Pathogenesis of Salmonella-induced Enteritis

Infections with Salmonella serotypes are a major cause of food-borne diseases worldwide. Animal models other than the mouse have been employed for the study of nontyphoidal Salmonella infections because the murine model is not suitable for the study of Salmonella-induced diarrhea. The microbe has developed mechanisms to exploit the host cell machinery to its own purpose. Bacterial proteins delivered directly into the host cell cytosol cause cytoskeletal changes and interfere with host cell signaling pathways, which ultimately enhance disease manifestation. Recently, marked advances have been made in our understanding of the molecular interactions between Salmonella serotypes and their hosts. Here, we discuss the molecular basis of the pathogenesis of Salmonella-induced enteritis.


Introduction
Salmonella serotypes have a broad host range and clinical manifestations that result from the combination between serotype and host species involved.Recently, a large amount of information on the molecular level of interactions between bacteria and host cells has allowed us to propose molecular mechanisms determining the pathologic and clinical manifestations of nontyphoidal Salmonella infections.For simplification, Salmonella enterica subsp.enterica ser.Typhimurium will be referred to hereafter as either S. typhimurium or serotype Typhimurium.
Salmonella infection is one of the most common food-borne infections worldwide.In the United States an estimated 1.41 mil-lion cases and more than 500 human deaths occur annually (1).Approximately 95% of the human Salmonella infections are foodborne, corresponding to approximately 30% of deaths caused by food-borne infections in the United States (1).Salmonella infection is even more detrimental in the developing world.

Experimental models for Salmonella infection
The mouse, which has been used for most of the studies addressing the various aspects of host-pathogen interaction in S. typhimurium infection, develops a systemic disease when infected with S. typhimurium, but no diarrhea.Murine infection with S. typhimu-rium results in a disease that is similar to human typhoid fever caused by infection with serotypes Typhi, Paratyphi A, B, and C, which are host-restricted serotypes that infect only man and few other primate species.Thus, murine infection with S. typhimurium has been employed as a model for human typhoid fever, but not Salmonella-induced diarrhea.In contrast, calves infected with S. typhimurium develop a diarrheic disease with clinical manifestations similar to those observed in human infections, which also result in diarrhea with a low mortality rate (reviewed in Refs. 2 and 3).
An important aspect of the pathology of Salmonella-induced enteritis is the same pattern of inflammatory reaction developed by calves after S. typhimurium infection, which is characterized by a marked infiltration of neutrophils, also observed in non-human primates in experimental infections, and in human infections.In sharp contrast, mice infected with S. typhimurium develop an inflammatory response with predominance of mononuclear leukocytes, which is not associated with diarrhea (reviewed in Ref. 3).
Interestingly, although several bacterial genes are required for disease progression and expression in both calves and mice, some genes that play a role in the murine typhoid model are not required for enteropathogenesis in cattle and vice versa (4).Many of the Salmonella virulence genes are clustered in certain areas of the chromosome known as "Salmonella pathogenicity islands" (SPI).To date, five SPI have been described and two of them, SPI-1 and SPI-2, encode type III secretion systems.The SPI-1-encoded type III secretion system translocates effector proteins into the cytosol of host cells.This system is required for invasion of nonphagocytic host cells (5) and enteropathogenesis (6), while the SPI-2-encoded type III secretion system is required for intracellular survival in murine macrophages (7).In the murine typhoid model, S. typhimurium strains having mutations in SPI-1 and SPI-2 are 50-fold and >10,000-fold attenuated, respectively, after oral infection (5,7).In contrast, SPI-2 does not play a major role in enteropathogenesis, whereas SPI-1 mutants are nonpathogenic for calves (6) as opposed to just a mild attenuation of these SPI-1 mutants in the mouse.

