scholarly journals Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Rudolph E. Sloup ◽  
Ashley E. Konal ◽  
Geoffrey B. Severin ◽  
Michelle L. Korir ◽  
Mira M. Bagdasarian ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Protein Complexes ◽  
Second Messenger ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface. IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.

scholarly journals Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Chelsea S. Rule ◽  
Young-Jun Park ◽  
Jaclyn R. Delarosa ◽  
Stewart Turley ◽  
Wim G. J. Hol ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Envelope ◽  
Envelope Proteins ◽  
Growth Defect ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Transport Pathway ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding

ABSTRACT The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae. Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC. IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

scholarly journals The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by σE

2014 ◽  
Vol 82 (7) ◽  
pp. 2788-2801 ◽  
Author(s):  
Ryszard A. Zielke ◽  
Ryan S. Simmons ◽  
Bo R. Park ◽  
Mariko Nonogaki ◽  
Sarah Emerson ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Growth Phase ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Transcriptional Expression ◽  
Content Type ◽  
Expression Of Genes ◽  
Cargo Proteins ◽  
Genes Encoding ◽  
Kinetics Of

ABSTRACTVibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughoutV. choleraegrowth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure ofV. choleraeto different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of aV. choleraegenomic library suggested that σEstress response, phosphate metabolism, and the second messenger 3′,5′-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σE, we discovered that the upstream region of the T2S operon possesses both the consensus σEand σ70signatures, and deletion of the σEbinding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σEstimulated transcription of T2S in wild-type and isogenic ΔrpoEstrains ofV. cholerae, providing additional support for the idea that the T2S complex belongs to the σEregulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness ofV. choleraein different ecological niches.

scholarly journals Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

2015 ◽  
Vol 11 (10) ◽  
pp. e1005232 ◽  
Author(s):  
Kevin G. Roelofs ◽  
Christopher J. Jones ◽  
Sarah R. Helman ◽  
Xiaoran Shang ◽  
Mona W. Orr ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Binding Proteins ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Systematic Identification

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 A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

2015 ◽  
Vol 197 (6) ◽  
pp. 1051-1064 ◽  
Author(s):  
Bo R. Park ◽  
Ryszard A. Zielke ◽  
Igor H. Wierzbicki ◽  
Kristie C. Mitchell ◽  
Jeffrey H. Withey ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Type I Collagen ◽  
Diarrheal Disease ◽  
Type Ii Secretion ◽  
Type I ◽  
Type Ii ◽  
Marine Animals ◽  
Natural Habitats ◽  
Content Type ◽  
Functional Components

Vibrio choleraeis autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence ofV. choleraein natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studiedV. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues andV. choleraehas been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of theV. choleraeputative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose thatV. choleraeis a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of theV. choleraecollagen utilization program.

scholarly journals YghG (GspSβ) Is a Novel Pilot Protein Required for Localization of the GspSβType II Secretion System Secretin of Enterotoxigenic Escherichia coli

2012 ◽  
Vol 80 (8) ◽  
pp. 2608-2622 ◽  
Author(s):  
Timothy G. Strozen ◽  
Gang Li ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Secretion System ◽  
Enterotoxigenic Escherichia Coli ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Content Type ◽  
Insertional Inactivation ◽  
Prepilin Peptidase

ABSTRACTThe enterotoxigenicEscherichia coli(ETEC) pathotype, characterized by the prototypical strain H10407, is a leading cause of morbidity and mortality in the developing world. A major virulence factor of ETEC is the type II secretion system (T2SS) responsible for secretion of the diarrheagenic heat-labile enterotoxin (LT). In this study, we have characterized the two type II secretion systems, designated alpha (T2SSα) and beta (T2SSβ), encoded in the H10407 genome and describe the prevalence of both systems in otherE. colipathotypes. Under laboratory conditions, the T2SSβis assembled and functional in the secretion of LT into culture supernatant, whereas the T2SSαis not. Insertional inactivation of the three genes located upstream ofgspCβ(yghJ,pppA, andyghG) in the atypical T2SSβoperon revealed that YghJ is not required for assembly of the GspDβsecretin or secretion of LT, that PppA is likely the prepilin peptidase required for the function of T2SSβ, and that YghG is required for assembly of the GspDβsecretin and thus function of the T2SSβ. Mutational and physiological analysis further demonstrated that YghG (redesignated GspSβ) is a novel outer membrane pilotin protein that is integral for assembly of the T2SSβby localizing GspDβto the outer membrane, whereupon GspDβforms the macromolecular secretin multimer through which T2SSβsubstrates are translocated.

scholarly journals Specificity of the Type II Secretion Systems of Enterotoxigenic Escherichia coli and Vibrio cholerae for Heat-Labile Enterotoxin and Cholera Toxin

2010 ◽  
Vol 192 (7) ◽  
pp. 1902-1911 ◽  
Author(s):  
Benjamin Mudrak ◽  
Meta J. Kuehn
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Vibrio Cholerae ◽  
Enterotoxigenic Escherichia Coli ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Wild Type ◽  
Secretion Systems ◽  
B Subunit ◽  
Heat Labile

ABSTRACT The Gram-negative type II secretion (T2S) system is a multiprotein complex mediating the release of virulence factors from a number of pathogens. While an understanding of the function of T2S components is emerging, little is known about what identifies substrates for export. To investigate T2S substrate recognition, we compared mutations affecting the secretion of two highly homologous substrates: heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli (ETEC) and cholera toxin (CT) from Vibrio cholerae. Each toxin consists of one enzymatic A subunit and a ring of five B subunits mediating the toxin's secretion. Here, we report two mutations in LT's B subunit (LTB) that reduce its secretion from ETEC without global effects on the toxin. The Q3K mutation reduced levels of secreted LT by half, and as with CT (T. D. Connell, D. J. Metzger, M. Wang, M. G. Jobling, and R. K. Holmes, Infect. Immun. 63:4091-4098, 1995), the E11K mutation impaired LT secretion. Results in vitro and in vivo show that these mutants are not degraded more readily than wild-type LT. The Q3K mutation did not significantly affect CT B subunit (CTB) secretion from V. cholerae, and the E11A mutation altered LT and CTB secretion to various extents, indicating that these toxins are identified as secretion substrates in different ways. The levels of mutant LTB expressed in V. cholerae were low or undetectable, but each CTB mutant expressed and secreted at wild-type levels in ETEC. Therefore, ETEC's T2S system seems to accommodate mutations in CTB that impair the secretion of LTB. Our results highlight the exquisitely fine-tuned relationship between T2S substrates and their coordinate secretion machineries in different bacterial species.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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