B-278. Glycogen Storage Plays a Role in the Survival and Pathogenesis of Vibrio cholerae

L. Bourassa, A. Camilli;
Tufts Univ. Sch. of Med. and Howard Hughes Med. Inst., Boston, MA.

Vibrio cholerae is a water-borne intestinal pathogen and native member of the aquatic environment. The ability to store carbon as glycogen for future utilization might facilitate the transition of V. cholerae from the aquatic environment to colonization of the human small intestine and/or the reverse transition. Glycogen, a branched glucose polymer, is a major energy storage compound that might be utilized when a bacterium encounters a carbon poor environment. The role of glycogen on V. cholerae survival and pathogenesis was investigated by constructing mutants that contain deletions of the ADP-glucose pyrophosphorylase gene (glgC) which catalyzes the first step of glycogen biosynthesis. V. cholerae El Tor N16961 contains two GlgC paralogues, VC1727 and VCA0699. Three null mutant strains, two single gene deletions of VC1727 and VCA0699 and a third double deletion were constructed. Glycogen stores could not be detected by enzymatic hydrolysis assays in the wild-type (wt) strain or the glgC mutant strains when grown on minimal medium plus glucose. However, growth under nitrogen limiting conditions resulted in comparable levels of glycogen storage in wt and in ΔVCA0699. Glycogen storage in ΔVC1727 was reduced to one-third the level of wt whereas no glycogen stores were detected in the double mutant. Quantitative RT-PCR analysis revealed that VCA0699 expression is 5-fold lower than VC1727 expression in wt V. cholerae. ΔVC1727 and the double mutant are attenuated for survival in competition assays with the wt strain in minimal media or pond water with little or no carbon source and when subjected to various environmental stresses. The double mutant was attenuated for virulence in the infant mouse model of infection when the inoculum was grown under nitrogen limiting conditions followed by a short starvation in minimal media lacking a carbon source. A role for glycogen in the life-cycle of pathogenic V. cholerae is further supported by the presence of glycogen granules in rice-water stool V. cholerae. We are continuing to investigate the regulation of VC1727 and VCA0699 and their role in the survival and pathogenesis of V. cholerae.