Invasion of epithelial cells by S. typhimurium
A remarkable aspect of Salmonella pathogenesis is its ability to invade nonphagocytic cells in a process that morphologically resembles phagocytosis.M cells located in the follicle-associated epithelium in the Peyer's patches are the primary intestinal epithelial cell type targeted for invasion by Salmonella in the mouse (8).In cattle, S. typhimurium is able to invade both M cells and enterocytes with no predilection for a particular cell type (9).
Upon contact with intestinal epithelial cells, S. typhimurium translocates bacterial effector proteins into the host cell cytosol via the SPI-1-encoded type III secretion system previously discussed.Some of these proteins have kinase, phosphatase, or actin-binding activity, and once in the epithelial cell cytosol, they alter host cell signaling pathways that promote changes in the cytoskeleton, with consequent bacterial internalization and changes in host gene expression (reviewed in Ref. 10).Mutant strains of S. typhimurium lacking structural components of the SPI-1-encoded type III secretion system, secreted proteins, or SPI-1 transcriptional regulators are unable to invade epithelial cells (11).
Salmonella senses environmental factors such as oxygen concentration, osmolarity, and pH that determine the expression of invasion genes in the intestinal lumen when their products are required for invasion of intestinal epithelial cells.The effects of these environmental factors are mediated by regulation of expression of the transcriptional regulator HilA (12).HilA-dependent regulation of expression is not restricted to SPI-1, since SPI-4-and SPI-5-encoded genes were found to be regulated by HilA, whose expression is regulated by SirA (13).
Studies in the early 1990's determined the morphologic features and dynamics of the interaction between S. typhimurium and intestinal epithelial cell monolayers.Shortly after Salmonella enters in contact with the apical surface of the epithelial monolayer, the epithelia develop cytoplasmic projections with disruption of the underlying cytoskeleton, and intracellular bacteria are detected 30 min after infection.Two hours post-infection, free bacteria are detected on the basolateral side of the monolayer (14).Additional studies indicated that S. typhimurium grown under conditions that favor expression of invasin induce morphologic changes in epithelial cells as quickly as 40 s after contact (15), which are associated with recruitment of cytoskeletal components (16).These morphological and cytoskeletal changes, characterized by formation of rufflelike structures, mediate bacterial internalization into epithelial cells (17).As shown in Figure 1, similar morphologic changes occur in calves infected with S. typhimurium in vivo.SipC is an SPI-1-encoded protein that acts as a translocase and is translocated itself into the host cytosol via the SPI-1-encoded type III secretion system.This protein bundles actin filaments and nucleates actin polymerization in vitro, which results in cytoskeletal rearrangements in vivo (18).SipA, which is not required for invasion, binds to F-actin inhibiting depolymerization (19).Thus, SipC is essential for actin nucleation and bundling of actin filaments whereas SipA acts by enhancing the efficiency of this process (20).
Although sopE is absent in many S. typhimurium strains, a homologue, sopE2, is present in all strains of S. typhimurium (21).Like SopE, SopE2 is also a guanine nucleotide exchange factor for Cdc42 and plays a role recruiting the actin-nucleating complex Arp2/3 to the membrane ruffles (21).Therefore, SopE2 is required for optimal invasion of cultured epithelial cells by S. typhimurium (21).SptP, a Salmonella protein that acts as a GTPase-activating factor for Rac-1 and Cdc42 and is also delivered into the epithelial cell cytosol via the SPI-1-encoded type III secretion system, has been shown to disrupt the actin cytoskeleton.Therefore it acts by reversing the cytoskeletal changes induced by the bacteria during invasion, restoring the normal cytoskeletal structure (22).The effect of SptP antagonizing the action of other bacterial effector proteins clearly indicates that S. typhimurium is able to finely regulate cellular pathways in favor of its own purposes.

