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

BACKGROUND: Two 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 FINDINGS: We 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. CONCLUSION: Our 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

Rabbit ileal loop assay.

<p>A) RIL response of partially purified protease (50 µg, NB) showing significant hemorrhagic fluid accumulation (FA ratio 1.2+/−0.2 n = 3) and its effect after inhibition with 25 mM PMSF and 10 mM EDTA (NB+PMSF+EDTA) shows significant decrease in fluid accumulation (FA ratio 0.3+/−0.05 n = 3). Twenty five mM Tris-HCl with 25 mM PMSF +10 mM EDTA was used as a negative control (FA ratio = 0.12+/−0.002, n = 3). B) RIL response with culture supernatants of C6709 (FA ratio 1.1+/−0.3, n = 3), CHA6.8 (FA ratio 1.08+/−0.2, n = 3), CHA6.8Δ<i>prtV</i> (FA ratio 1.02+/−0.2, n = 3), CHA6.8Δ<i>prtV</i>ΔVC1649 (FA ratio 0.11+/−0.005, n = 3) and Tryptic soy broth as negative control (FA ratio 0.09+/−0.002, n = 3).</p

Partial purification and identification of protease.

<p>Chromatographic profile of ammonium sulphate precipitated crude proteins from culture supernatants of CHA6.8Δ<i>prtV</i> strain loaded onto an anion exchange column (DE-52). A) Proteins eluted in the non-binding fraction (NB), B) proteins eluted with 0.1 M NaCl, C) proteins eluted with 0.3 M NaCl, +/− shows presence or absence of protease activity, D) azocasein assay with pooled samples (30 µg) NB, 0.1 M#1, 0.1 M #2, 0.3 M and crude proteins. E) Native PAGE profile (lane 1) of crude proteins of CHA6.8Δ<i>prtV</i> strain and (lane 2) of partially purified protease (NB) from DE-52 column. The marked protein band was analyzed by MS/MS sequencing and the peptides highlighted showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. F) The underlined GDSGGP are the amino acid sequences around the serine residue present in trypsin-like serine proteases. G) Protease inhibition test of NB fraction (5 µg) with protease inhibitors 10 mM EDTA, 25 mM PMSF, 25 mM PMSF and 10 mM EDTA, 10 mM EDTA and 20 mM CaCl₂, 10 mM EGTA, 1 µg/ml aprotinin, 28 mM E64, 1 µg/ml leupeptin and 10 mM 1,10-phenanthroline incubated for 30 mins at 37°C. Residual protease activity was assayed by azocasein assay. Twenty-five mM Tris-HCl was used as a negative control. The values shown are the means with standard deviations from three experiments.</p

Histopathological study of ileal tissues.

<p>Panels show photomicrographs of histology of rabbit ileal loop tissue after treatment with A) Partially purified serine protease from <i>V. cholerae</i> strain CHA6.8Δ<i>prtV</i> showing hemorrhagic fluid accumulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, NB). Gross damage of the villus surface structure was observed with hemorrhage in all layers of the mucosa. Magnification, 20X. B) Almost normal villous architecture observed in ileal tissues treated with 50 µg of partially purified protease inhibited with 25 mM PMSF and 10 mM EDTA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, NB+PMSF+EDTA). This photomicrograph shows no gross alteration in villus structure but villus lamina propria are slightly dilated and RBC have accumulated at a few places in the basal area. Magnification, 20X. C) Ileal tissues treated with 25 mM Tris-HCl buffer with PMSF and EDTA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-g004" target="_blank">Fig. 4A</a>, control) showed normal villus structure. Magnification 20X. (D) ileal tissues treated with culture supernatant from C6709 strain showed presence of hemorrhage in all layers of the gut mucosa specially in the submucosal layer, Magnification 20X. E) ileal tissues treated with culture supernatant from CHA6.8 strain showed widely dialated villi with rupture at places with gross hemorrhage and inflammatory cells in mucosa and submucosa, Magnification 20X. F) Ileal tissues treated with culture supernatant of CHA6.8Δ<i>prtV</i> strain also showing dilated villi with gross hemorrhage in all layers of the mucosa. Magnification 20X. G) The same section in higher magnification 40X showing ruptured villi with hemorrhage and inflammatory cells in mucosa and submucosa. (H) ileal tissues treated with culture supernatant from CHA6.8Δ<i>prtV</i>ΔVC1649 strain showing villous architecture almost normal with minimum hemorrhage in mucosa and submucosa. (I) TSB treated ileal tissue showing normal gut mucosa.</p

Protease activity assay.

<p>A) Azocasein assay with 30 µg of ammonium sulphate precipitated proteins from culture supernatants of C6709, CHA6.8, CHA6.8Δ<i>prtV</i> and CHA6.8Δ<i>prtV</i>ΔVC<i>1649</i> and inhibition test with 25 mM PMSF, 10 mM EDTA and 10 mM 1,10- phenanthroline. Negative controls were (1) 25 mM Tris-HCl and 25 mM Tris-HCl in the presence of (2) 25 mM PMSF, (3) 10 mM EDTA and (4) 10 mM 1,10- phenanthroline. The values shown are the means with standard deviations from three experiments. B) Skim milk assay for detection of protease in C6709, CHA6.8, CHA6.8Δ<i>prtV</i> and CHA6.8Δ<i>prtV</i>ΔVC<i>1649</i> strains.</p

Bacterial strains, plasmids, primers and oligonucleotides used in this study.

<p>Bacterial strains, plasmids, primers and oligonucleotides used in this study.</p

Confirmation of knock out mutant.

<p>A) PCR amplification with internal primers for <i>ctx</i> (lane 1), <i>hapA</i> (lane 3), <i>prtV</i> (lane 5) and VC1649 (lane 7) in strain C6709 and for <i>ctx</i> (lane 2), <i>hapA</i> (lane 4), <i>prtV</i> (lane 6) and VC1649 (lane 8) in strain CHA6.8Δ<i>prtV</i>ΔVC1649. The primer sequence used in the above experiment is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013122#pone-0013122-t001" target="_blank">Table 1</a>. (MW) denotes molecular weight marker (20 kb-75 bp marker, Fermentas). B) Native PAGE (10%) with ammonium sulphate precipitated proteins of C6709 (lane 1), CHA6.8 (lane 2), CHA6.8Δ<i>prtV</i> (lane 3) and CHA6.8Δ<i>prtV</i>ΔVC1649 (lane 4). The * shows the protein band with sequence homology to the 59-kDa serine protease (VC1649). This band is absent in strain CHA6.8Δ<i>prtV</i>ΔVC1649 (lane 4).</p