Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Syngkon, Aurelia; Elluri, Sridhar; Koley, Hemanta; Rompikuntal, Pramod Kumar; Saha, Dhira Rani; Chakrabarti, Manoj K.; Bhadra, Rupak K.; Wai, Sun Nyunt; Pal, Amit

doi:10.1371/journal.pone.0013122

Cited by 41 publications

(28 citation statements)

References 41 publications

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“…These data indicated that all three proteases displayed enzymatic activities and were capable of hydrolyzing the synthetic peptide, albeit to different degrees. Consistent with our findings, during the preparation of this work, the enzymatic activity of VesC toward azocasein was reported (73). Next, to obtain further support for the suggestion that the Ves proteins are serine proteases, we examined the effects of AEBSF (serine protease inhibitor) and leupeptin (serine/cysteine protease inhibitor) on the proteolytic activity of VesA, VesB, and VesC.…”

Section: Cholerae Secretes a Variety Of Proteins In Rich Medium-supporting

confidence: 86%

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Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Sikora¹,

Zielke²,

Lawrence

et al. 2011

Journal of Biological Chemistry

107

143

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The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many Gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase. Here, for the first time, we have applied proteomic approaches, including isotope tagging for relative and absolute quantification coupled with multidimensional liquid chromatography and tandem mass spectrometry, to perform an unbiased and comprehensive analysis of proteins secreted by the T2S apparatus of the V. cholerae El Tor strain N16961 under standard laboratory growth conditions. This analysis identified 16 new putative T2S substrates, including sialidase, several proteins participating in chitin utilization, two aminopeptidases, TagA-related protein, cytolysin, RbmC, three hypothetical proteins encoded by VCA0583, VCA0738, and VC2298, and three serine proteases VesA, VesB, and VesC. Focusing on the initial characterization of VesA, VesB, and VesC, we have confirmed enzymatic activities and T2S-dependent transport for each of these proteases. In addition, analysis of single, double, and triple protease knock-out strains indicated that VesA is the primary protease responsible for processing the A subunit of cholera toxin during in vitro growth of the V. cholerae strain N16961.Gram-negative bacteria have evolved at least six secretion pathways devoted to the transport of proteins through the cell envelope into either the extracellular environment or directly into host cells (1, 2). The type II secretion (T2S) 2 system was first discovered in Klebsiella oxytoca and has been shown to be widely distributed among ␥-proteobacteria (3-6). Depending on the bacterial species, the T2S complex consists of 12-16 different constituents that form a multiprotein apparatus spanning the entire cell envelope (7,8). The conserved components of the T2S machinery include the cytoplasmic ATPase (T2S E), the inner membrane platform (T2S C, F, L, and M), a pilus-like structure (T2S G-K), a protein responsible for the processing of pseudopilins (T2S O), and the secretion pore (T2S D) embedded in the outer membrane (9). The exoprotein precursors are synthesized with N-terminal signal peptides that direct them into the periplasmic space via either the Sec or Tat transport systems (10, 11). After obtaining tertiary conformation, the exoproteins enter the T2S machinery and are subsequently translocated into the extracellular milieu (12, 13). Many key steps in the secretion process are still not well understood, including how the exoproteins are recognized by the T2S system, and a specific secretion signal common to known substrates has not yet been identified....

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Section: Cholerae Secretes a Variety Of Proteins In Rich Medium-supporting

confidence: 86%

“…5 and 6). However, a very recent study has shown that VesC induces a hemorrhagic response when injected into rabbit ileal loops suggesting that it may play a role in pathogenesis (73). As we have showed here; however, this activity does not seem to be required for intestinal colonization of mice.…”

Section: Discussioncontrasting

confidence: 43%

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Sikora¹,

Zielke²,

Lawrence

et al. 2011

Journal of Biological Chemistry

107

143

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“…Initially ligated loops of rabbit small intestine were used as a model to assess the contribution of putative virulence factors to bacterial pathogen-induced diarrhea. This model was used for Vibrio cholerae, where the injection of whole cultures (24,25), culture supernatants (26), and cell extracts (27) caused dilation of the loop due to fluid accumulation. The rabbit ileal loop model also has been used to study pathogenesis of Escherichia coli (28)(29)(30), Salmonella (20,31,32), Shigella (33), Pseudomonas aeruginosa (34), Clostridium perfringens (35), Vibrio parahaemolyticus, Vibrio alcaligenes (36), and Bacillus cereus (37).…”

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confidence: 99%

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

et al. 2014

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e Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown. Edwardsiella ictaluri is a primitive member of the Enterobacteriaceae family that causes enteric septicemia in channel catfish (Ictalurus punctatus). E. ictaluri infects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level, E. ictaluri pathogenesis is relatively well characterized, but our knowledge about E. ictaluri intestinal interaction is limited. Recently, we observed that E. ictaluri oligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects of E. ictaluri O-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated with Salmonella enterica serovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses to E. ictaluri and S. Typhimurium LPS. We determined that catfish respond to E. ictaluri LPS but not to S. Typhimurium LPS. We also determined that E. ictaluri inhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. The E. ictaluri wild type and ⌬wibT LPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast to E. ictaluri ⌬gne and ⌬ugd LPS mutants. We concluded that the E. ictaluri O-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure of E. ictaluri mutants is needed to understand the mechanism of interaction.

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“…In contrast, at high cell density (HCD), the intracellular HapR concentration is increased, leading to repression of the above-described processes and upregulation of expression of the hemagglutinin protease gene hapA, which codes for the major protease HAP of V. cholerae. Several reports indicate that HAP is most likely involved in V. cholerae's pathogenesis program (7,16,35,47), including its role in detaching adhered V. cholerae cells on intestinal epithelial cell surfaces.…”

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confidence: 99%

Functional Characterization of the Stringent Response Regulatory GenedksAof Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes

et al. 2012

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In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA Vc ), coding for the protein DksA Vc . Extensive genetic analyses of the ⌬dksA Vc mutants suggest that DksA Vc is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA Vc positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA Vc upregulates expression of the sigma factor FliA ( 28 ), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA Vc also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

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Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Cited by 41 publications

References 41 publications

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut

Functional Characterization of the Stringent Response Regulatory GenedksAof Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes

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