Inflammatory response during S. typhimurium infection
Although the complete mechanism of Salmonella-induced diarrhea is still not clear, some previous reports indicate that it is distinct from secretory diarrheas such as those caused by cholera toxin (23,24).As discussed above, infection of calves with S. typhimurium results in enteritis in which neutrophils are the primary inflammatory cells involved.On the other hand, the mouse, which does not develop diarrhea, responds to the infection mostly with a mononuclear infiltrate in the intestine (3).Furthermore, experimental depletion of the polymorphonuclear leukocyte pool by administration of nitrogen mustard to rabbits results in a significant decrease in intestinal fluid secretion induced by S. typhimurium infection (23).In addition, administration of indomethacin, an anti-inflammatory agent, completely abolishes fluid secretion in rabbit intestinal loops inoculated with S. typhimurium (24).Therefore, neutrophils are proposed to play a very important role in the pathogenesis of Salmonella-induced diarrhea.Some of our recent experimental findings corroborate this hypothesis since the inflammatory response, characterized by neutrophil infiltration, precedes intestinal fluid secretion after infection with S. typhimurium in calves (25).Several Salmonella virulence factors required for enteropathogenicity in calves, which are involved in eliciting neutrophil influx, are also required for fluid secretion (26,27), reinforcing the significance of neutrophils in this process.
Following oral infection, intestinal epithelial cells are the first barrier to be crossed by S. typhimurium in order to invade and colonize the intestinal tissues and other organs.Current data indicate that epithelial cells play an important role in the outcome of infection by influencing the host inflammatory response.Supporting this notion, a study demonstrated that Salmonella strains and serotypes that elicit enteritis and diarrhea are also able to induce transepithelial signaling for neutrophil migration across epithelial cell layers, whereas strains that do not cause diarrhea failed to trigger neutrophil transepithelial migration (28).Invasion of cultured epithelial cells by bacteria, including Salmonella, results in expression and secretion of interleukin 8 (IL-8), a chemoattractant for neutrophils (29).Salmonellainduced IL-8 secretion by epithelial cells is dependent on the mitogen-activated protein kinase pathway and activation of the transcription factor NF-kB and requires a functional SPI-1-encoded type III secretion system (30).Cultured epithelial cells also respond to S. typhimurium invasion with an increase in the cytosolic concentration of calcium, which is absolutely required for NF-kB activation and IL-8 expression (31).
Infiltration of neutrophils into the lamina propria occurs shortly after infection with S. typhimurium, which is followed by a massive migration of neutrophils through the epithelium into the intestinal lumen (9,25).Thus, one can expect that chemoattractants for neutrophils are secreted into the intestinal lumen at the early stages of infection.Interestingly, experiments with polarized intestinal epithelial cell monolayers indicate that IL-8 is secreted at the basolateral aspect of the epithelium, which implies that the role of IL-8 is primarily recruitment of neutrophils to the subepithelial space rather than transepithelial migration into the intestinal lumen (32).Further experiments have led to the identification of a pathogen-elicited epithelial chemoattractant (PEEC) bioactivity, which is released in a polarized fashion towards the apical aspect of the epithelial monolayer.Once secreted on the apical side of the epithelial cell, PEEC induces direct migration of neutrophils across cultured intestinal epithelial cell monolayers (33).PEEC has a 1-to 3-kDa mass, stimulates neutrophils via a pertussis toxin-sensitive receptor and elicits the Ca 2+ signal, but its molecular nature is Salmonella-induced enteritis still uncharacterized (33).Actual invasion of epithelial cells is not required for induction of epithelial promotion of neutrophil transepithelial migration since treatment of the monolayers with cytochalasin D, which blocks S. typhimurium invasion, does not reduce the promotion of neutrophil migration (34).Importantly, although IL-8 and PEEC act in concert to promote neutrophil migration through the lamina propria and epithelia, respectively, secretion of these two chemotactic factors is mediated by distinct signaling pathways.In contrast to IL-8 expression and secretion, PEEC activity is not dependent on NF-kB activation (34).It has been recently demonstrated that the bacterial protein SipA, an SPI-1-encoded effector protein translocated into the cytosol of the host cell via the SPI-1 type III secretion system, is sufficient to trigger neutrophil transepithelial migration in cultured intestinal epithelial monolayers (35).Another Salmonella protein secreted by the SPI-1-encoded type III secretion system, SopA, is also involved in the induction of neutrophil transepithelial migration (36).
Although some recent studies have addressed in vivo cytokine production in response to Salmonella infection, most of these studies were performed on mice.We have demonstrated that there is a marked increase in expression of CXC chemokines such as IL-8, GROa/g and GCP2, and the proinflammatory cytokine IL-1ß in bovine Peyer's patches as early as 1 h post-infection, increasing continuously until at least 5 h postinfection (9).Interestingly, anti-inflammatory cytokines such as IL-4 and the IL-1 receptor antagonist (IL-1Ra) are also upregulated in bovine Peyer's patches in vivo after infection with S. typhimurium (9).

