Analysis of Salmonella typhi-host cell interaction

Project Associates / Assistants
Huma Mustafa

Ph D Students
Amita Sharma
Naeha Subramanian
Srikanth KS

The project is aimed at deciphering Salmonella typhi-specific host-pathogen interactions, and understanding the molecular basis of the two-way biochemical cross talk that is initiated following interaction of cells with this pathogen.

Role of outer capsule in S.typhi-host cell interaction

We have shown earlier that S. typhi can engage its capsule, the Vi polysaccharide, to interact with host cells through specific recognition molecules of about 30 and 35KDa. The capsule is absent in the closely related mouse pathogen, S. typhimurium. The identity of Vi-binding molecules was established by mass spectrometry and immunoblot. Analysis of the 35KDa Vi-interacting molecule immunoprecipitated from a human intestinal epithelial cell line Caco-2, by mass spectrometry, revealed that this molecule contained peptides corresponding to BAP-37, a member of the recently identified evolutionary conserved proteins called prohibitins. Immunoblot with anti-prohibitin antibodies showed strong reactivity with the 30KDa-molecule immunoprecipitated with Vi suggesting that this molecule was prohibitin. The antibodies also recognised, though weakly, the BAP-37 molecule, and showed minor reactivity with a 68KDa protein present in the immunoprecipitated complex. Prohibitin is a potential tumor suppressor molecule and has been assigned a number of functions including regulation of cells cycle, replicative senescence and stabilisation of mitochondrial proteins. Prohibitin and BAP-37 have been reported to interact physically with each other. The two molecules are remarkably similar in the middle region and contain a series of repeated leucine residues; the N and C termini show no homology. The high degree of similarity between the two proteins explains the cross reactivity seen with anti-prohibitin antibodies. Western blot with Triton X-100 insoluble membranes prepared from Caco-2 cells showed that prohibitins were enriched in lipid rafts, suggesting that these proteins might be engaged in a cross talk with molecules involved in intracellular signaling. Prohibitin has been shown to interact with a number of molecules including retinoblastoma protein (Rb), signaling kinase Raf-1 and transcription factor E2F. To analyse the significance of Vi-intestinal epithelial cell interaction, we studied the effect of stimulation with Vi on the ability of Caco-2 cells to respond to subsequent activation with flagellin or PMA. Flagellin triggers secretion of IL-8 and many other cytokines from intestinal epithelial cells and mononuclear phagocytes through engagement of TLR-5. The results showed that prior engagement of Caco-2 cells with Vi could significantly inhibit IL-8 secretion by these cells. We have initiated studies to analyse intracellular events including activation of NF-kB, a key regulator of IL-8 secretion, that might get modulated following interaction of cells with Vi. This analysis should give important insights into the role of prohibitin and prohibitin related molecules in infection with S.typhi. Our study demonstrates that Vi can bind to intestinal epithelial cells through a specific receptor complex containing prohibitin and prohibitin related molecule(s). Engagement of these molecules by Vi can modulate early inflammatory/innate immune responses that might play a crucial role in the establishment of S. typhi infection.

Modulation of flagellin (TLR-5 ligand) secretion from salmonella by host stimulus

Salmonella typhi and many pathogenic bacteria possess a specialized protein secretion apparatus, which is activated following contact with host cells. This enables these pathogens to translocate bacterial virulence proteins into the host cell and initiate a two-way biochemical cross-talk between the bacterium and the host cell. However, the identity of host stimuli involved in triggering translocation of these effector molecules remains unknown. We have reported earlier that contact of S. typhi with a human intestinal epithelial cell line Caco-2 or fetal calf serum (FCS) can trigger the bacteria to release flagellin. Bacterial flagellin is the ligand for Toll-like receptor-5 (TLR-5) and is known to induce a variety of innate immune responses. Flagellin-containing supernatants from S. typhi following contact with cells, serum or supernatant from Caco-2 cells grown under serum-free conditions, induced intestinal epithelial cell monolayers to release the chemokine IL-8. The effect of these stimuli could be efficiently reproduced with lysophosphatidic acid (LPA), a bioactive lipid mediator, which is an abundant constituent of serum, where it is present in an albumin-bound form. LPA generated from cells through hydrolysis of existing phospholipids by phospholipaseA2 (PLA2) is known to be upregulated during certain inflammatory responses. The secretion of proinflammatory flagellin by S. typhi was triggered early in a dose-dependent fashion and did not require de novo protein synthesis, but required live bacteria. Our study identifies for the first time, a well-defined host stimulus that can modulate the secretion of flagellin, one of the key Toll ligands involved in initiating innate immune responses. It also assigns a novel function to LPA, which is otherwise known to regulate a variety of biological responses in mammalian cells.