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

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

doi:10.1371/journal.pone.0013122

Aurelia Syngkon, Rupak K Bhadra + Show 8 more

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https://doi.org/10.1371/journal.pone.0013122

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Abstract

BackgroundTwo well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).Methodology/Principal FindingsWe partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.ConclusionOur results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

Highlights

Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins or peptides
We have reported that hemagglutinin protease (HAP) may play a role in the pathogenesis of a ctx-negative V. cholerae non-O1, non-O139 strain by inducing a hemorrhagic fluid response in the rabbit ileal loop model (RIL) assay [12]
The proteins from culture supernatants of the above strains were precipitated with ammonium sulphate and after dialysis, 30 mg of crude proteins were tested for protease activity by azocasein assay

Summary

Introduction

Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins or peptides. Microbial peptides are predominantly secreted enzymes and can be classified based on the essential catalytic residue at their active site. They include serine proteases, cysteine proteases, aspartate proteases and metalloproteases. The protease activity in V. cholerae vaccine strains reduced the transcellular epithelial resistance of polarized T84 intestinal epithelial cells [6]. These results suggested a role of HAP in reactogenicity, including inflammatory diarrhea. We initiated this study to characterize the protease present in CHA6.8DprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL)

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