showed that EscK localizes to the inner membrane independently of the presence of any other T3SS component. Combining yeast two-hybrid screening and pulldown assays, we identified an interaction between EscK and the C-ring/sorting platform component EscQ. Site-directed mutagenesis of conserved residues revealed amino acids that are critical for EscK function and for its interaction with EscQ. In addition, we found that T3S substrate overproduction is capable of compensating for the absence of EscK. Overall, our data suggest that EscK is a structural component of the EPEC T3SS sorting platform, playing a central role in the recruitment of T3S substrates for boosting the efficiency of the protein translocation process. IMPORTANCE The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

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

2016 ◽  
Vol 198 (6) ◽  
pp. 973-985 ◽  
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.

scholarly journals A Small Unstructured Region in Vibrio cholerae ToxT Mediates the Response to Positive and Negative Effectors and ToxT Proteolysis

2014 ◽  
Vol 197 (3) ◽  
pp. 654-668 ◽  
Author(s):  
Joshua J. Thomson ◽  
Sarah C. Plecha ◽  
Jeffrey H. Withey
Keyword(s):  
Vibrio Cholerae ◽  
Virulence Factors ◽  
Unsaturated Fatty Acids ◽  
Diarrheal Disease ◽  
Virulence Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Terminal Domain ◽  
Virulence Gene Regulation ◽  
Host Signals

Vibrio choleraeis the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

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