Salmonella-induced host cell death
Although apoptosis has been defined classically as a form of cell death that does not elicit an inflammatory reaction, under spe-cific conditions this process may ultimately act as a proinflammatory signal.Several groups have reported that murine macrophages and macrophage-like cell lines undergo cell death when infected with S. typhimurium (37)(38)(39).A previous report indicated that Salmonella-induced macrophage apoptosis is associated with marked IL-1 release (40).Thus, since IL-1 is a potent pro-inflammatory cytokine, this was the first indication of a possible link between Salmonella-induced cell death and inflammation.Salmonella-induced macrophage cell death is largely due to expression of genes associated with invasion, since mutant strains lacking functional SPI-1 or grown under conditions that prevent SPI-1 expression do not cause rapid cell death after infection of macrophages (37,39,41,42), although SPI-1-independent cell death has also been described (38,42,43).Further investigation has led to the identification of the SipB protein as the bacterial effector responsible for induction of apoptosis (44).SipB is translocated into the host cell cytosol via the SPI-1-encoded type III secretion system, where it binds to and activates caspase-1, an intracellular cysteine protease also known as IL-1ß converting enzyme.Once activated, caspase-1 cleaves and activates IL-1ß (44).Caspase-1 is also responsible for triggering apoptosis in Salmonella-infected macrophages, since a specific caspase-1 inhibitor blocks this mechanism of cell death (44).Infection of macrophages with S. typhimurium also results in degradation of the host protein Raf-1 in a SipB-and caspase-1-dependent manner, which favors the cytotoxic effect of SipB since Raf-1 acts by antagonizing the caspase-1-mediated cell death (45).Thus, Salmonella-induced macrophage apoptosis results in release of active IL-1ß, which is thought to play a significant role in Salmonella-elicited inflammation.This mechanism appears to be conserved since a similar mechanism of cell death, mediated by SipB and caspase-1 activity, occurs in bovine mac-rophages infected with S. typhimurium (42).
Although all the initial papers described Salmonella-induced cell death as apoptotic in nature, more recent publications argue that it is a necrotic rather that an apoptotic mechanism of cell death.The conclusions of these reports are based on either failure to detect DNA fragmentation and morphologic features of apoptosis (46) or on the effect of glycine blocking Salmonella-induced cytotoxicity (47).However, while the definition of a proper classification and terminology for this mechanism is still debatable, the requirement for caspase-1 activation and the proinflammatory nature of this mechanism is a consensus among different laboratories and has lead to the proposition of "pyroptosis" as a new term to describe proinflammatory programmed cell death (48).
In spite of the increasing amount of information on the interaction between Salmonella and macrophage in cell culture systems, there are few data available regarding the significance of macrophage cell death and its putative proinflammatory effect on the outcome of Salmonella infections in vivo.The ability of S. typhimurium to induce macrophage cell death has been demonstrated in vivo in mice intravenously inoculated with small infectious doses (49).In addition, caspase-1 is required for colonization of Peyer's patches and induction of systemic infection in mice orally inoculated with S. typhimurium (50).Although quite valuable, these data are not applicable to the pathogenesis of Salmonella-induced diarrhea.Indeed, we have demonstrated that Salmonella-induced host cell death is not sufficient to trigger the inflammatory response after S. typhimurium infection in calves (25).

