The human intestine is home to a plethora of mutualistic microorganisms, dominated mostly by Lactobacillus, Bacteroides, and Firmicutes (Backhed et al., 2005). Among other roles, these organisms assist immune system development and promote epithelial homeostasis (Rakoff-Nahoum et al., 2004; Mazmanian et al., 2005). Beyond these regulatory roles, the microbiome also functions as a critical barrier to invading pathogens. The population of host microbes in the gut physically blocks pathogen access to the epithelial layer, and also outcompetes pathogens for nutrients, thus reducing the survival and invasiveness of intestinal pathogens. Nevertheless, S. Typhimurium is capable of maneuvering through the microbiome to reach intestinal epithelial cells. In mouse experiments with wild-type and avirulent S. Typhimurium, Stecher et al. demonstrate that an inflammatory state in the intestines not only permits wild-type S. Typhimurium to outcompete the microbiome during colonization, but also allows a normally avirulent strain to colonize the intestines in the presence of intestinal microbiota (Stecher et al., 2007). These data unveil an intriguing concept that S. Typhimurium utilizes inflammation to outcompete host microbiota in mouse models of salmonellosis.

Host microbiota may also regulate the degree of S. Typhimurium shedding by infected mice. Following infection with 10⁸ CFU of bacteria, mice were classified into three groups corresponding to the level of fecal shedding: supershedders (>10⁸ CFU/g feces), moderate shedders (10⁴–10⁸ CFU/g feces), and low shedders (<10⁴ CFU/g feces). Treating low shedders with streptomycin converted these mice to supershedders (Lawley et al., 2008), suggesting that the absence of host microbes may have permitted Salmonella to colonize more heavily, thus elevating the level of shed bacteria. The significance of a gut microbiota barrier to Salmonella infection was also explored by Croswell et al. (2009). The authors found that treatment of mice with streptomycin, streptomycin–bacitracin, or ampicillin–vancomycin–neomycin–metronidazole (AVNM) reduced gut microflora populations, and that each treatment was subsequently associated with an increase in Salmonella colonization and Salmonella-induced inflammation compared to untreated mice. The authors further show that allowing 3 weeks between antibiotic treatment and infection still predisposed mice to greater levels of inflammation in response to Salmonella infection than was seen in mice that were never treated with antibiotics. These results suggest that antibiotics have long-lasting effects on gut microflora populations, and that the presence and composition of gut microfloral populations may play an important role in controlling Salmonella colonization.

Additionally, Barman et al. (2008) showed that salmonellosis may alter the microfloral population in FvB mice. Infected mice showed disrupted numbers of Bacteroides, Lactobacillus/Enterococcus, and Eubacterium rectale/Clostridium coccoides groups and of the C. perfringens group compared to uninfected mice. However, Salmonella did not replace these populations, and wild-type population numbers returned after Salmonella clearance, suggesting any effect salmonellosis has on the intestinal microbiota is not permanent.

Key to the mucosal epithelium is the formation of a mucus layer along the luminal lining of the gastrointestinal tract. Mucosal epithelial cells, specifically goblet cells, secrete glycosylated transmembrane proteins called mucins at the cell surface. These proteins generate a layer of large complexes containing thread-like structures and oligosaccharides. The resulting gelatinous layer blocks contact between the underlying epithelial monolayer and large particles, including bacteria. The mucus layer also contains trefoil factors, which are peptides produced in tissues containing mucus-producing cells, such as the intestinal epithelia. Their various proposed functions include limiting intestinal inflammation (Playford et al., 1996) and regulation of immune system responses (Baus-Loncar et al., 2005). Adhering to the mucosal layer would be necessary for Salmonella to avoid being washed out of the intestines. Indeed, there is evidence of Salmonella binding to mucus, although increased adherence does not necessarily correlate with increased mucosal penetration (Nevola et al., 1987; McCormick et al., 1988; Vimal et al., 2000).

The epithelial monolayer mediates interactions between triggers of immune responses, and the gut-associated lymphoid tissue (GALT), where immune responses originate (MacDonald and Monteleone, 2005; Turner, 2009). A follicle-associated epithelial (FAE) layer of columnar epithelial cells blankets lymphoid tissue within the intestinal wall. The intestinal mucosa contains a variety of cell types with unique functions including enterocytes, entero-endocrine cells, goblet cells, Paneth cells, and microfold (M) cells (Table 2). While all the unique epithelial cells found within the intestine are important for establishing the functional intestinal mucosal epithelium, in mice the M cells are a key route of invasion by Salmonella, and are thus discussed in greater detail here.

