Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio...

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Vibrio parahaemolyticus is a significant pathogen causing foodborne illnesses and wound infections.
• The bacterium possesses two putative collagenase genes: vppC and prtV.
• Understanding collagenase production is key to V. parahaemolyticus virulence.

Purpose of the Study:

• To identify and characterize the primary extracellular collagenase produced by V. parahaemolyticus.
• To investigate the role of the vppC gene in collagenase secretion.
• To differentiate between collagenases encoded by vppC and prtV.

Main Methods:

• Cultivation of V. parahaemolyticus under specific conditions (26°C, 3.0% NaCl).
• Detection of collagenolytic activity in culture supernatants.
• Protein analysis using Native polyacrylamide gel electrophoresis (Native-PAGE).
• N-terminal amino acid sequencing of the active collagenase.
• Gene expression analysis using reverse transcriptase PCR (RT-PCR).
• Construction and analysis of a vppC-negative mutant.

Main Results:

• Significant extracellular collagenolytic activity was detected in the early stationary phase.
• A 90-kDa collagenolytic protein, identified as VppC, was purified and sequenced.
• VppC is a truncated form of the full-length protein, lacking 72 N-terminal residues.
• RT-PCR confirmed significant expression of the vppC gene.
• A vppC-negative mutant produced a 50-kDa collagenolytic enzyme, identified as a previously reported serine protease.

Conclusions:

• VppC is the primary extracellular collagenase secreted by V. parahaemolyticus.
• The vppC gene is responsible for the production of this major collagenase.
• VppC likely plays a critical role in the pathogenesis of V. parahaemolyticus infections.