Intestinal innate immunity and the pathogenesis of Salmonella enteritis.
ABSTRACT Acute gastroenteritis caused by Salmonella typhimurium infection is a clinical problem with significant public health impact. The availability of several experimental models of this condition has allowed detailed investigation of the cellular and molecular interactions involved in its pathogenesis. Such studies have shed light on the roles played by bacterial virulence factors and host innate immune mechanisms in the development of intestinal inflammation.
Article: Cooperative interactions between flagellin and SopE2 in the epithelial interleukin-8 response to Salmonella enterica serovar typhimurium infection.[show abstract] [hide abstract]
ABSTRACT: Flagellin is an important stimulus for epithelial interleukin-8 (IL-8) secretion because of its ability to activate Toll-like receptor 5 (TLR5). SopE2, a Salmonella guanine nucleotide exchange factor (GEF), is also involved in intestinal inflammation. To clarify the proinflammatory mechanisms of these proteins, we examined their effects on IL-8 secretion and intracellular signaling in T84 epithelial cells. A Salmonella strain lacking SopE2 (and its homolog SopE) induced lower levels of IL-8 than the wild type and exhibited reduced activation of mitogen-activated protein kinases (MAPKs). Overexpression of wild-type SopE2 in this strain restored MAPK activation and augmented IL-8 production, whereas a mutant lacking GEF activity failed to increase IL-8 expression. Additional effects on signaling were demonstrated in transient transfection experiments, in which SopE2 enhanced the ability of TRAF6, a signal transducer downstream of TLR5, to activate the NF-kappaB transcription factor in 293 cells. Flagellin was also found to be required for IL-8 induction in T84 cells. In its absence, the ability of SopE2 overexpression to increase IL-8 secretion was impaired. Part of this impairment was related to the decreased motility of the flagellin-deficient strain, but lack of flagellin also affected translocation of SopE2 into the infected cells. Our results indicate that flagellin and SopE2 interact functionally at multiple levels to increase IL-8 secretion by epithelial cells-flagellin facilitating the translocation of SopE2, and SopE2 enhancing signaling pathways activated by flagellin. These observations offer a mechanistic explanation for the involvement of these proteins in the pathogenesis of Salmonella-induced gastroenteritis.Infection and Immunity 10/2004; 72(9):5052-62. · 4.16 Impact Factor
Article: Stimulation of Toll-like receptor 4 by lipopolysaccharide during cellular invasion by live Salmonella typhimurium is a critical but not exclusive event leading to macrophage responses.[show abstract] [hide abstract]
ABSTRACT: Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4(-/-) (TLR-4(-/-)) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-alpha. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-kappa B is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-kappa B, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-alpha is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.The Journal of Immunology 07/2003; 170(11):5445-54. · 5.79 Impact Factor
Article: Overexpression of the inositol phosphatase SopB in human 293 cells stimulates cellular chloride influx and inhibits nuclear mRNA export.[show abstract] [hide abstract]
ABSTRACT: SopB is an inositol phosphate phosphatase that is a virulence factor in Salmonella species. We have overexpressed SopB cDNA in a tetracycline-dependent system in human embryonic 293 cells, and used this model system to directly analyze the role of SopB in altering inositol metabolite levels in vivo. Addition of tetracycline to these cells resulted in the rapid induction of SopB expression, which was coincident with perturbations in the cellular levels of multiple soluble inositol phosphates. All of the changes induced by SopB expression were reversed within 24 h on removal of tetracycline from media. Specifically, cellular inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) levels were depleted within 4 to 6 h after inducing SopB expression. A transient rise in cellular inositol 1,4,5,6-tetrakisphosphate was also observed and was accompanied by increased chloride channel activity. This indicates that SopB alone is sufficient for changes in chloride channel function in cells infected with Salmonella organisms. Depletion of inositol phosphates, including InsP(5) and InsP(6) metabolites, was coincident with the accumulation of polyadenylated RNA in the nucleus. This suggested that a defect in nuclear export had occurred. Moreover, the penetrance of the export defect required localization of SopB to the nucleus. These results provide evidence that inositol phosphate productions may be required for efficient mRNA export in mammalian cells.Proceedings of the National Academy of Sciences 02/2001; 98(3):875-9. · 9.68 Impact Factor