Microfold cells are specialized intestinal epithelial cells that are found in the FAE overlying mucosa-associated lymphoid tissue (MALT). Their primary role is to sample mucosal contents and transfer antigens from the lumen to underlying Peyer's patches (Tam et al., 2008), and thus act as sentinels of the intestinal epithelium. The overall structure of M cells differs in various ways from that of enterocytes. For example, the apical (mucosal) surface of M cells is not covered by the mucus layer observed over other cells in the intestines (Frey et al., 1996). Additionally, the apical brush border characteristic of enterocytes is absent from M cells, which instead contain microfolds. These features promote the sampling role of M cells, but also inadvertently provide opportunities for enteropathogen docking and invasion.

An additional trait of M cells that promotes both their role as intestinal sentinels and promotes enteropathogen invasion is their use of a transcytotic pathway to shuttle luminal contents to lymphoid tissue (Kraehenbuhl and Neutra, 2000). Following endocytosis of extra-cellular material (including invading pathogens), M cells transfer the endosomal contents to the basolateral (serosal) surface and to the underlying MALT. This action results in appropriate immune responses to infection. Likewise, bacteria engulfed at the apical surface can escape the M cell through this same pathway and disseminate to other areas.

M cells provide a key route of invasion by S. Typhimurium (Jensen et al., 1998). SPI-1 genes help regulate Salmonella invasion through M cells, although SPI-1-independent processes are also proposed to regulate M cell invasion (Clark et al., 1996). For example, Salmonella defective for invA are less able to invade M cells (Clark et al., 1998), but SPI-1 mutants are still capable of invading M cells (Martinez-Argudo and Jepson, 2008). These results suggest that while SPI-1 genes are needed for wild-type levels of invasion, SPI-1-independent mechanisms are also important for Salmonella invasion through M cells.

The lymphoid tissue underlying M cells contains Peyer's patches, which are large aggregates of B lymphocyte follicles that contain germinal centers, and can be found along the length of the intestine. Here, the antigens translocated by M cells are processed by local dendritic cells. Lysozyme-producing dendritic cells associated with Peyer's patches mediate uptake of S. Typhimurium (LeLouard et al., 2010). Peyer's patches also play a role in up-regulating intestinal IgA as a result of Salmonella infection (Hashizume et al., 2008).

The barrier function of the epithelial monolayer results from a variety of proteins that maintain close intercellular interactions. Desmosomes are composed of cadherins, which form an adhesive interface (Koch and Franke, 1994; Green and Simpson, 2007), and varying structural proteins, notably plakoglobin and desmoplakin (Hatsell and Cowin, 2001), all of which help disperse forces from physical stress. Meanwhile, the paracellular pathway, or area between adjacent epithelial cells, is kept closed by tight junctions and adherens junctions. Bacteria attempting to penetrate the epithelial monolayer must possess mechanisms for overcoming these strong interactions. As such, Salmonella possess several effectors that mediate disruption of the epithelial barrier and subsequent uptake into the non-phagocytic epithelial cells lining the intestinal lumen. These actions are carried out largely through the alteration of junctional protein localization and of Rho GTPase activity. The latter mechanism is especially important in the Salmonella-mediated modulation of tight junctions. Dis-regulating the Rho-GTPases leads to changes in organization of junctions and of actin at the cell membrane, which subsequently alters the membrane morphology in a manner referred to as “ruffling” (Finlay et al., 1991), and promotes Salmonella entry (Figure 2).

Interactions between classical (Ca²⁺ dependent) cadherins and catenin family proteins are the foundation of adherens junctions. E-cadherin (epithelial cadherin) is a single pass transmembrane protein and its ability to homodimerize with E-cadherins on neighboring cells makes it a key component of adherens junctions (Takeichi, 1991). E-cadherins form small, intercellular clusters that quickly associate with actin and, overtime, expand into larger bundles that strengthen cell–cell adhesion (Adams et al., 1998). p120-catenin stabilizes E-cadherin at the cell surface, and its loss induces down-regulation of E-cadherin (Davis et al., 2003). Further, beta-catenin, a transcription factor involved in cell proliferation and differentiation, can bind to the cytoplasmic domain of E-cadherin (Hartsock and Nelson, 2008), and direct the localization of E-cadherin from the ER to the plasma membrane (Chen et al., 1999). Phosphorylation of beta-catenin promotes its ubiquitination and subsequent degradation (Aberle et al., 1997). Duan et al. (2007) demonstrated that cultured epithelial cells infected with S. Typhimurium display increased phosphorylated beta-catenin. This increase in phosphorylated beta-catenin resulting from Salmonella infection could increase the level of beta-catenin degradation, and thus could limit E-cadherin translocation to the plasma membrane. Such an action would result in weakened adherens junctions and promote Salmonella invasion through the epithelial monolayer.

