Enterotoxigenicity of mature 45-kilodalton and processed 35-kilodalton forms of hemagglutinin protease purified from a cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain

Abstract

Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 microg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 microg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21.

FIG. 1.

Chromatographic profiles showing purification of 35-kDa HAP from the culture supernatant of V. cholerae PL-21 strain. (a) Gel filtration chromatography column on G-200 column with ammonium sulfate-precipitated proteins. (b) Anion-exchange chromatography on DE-52 column of pooled fractions showing HA, protease, and LAP activity from G-200 column. (c) Gel filtration chromatography on G-75 column of pooled fractions of the second peak from DE-52 column. The presence of HA (++), protease (00), aminopeptidase (**), and lipopolysaccharide (xx) activity in the peaks is indicated. Absorbance is shown in arbitrary units (AU). (d) SDS-PAGE (12.5%) of the proteins from the two peaks in Fig. 1b. Lanes 1 and 2 contain the proteins from the first and second peaks, respectively. (e) SDS-PAGE (12.5%) of the protein from the single peak in Fig. 1c. The positions (in kilodaltons) of molecular mass markers (MM) are shown to the left of the gels.

FIG. 2.

Chromatographic profiles showing purification of 35-kDa HAP-B from DE-52 column (run in the absence of EDTA) by NaCl gradient (0 to 1 M). (a) The first peak eluted showed HA (++), protease (00), and LAP (**) activity. Absorbance is shown in arbitrary units (AU). (b) SDS-PAGE (12.5%) of the proteins from the first peak showed the presence of a single 35-kDa band. The positions (in kilodaltons) of molecular mass markers (MM) are shown to the left of the gel.

FIG. 3.

Chromatographic profiles showing purification of 45-kDa HAP purified in the presence of EDTA. (a) Gel filtration chromatography on G-75 column of the pooled fractions from successively chromatographed proteins by G-200 and DE-52 columns. Absorbance is shown in arbitrary units (AU). (b) SDS-PAGE (12.5%) of the major peak showed the presence of 45- and 42-kDa bands. The positions (in kilodaltons) of molecular mass markers (MM) are shown to the left of the gel.

FIG. 4.

RIL response of HAP purified from V. cholerae non-O1, non-O139 strain PL-21. Dose-dependent response with G-200 pool 2 (A), 35-kDa HAP (B), 35-kDa HAP-B (C), and 45-kDa HAP (D). Values are means and standard deviations (error bars) for two experiments. The absence of hemorrhagic FA (*) is indicated.

FIG. 5.

Effect of purified HAP in the RIL assay. Purified HAP-treated ileal tissues were processed for histopathological analysis, and photomicrographs were taken. (A) Villous architecture observed in ileal tissues treated with 25 mM Tris-HCl. This photomicrograph shows normal villous structure. Magnification, ×10. (B) Rabbit ileal tissues treated with 40 μg of purified 35-kDa HAP show disruption of normal villous architecture with shortening of the villi. Magnification, ×20. (C) Magnified view of Fig. 4B shows infiltration of polymorphonuclear neutrophils, eosinophils, and erythrocytes. Magnification, ×40. (D) The black box in Fig. 4C was further magnified to show the presence of red blood cells (R), neutrophils (N), and eosinophils (E). Magnification, ×200.

FIG. 6.

Dose-dependent response of HAP on the intestinal short-circuit current of rat ileum loaded in a Ussing chamber. HAP was added in the absence or presence of EDTA to the Ringer's solution in an Ussing chamber, and the change in Isc was recorded for the next 2 h from the time the sample was added. (a) Response of Isc to increasing doses of 35-kDa HAP. (b) Response of Isc to increasing doses of 45-kDa HAP. There is an initial increase, but the Isc falls as the dose reaches beyond 40 μg/ml. Values are the means ± standard deviations (error bars) for triplicate experiments. Proteins purified from E. coli DH5α were used as controls.

FIG. 7.

Effects of HAP on HeLa cells. HeLa cells grown in tissue culture dishes were treated overnight with different concentrations of either the 35-kDa HAP or the 45-kDa HAP. Cells were observed with a phase-contrast microscope. (A) Cell rounding effect of 35-kDa HAP and a higher concentration of 45-kDa HAP. (B) Cell distending effect of a lower concentration of 45-kDa HAP (concentrations given in Table 1). (C) Normal HeLa cells grown in tissue culture plates.