Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses

Abstract

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.

FIG. 1.

(A) Fresh collected chironomid egg mass. The eggs are embedded in a gelatinous material. The eggs are in a chain, which is folded into loops that form a spiral. (B) Egg mass incubated for 2 h with the supernatant of a 48-h culture of V. cholerae O9. The eggs were freed from the degraded gelatinous matrix. Original magnifications, ×40 (egg mass size, ca. 5 by 20 mm).

FIG. 2.

Egg mass degradation bioassay in a 96-well microplate. An egg mass incubated at 37°C with V. cholerae O1 supernatant for 2 h is shown on the left, and an egg mass incubated at 37°C with V. cholerae O1 HA/P null mutant 638 supernatant for 2 h is shown on the right.

FIG. 3.

(a and b) SDS-PAGE gel stained with ASR (a) and silver stain (b). (c) Western blot analysis with anti-HA/P antibody. After SDS-PAGE, the samples were transferred onto a nitrocellulose membrane, and HA/P was detected with the antibody against HA/P from V. cholerae O9. Lane 1, egg mass control; lanes 2 to 4, V. cholerae O1 supernatant incubated at 37°C (lane 2, supernatant control without egg mass; lane 3, supernatant with egg mass incubated for 10 min; lane 4, supernatant with egg mass incubated for 120 min); lane 5, broad-range size markers (200, 116, 97, 66, 45, 31, 21, and 14 kDa; Bio-Rad); lanes 6 to 8, V. cholerae O1 HA/P null mutant 638 supernatant incubated at 37°C (lane 6, supernatant control without egg mass; lane 7, supernatant with egg mass incubated for 10 min; lane 8, supernatant with egg mass incubated for 120 min). All V. cholerae supernatant samples contained 20 μg of protein.

FIG. 4.

Chironomid egg mass stained with ASR (egg size, ca. 70 by 250 μm).