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Infection and Immunity, May 2006, p. 2937-2946, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2937-2946.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

A. Ghosh,1 D. R. Saha,2 K. M. Hoque,1 M. Asakuna,5 S. Yamasaki,5 H. Koley,3 S. S. Das,4 M. K. Chakrabarti,1 and A. Pal1*

Divisions of Pathophysiology,1 Histology and Electron Microscopy,2 Microbiology,3 Clinical Medicine, National Institute of Cholera and Enteric Diseases, Calcutta, India,4 Laboratory of International Prevention of Epidemics, Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan5

Received 27 April 2005/ Returned for modification 14 October 2005/ Accepted 14 February 2006

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 µg 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 µg 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.

* Corresponding author. Mailing address: Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme XM, Calcutta 700010, West Bengal, India. Phone: 91-33-350-5533. Fax: 91-33-350-5066. E-mail: tumpa_p2001{at}yahoo.co.in.

Editor: V. J. DiRita

Infection and Immunity, May 2006, p. 2937-2946, Vol. 74, No. 5
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.5.2937-2946.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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