The tight junction is an important regulator of epithelial monolayer permeability (Martinez-Palomo and Erlij, 1975), and of cell polarity by preventing mixing of apical (mucosal) and basolateral (serosal) components. The “leak pathway” permits passage of larger solutes, such as bacterial peptides (but not whole bacteria), while the “small pore pathway” excludes solutes larger than 4 Å, and exhibits some charge selectivity (Forster, 2008; Turner, 2009). The core of tight junction complexes is composed of occludin, ZO, and claudin proteins. Occludin binds to several crucial tight junction proteins including ZO-1 (Furuse et al., 1994), ZO-2 (Itoh et al., 1999), and ZO-3 (Haskins et al., 1998) and its activity is regulated by PKC-mediated phosphorylation (Andreeva et al., 2001). ZO-1, ZO-2, and ZO-3 are members of the membrane-associated guanylate kinase (MAGUK) family (Gonzalez-Mariscal et al., 2000). ZO-1 and ZO-2 assist in the polymerization of claudins, which permits extension of tight junctions, and also recruit ZO-3 to the tight junction (Umeda et al., 2006; Tsukita et al., 2009). A specific role for ZO-3 has yet to be determined, although it is shown to not be required for tight junction formation (Adachi et al., 2006). Claudins, which are tetraspan transmembrane proteins, are key structural elements, as they recruit occludin (Furuse et al., 1998). Another class of tight junction proteins include junction-associated adhesion molecules (JAMs), or which are integral membrane proteins that belong to the immunoglobin superfamily.

Cytoskeletal regulators of tight junctions include myosin ATPase, AMP-activated protein kinases, and especially Rho-GTPases (Turner, 2009). Rho-GTPases regulate various cell functions and are activated by guanine-nucleotide exchange factors (GEFs), which facilitate the active, GTP-bound state. GTPase activating protein (GAPs) inhibit Rho-GTPases by activating GTP hydrolysis, thereby inducing the inactive, GDP-bound state (Etienne-Manneville and Hall, 2002). Rho-GTPases are regulated by several proteins, the best-studied being Rho, Cdc42, and Rac. Rho is required for formation of focal adhesions and stress fibers, while constitutive Rac causes formation of plasma membrane extensions (Ridley and Hall, 1992; Ridley et al., 1992). Cdc42 activity promotes finger-like extrusions called filopodia (Nobes and Hall, 1995).

The tight junction is a key target of Salmonella, as infection with S. Typhimurium induces altered localization of ZO-2 and claudin-1, degradation of ZO-1, and promotes dephosphorylation of occludin in T84 cells (Kohler et al., 2007). Salmonella SPI-1 effectors SopB, SopE, SopE2, and SipA each play various roles in inducing ruffling and bacterial uptake by changing the localization and expression of ZO-1 and occludin, although SipA and SopB alone are not sufficient for this process (Boyle et al., 2006). These same effectors were also associated with altered epithelial cell polarity. Specifically, SopB (or SopB/SigD) is a phosphoinositide phosphatase that promotes formation of macropinosomes (Hernandez et al., 2004). This effector is capable of indirectly stimulating Cdc42-dependent cytoskeletal rearrangements (Zhou et al., 2001), which can lead to extension of the cell membrane around the bacterium. Similarly, SopE and SopE2 act like GEFs, and activate Cdc42 and Rac1 GTPases to induce cytoskeletal rearrangements that favor bacterial uptake (Stender et al., 2000). SipA and SipC associate with actin and promote bundling of actin filaments so as to facilitate macropinocytosis of Salmonella (McGhie et al., 2001).

As control mechanisms, Salmonella also secrete SptP and AvrA. SptP acts like a GAP, and essentially reverses the cytoskeletal rearrangements that occur during Salmonella uptake (Fu and Galan, 1999). AvrA, which stabilizes tight junctions (Liao et al., 2008) and inhibits NF-kB activation (Collier-Hyams et al., 2002), keeps the Salmonella-induced inflammation response in check.

The release of IL-8 resulting from pathogen-induced immune signaling stimulates the recruitment of neutrophils from blood vessels to the basolateral (serosal) surface (Figure 2). Neutrophils are phagocytic white blood cells that are among the first line of the innate immune defense in response to pathogens. Recruitment of neutrophils to the apical (mucosal) surface is mediated by release of hepoxilin A₃ (HXA₃), a potent neutrophil chemoattractant, and a metabolite of the arachidonic acid pathway (McCormick, 2007). SipA at the apical surface induces a lipid signal cascade that includes the activation of PKC, which causes the release of arachidonic acid from the plasma membrane (Wall et al., 2007). Arachidonic acid is then converted to HXA₃ by 12-lipoxygenase activity. Release of HXA₃ is facilitated by the ABC transporter, MRP2 (Pazos et al., 2008), and generates a concentration gradient across the tight junction and through the paracellular space. The recruitment of neutrophils superficially seems solely an act of host defense; however the rapid migration of these cells through the epithelial monolayer may actually loosen the epithelial cell–cell interactions, and thus create space through which Salmonella can invade, further facilitating PMN infiltration (Kohler et al., 2007). Indeed, PMN transmigration has been demonstrated to reduce epithelial monolayer resistance in vitro (Nash et al., 1987).