Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon, which is characterized by an acute polymorphonuclear leukocyte (PMNL)-rich inflammation. Our recent studies demonstrated that cadaverine, a polyamine, specifically acts to abrogate transepithelial signaling to PMNL induced by S. flexneri. Here, insight is provided into the cellular mechanisms by which cadaverine attenuates the ability of Shigella species to induce PMNL signaling. It was found that cadaverine retards the lysis of the Shigella species-containing vacuole, suggesting that a blockade is established, in which the pathogen is prevented from adequately interacting with the cytoskeleton. Furthermore, an IcsA mutant of S. flexneri that cannot interact with the cytoskeleton and spreads intercellularly fails to induce transmigration of PMNL. Results indicate that cadaverine-induced compartmentalization of Shigella species to the phagolysosome might be a protective response of the host that directly contributes to the diminished ability of PMNL to transmigrate across model intestinal epithelia.

A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration.

In response to Salmonella typhimurium, the intestinal epithelium generates an intense inflammatory response consisting largely of polymorphonuclear leukocytes (neutrophils, PMN) migrating toward and ultimately across the epithelial monolayer into the intestinal lumen. It has been shown that bacterial-epithelial cell interactions elicit the production of inflammatory regulators that promote transepithelial PMN migration. Although S. typhimurium can enter intestinal epithelial cells, bacterial internalization is not required for the signaling mechanisms that induce PMN movement. Here, we sought to determine which S. typhimurium factors and intestinal epithelial signaling pathways elicit the production of PMN chemoattractants by enterocytes. Our results suggest that S. typhimurium activates a protein kinase C-dependent signal transduction pathway that orchestrates transepithelial PMN movement. We show that the type III effector protein, SipA, is not only necessary but is sufficient to induce this proinflammatory response in epithelial cells. Our results force us to reconsider the long-held view that Salmonella effector proteins must be directly delivered into host cells from bacterial cells.

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis.

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization.

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.

Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine.

Dysentery caused by Shigella species is characterized by infiltration of polymorphonuclear leucocytes (PMNs) into the colonic mucosa. Shigella spp. evolved into pathogens by the acquisition of virulence genes and by the deletion of 'antivirulence' genes detrimental to its pathogenic lifestyle. An example is cadA (encoding lysine decarboxylase), which is uniformly absent in Shigella spp., whereas it is present in nearly all isolates of the closely related non-pathogen Escherichia coli. Here, using monolayers of T84 cells to model the human intestinal epithelium, we determined that the introduction of cadA into S. flexneri and the expression of lysine decarboxylase attenuated the bacteria's ability to induce PMN influx across model intestinal epithelium. Such inhibition was caused by cadaverine generated from the decarboxylation of lysine. Cadaverine treatment of model intestinal epithelia specifically inhibited S. flexneri induction of PMN transepithelial migration, while having no effect on the ability of Salmonella or enteropathogenic E. coli (EPEC) to induce PMN migration. These observations not only provide insight into mechanisms of S. flexneri pathogen evolution and pathogenesis, but also suggest a potential for the use of cadaverine in the treatment of dysentery.

Requirement of the Shigella flexneri virulence plasmid in the ability to induce trafficking of neutrophils across polarized monolayers of the intestinal epithelium.

Attachment of an array of enteric pathogens to epithelial surfaces is accompanied by recruitment of polymorphonuclear leukocytes (PMN) across the intestinal epithelium. In this report, we examine how Shigella-intestinal epithelium interactions evoke the mucosal inflammatory response. We modeled these interactions in vitro by using polarized monolayers of the human intestinal epithelial cell line, T84, isolated human PMNs, and Shigella flexneri. We show that Shigella attachment to T84-cell basolateral membranes was a necessary component in the signaling cascade for induction of basolateral-to-apical directed transepithelial PMN migration, the direction of PMN transepithelial migration in vivo. In contrast, attachment of Shigella to the T84-cell apical membrane failed to stimulate a directed PMN transepithelial migration response. Importantly, the ability of Shigella to induce PMN migration across epithelial monolayers was dependent on the presence of the 220-kb virulence plasmid. Moreover, examination of Shigella genes necessary to signal subepithelial neutrophils established the requirement of a functional type III secretion system. Our results indicate that the ability of Shigella to elicit transepithelial signaling to neutrophils from the basolateral membrane of epithelial cells represents a mechanism involved in Shigella-elicited enteritis in humans.