Distinct Isoforms of Phospholipase A2 Mediate the Ability of Salmonella enterica Serotype Typhimurium and Shigella flexneri To Induce the Transepithelial Migration of Neutrophils

Salmonella spp. and Shigella spp. are frequently implicated in outbreaks of enteric disease due to the consumption of contaminated food. In the United States alone, the number of prevalent cases of salmonellosis is estimated to range from 80,000 to 3,700,000 annually (7). Moreover, Shigella sp. infections are responsible for ∼163 million cases of dysentery and 1 million deaths each year worldwide (27). While the majority of infections occur in third-world countries, where contaminated food and drinking water are common (27), even developed nations cannot escape several outbreaks in a given year (65). Therefore, studying the molecular pathogenesis of these gram-negative enteric pathogens is of paramount importance, especially taking into consideration emerging antibiotic resistance and the lack of appropriate vaccines. Salmonella enterica serotype Typhimurium and Shigella flexneri interact with the intestinal epithelium to induce intestinal inflammation characterized by the transepithelial migration of neutrophils (2, 12, 33, 41, 49). Symptoms of salmonellosis manifest within 48 h of the ingestion of contaminated food or water and include nausea, vomiting, and acute, mostly self-limiting diarrhea. Following its ingestion, this motile pathogen colonizes and penetrates the intestinal epithelium and gains access to systemic sites, such as the liver and spleen, through lymphatic and blood circulation (70). The ability of S. enterica serovar Typhimurium to cause disease in humans is related to the acquisition of virulence genes termed pathogenicity islands. Located on a 40-kb segment of the bacterial chromosome termed SPI-1 (Salmonella pathogenicity island 1) are 25 virulence genes that encode structural components and secreted substrates of a type III secretion system (TTSS) (11, 21). This TTSS is essential for the ability of S. enterica serovar Typhimurium to invade and induce proinflammatory responses (21). Like S. enterica serovar Typhimurium, Shigella flexneri enters the human host by being ingested via contaminated food or water, passaging through the stomach, and infecting the large intestine. Infection of the colonic mucosa by Shigella results in bacillary dysentery (shigellosis), an acute inflammatory disease characterized by abdominal cramps, fever, and severe diarrhea often containing blood and mucus (66). In contrast to what is the case for Salmonella, humans (and certain higher primates) serve as the only natural hosts and reservoirs for Shigella (66). Thus, this organism has evolved to be highly host specific as well as efficient, with a particularly low infectious dose in comparison to other enteric pathogens (77). Also distinct from Salmonella, Shigella has a unique mode of entry requiring access to the basolateral surface. It has been established that Shigella alters the tight junctional complex, thereby allowing the pathogen to traverse the paracellular space and access the basolateral surface, an event that also decreases barrier function (64). Once at the basolateral surface, Shigella rapidly invades and disseminates through the epithelium, a consequence of the TTSS and additional proteins encoded in a 31-kb region (the mxi-spa locus) of the large 220-kb virulence plasmid (5, 69). While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in the massive transepithelial migration of neutrophils across the intestinal mucosa (2, 12, 33, 41, 49). Both S. enterica serovar Typhimurium and S. flexneri induce epithelial cells to secrete a repertoire of chemokines that play an active role in recruiting polymorphonuclear leukocytes (PMNs) from the peripheral circulation and directing them across the epithelium to the intestinal lumen (15, 25, 43, 44, 61, 63, 68, 74). In the case of S. enterica serovar Typhimurium, the effector protein SipA is necessary to initiate the cellular events that lead to PMN transepithelial migration during acute states of active intestinal inflammation (10, 35, 73). Regarding the molecular mechanism underlying these cellular events, it has been revealed so far that SipA activates a novel ADP ribosylation factor 6- and phospholipase D-dependent lipid signaling cascade (10) that in turn activates protein kinase C α (PKC α) (73), events that ultimately lead to the apical secretion of a potent PMN chemoattractant, eicosanoid hepoxilin A3 (HXA3; 8-hydroxy-11,12-epoxy-eicosatetraenoic acid) (51). HXA3, an endogenous 12/15-lipoxygenase (12/15-LOX) product, forms a chemotactic gradient across the epithelial tight junctional complex that directs PMNs across the intestinal epithelium to the luminal surface (51). The mechanisms underlying Shigella-induced PMN transepithelial migration are less defined. Studies addressing PMN movement across model intestinal epithelia indicate that PMN transmigration does occur as a result of Shigella infection and that this event further facilitates bacterial invasion (48, 62). Furthermore, the induction of PMN transepithelial migration is dependent not only on a functional TTSS and invasive ability (24, 67) but also on intercellular spread (16). Recently, lipopolysaccharide and the S. flexneri Osp proteins have been shown to be involved in the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway (26, 85, 86). In turn, ERK1/2 phosphorylation and its nuclear localization have been shown to play a role in PMN transepithelial migration (26). The purpose of this study was to determine if S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce PMN transepithelial migration (PKC and ERK1/2, respectively). We found that S. flexneri, similarly to S. enterica serovar Typhimurium, induced the apical secretion of the potent PMN chemoattractant HXA3 from model intestinal epithelia. In addition, PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA3. Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A2 (PLA2), a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA3.