Analysis of Salmonella typhi-host cell interaction

Principal Investigator :  Ayub Qadri

Ph D Student
Amita Sharma

This project is aimed at understanding host-pathogen interaction during infection with Salmonella typhi. Our current understanding of how this pathogen establishes itself within the host is primarily based on studies carried out with Salmonella typhimurium, which causes typhoid like disease in mice. A large number of genes and proteins have been identified in S.typhimurium that are involved in adhesion to and invasion of epithelial cells, intracellular survival of bacteria and systemic infection of the pathogen in mice; majority of these molecules are components of type III secretion system. Inspite of a high degree of homology between S.typhimurium and S.typhi invasion genes identified to date, recent studies suggest that S.typhi might employ different molecules or mechanisms to interact with host cells. The distinction between these two closely related organisms could be at the level of initial contact involving unique cell wall and/or its associated components or it could be at the level of effector molecules secreted by the organisms following contact with host cells or at both the levels. In our laboratory, we would like to identify specific interactions which S.typhi engages itself during its encounter with host cells.

The role of Vi in S.typhi-host cell interaction was examined by analyzing infection of a macrophage cell line with Vi positive S.typhi and comparing it with Vi negative S.typhi. The expression of Vi on the bacteria was established by reactivity with an anti-Vi monoclonal antibody previously generated in the laboratory. The reactivity of the two isolates with three other anti-S.typhi monoclonal antibodies, two directed against the O-antigen and the third against flagellar antigen, was comparable. The intracellular growth of bacteria was monitored by gentamycin protection assay and cytotoxicity was measured by LDH release and trypan blue exclusion. Vi negative S.typhi showed significantly higher intracellular growth and induced considerable cellular cytotoxicity. As the degree of infection is governed by invasion promoting molecules, which are secreted by salmonellae in response to contact with host cells or other stimuli, we analyzed proteins secreted by the two S.typhi isolates. Vi negative S.typhi showed higher amounts of protein in the culture supernatant as compared to Vi positive S.typhi, indicating that Vi might regulate secretion of these proteins either by acting as a physical barrier or by some other mechanism. In our previous analysis, we have shown that Vi can bind intestinal epithelial cells as well as macrophages and also undergo endocytosis following binding to cells. In order to establish that Vi associated with bacteria was able to interact with host cells in a fashion similar to purified polysaccharide, we analyzed binding of Vi positive and Vi negative S.typhi to an intestinal epithelial cell line at 4°C. Significantly higher number of Vi positive S.typhi were consistently found to be associated with these cells strongly supporting a role for Vi in adhesion of S.typhi to intestinal cells which is an essential first step for subsequent invasion. To demonstrate that the interaction of Vi with cells was mediated through binding with specific molecules, we carried out immunoprecipitation using surface biotinylated cells. The polysaccharide bound to two cell surface associated entities having molecular weights between 25 and 35kDa. Our results suggest that Vi capsular polysaccharide might be one of the key molecules which S.typhi employs to contact and gain entry into host cells.

Publications

Original peer-reviewed articles

1.   Qadri A, Radu CG, Thatte J, Cianga P, Ober BT, Ober RJ and Ward ES (2000) A role for the region encompassing the c” strand of a TCR V alpha domain in T cell activation events. J Immunol 165:820-829.