Salmonella virulence factors involved in enteropathogenesis
There is clearly a difference between Salmonella genes required for virulence in mice, where the Salmonella virulence plas-mid and SPI-2-encoded genes are essential for virulence, and those involved in eliciting diarrhea, in which SPI-1-encoded genes are essential but SPI-2 and the Salmonella virulence plasmid play only minor roles (6,13,26, 51,52).Disruption of the SPI-1 type III secretion decreases or abolishes the ability of S. typhimurium to invade the intestinal epithelium, which correlates with its ability to elicit an inflammatory response and thereby induce diarrhea (6,13,26,51).
A fifth SPI has been identified and linked to the pathogenesis of diarrhea (27,53).An SPI-5-encoded gene, sopB, has been extensively studied.SopB, also known as SigD, is secreted via the SPI-1-encoded type III secretion system and its expression is dependent on the regulator sirA, which is also an activator of the SPI-1 regulator hilA (54).A sopB mutant of S. dublin has a significantly reduced ability to elicit inflammation and fluid secretion in bovine ligated ileal loops in spite of displaying wild-type levels of invasion in the Peyer's patches (27).Similar results were observed with a sopB mutant of S. typhimurium (25).SopB is an inositol phosphate phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate, which is an inhibitor of chloride secretion.In addition, SopB hydrolyzes inositol 1,3,4,5,6 pentakisphosphate, generating inositol 1,4,5,6 tetrakisphosphate (55), which may be involved in increasing chloride secretion (56).Thus, SopB is thought to mediate fluid secretion by increasing chloride secretion.However, changes in chloride secretion alone are not compatible with the pathologic features of Salmonella-induced diarrhea, which is associated with a severe acute neutrophilic infiltration.The most significant events in the pathogenesis of Salmonella-induced enteritis are illustrated in Figure 2. Interestingly, SopB also affects host cell signaling pathways that may be involved in regulation of cytokine expression such as activation of the serine-threonine kinase Akt (57).The Salmonella protein SopD, which is also se-creted in an SPI-1-dependent manner, has an additive effect to SopB in the induction of enteritis (58), whereas SopA influences the inflammatory response by a mechanism distinct from SopB and SopD.SopA is involved in induction of transepithelial migration of neutrophils, a phenomenon that is not influenced by SopB or SopD (36).Recent experimental findings from our laboratory indicate that the secreted effectors SipA, SopA, SopB, SopD, and SopE2 act in concert to induce diarrhea, since a strain lacking all of these genes (DsipAsopABDE) had additive attenuation when compared to the single gene mutants.The quintuplet mutant (DsipAsopABDE) was as attenuated as a mutant with a defective SPI-1-encoded type III secretion system in the bovine ileal loop model (59).The role of Salmonella virulence genes in enteropathogenesis is summarized in Table 1.
Salmonella-induced diarrhea is an effi-  cient way of spreading the organism in the environment.Recently, a gene, named shdA, found only in strains of Salmonella adapted to warm blooded-organisms (subspecies I) has been demonstrated to be involved in prolonged shedding of S. typhimurium in mice.Mutation of shdA causes a decrease in the number of organisms shed in feces and the duration of shedding (61).Subsequent studies indicated that the shdA gene product binds to extracellular matrix proteins, particularly fibronectin (62).From these discussions of the mechanisms of Salmonella-induced diarrhea, it is exceedingly clear that there is an intensive and intricate series of highly regulated adaptive gene expression events by both the host and the Salmonella microbe.Unraveling the intricacies of the molecular basis and the regulation of these interactions holds great promise for developing new vaccination strategies as well as improved therapeutic rationales.

Figure 1 .
Figure 1.Transmission electron micrograph showing invasion of bovine enterocytes in the Peyer's patches by Salmonella typhimurium.Intracellular bacteria are located within membrane bound vacuoles (arrows).Bacteria being internalized by cytoplasmic projections of the apical surface of the enterocyte (arrowhead).Bar = 1 µm.

Figure 2 .
Figure 2. Schematic representation of the pathogenesis of Salmonella-induced enteritis, with the most significant events described from A through H.

Table 1 .
Role of virulence genes in enteropathogenesis.