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 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 Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

2015 ◽  
Vol 83 (4) ◽  
pp. 1246-1256 ◽  
Author(s):  
Amable J. Rivas ◽  
Ana Vences ◽  
Matthias Husmann ◽  
Manuel L. Lemos ◽  
Carlos R. Osorio
Keyword(s):  
Secretion System ◽  
Transcriptional Level ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Marine Animals ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Photobacterium Damselae ◽  
Prepilin Peptidase

Photobacterium damselaesubsp.damselaeis a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion inepsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement ofepsLin HlyAchsecretion. In addition, mutation ofepsLin a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating thatepsLplays a major role in secretion of the plasmid-encoded HlyApland Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase genepilDseverely affected hemolysis, which dropped at levels inferior to those ofepsLmutants. Promoter expression analyses suggested that impairment of hemolysin secretion inepsLandpilDmutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, singleepsLandpilDmutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS andpilDinP. damselaesubsp.damselaevirulence.

scholarly journals Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton

2016 ◽  
Vol 82 (14) ◽  
pp. 4441-4452 ◽  
Author(s):  
Loni Townsley ◽  
Marilou P. Sison Mangus ◽  
Sanjin Mehic ◽  
Fitnat H. Yildiz
Keyword(s):  
Low Temperature ◽  
Vibrio Cholerae ◽  
Causative Agent ◽  
Biofilm Formation ◽  
Cold Shock ◽  
Diarrheal Disease ◽  
Dependent Manner ◽  
Natural Habitats ◽  
Content Type ◽  
Type Vi Secretion

ABSTRACTThe ability to sense and adapt to temperature fluctuation is critical to the aquatic survival, transmission, and infectivity ofVibrio cholerae, the causative agent of the disease cholera. Little information is available on the physiological changes that occur whenV. choleraeexperiences temperature shifts. The genome-wide transcriptional profile ofV. choleraeupon a shift in human body temperature (37°C) to lower temperatures, 15°C and 25°C, which mimic those found in the aquatic environment, was determined. Differentially expressed genes included those involved in the cold shock response, biofilm formation, type VI secretion, and virulence. Analysis of a mutant lacking the cold shock genecspV, which was upregulated >50-fold upon a low-temperature shift, revealed that it regulates genes involved in biofilm formation and type VI secretion. CspV controls biofilm formation through modulation of the second messenger cyclic diguanylate and regulates type VI-mediated interspecies killing in a temperature-dependent manner. Furthermore, a strain lackingcspVhad significant defects for attachment and type VI-mediated killing on the surface of the aquatic crustaceanDaphnia magna. Collectively, these studies reveal thatcspVis a major regulator of the temperature downshift response and plays an important role in controlling cellular processes crucial to the infectious cycle ofV. cholerae.IMPORTANCELittle is known about how human pathogens respond and adapt to ever-changing parameters of natural habitats outside the human host and how environmental adaptation alters dissemination.Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, experiences fluctuations in temperature in its natural aquatic habitats and during the infection process. Furthermore, temperature is a critical environmental signal governing the occurrence ofV. choleraeand cholera outbreaks. In this study, we showed thatV. choleraereprograms its transcriptome in response to fluctuations in temperature, which results in changes to biofilm formation and type VI secretion system activation. These processes in turn impact environmental survival and the virulence potential of this pathogen.

scholarly journals Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2

2015 ◽  
Vol 83 (10) ◽  
pp. 3825-3837 ◽  
Author(s):  
Ashley L. DuMont ◽  
Sara M. Karaba ◽  
Nicholas P. Cianciotto
Keyword(s):  
Serine Proteases ◽  
Stenotrophomonas Maltophilia ◽  
A549 Cells ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Cell Rounding ◽  
Type Ii Secretion System

Stenotrophomonas maltophiliais an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported thatS. maltophiliastrain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps- and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of theS. maltophiliaXps type II secretion system and its StmPr1 and StmPr2 substrates.

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 Intracellular and Extracellular Markers of Lethality in Osteogenesis Imperfecta: A Quantitative Proteomic Approach

2021 ◽  
Vol 22 (1) ◽  
pp. 429
Author(s):  
Luca Bini ◽  
Domitille Schvartz ◽  
Chiara Carnemolla ◽  
Roberta Besio ◽  
Nadia Garibaldi ◽  
...  
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Type I ◽  
Tandem Mass ◽  
Type Ii ◽  
Type Iii ◽  
Bioinformatic Tools ◽  
Proteomic Approach ◽  
Fibrillin 1 ◽  
Heritable Disorder

Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen α chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in α1 or α2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non–collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.

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