In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.     

Role of Salmonella Pathogenicity Island 1 Protein IacP in Salmonella enterica Serovar Typhimurium Pathogenesis

Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.The injection of bacterial proteins by the type III secretion system (T3SS) into the host cytoplasm has been broadly applied to study pathogen-host interactions ranging from the invasion of plant and animal pathogens to a symbiont interaction of Rhizobium (22, 42). The T3SS is composed of more than 20 different structural proteins that form needle-like appendages through which effector proteins are delivered directly into host cells to manipulate various host cell signaling events. Moreover, cytoplasmic chaperones are involved in the stability and efficient translocation of effector proteins (14). Salmonella enterica serovar Typhimurium, a facultative intracellular pathogen, has evolved two distinct T3SSs encoded by Salmonella pathogenicity island 1 (SPI-1), responsible for the invasion of nonphagocytic cells, and by SPI-2, required for intracellular survival and replication inside the Salmonella-containing vacuole (SCV). The expressions of the two T3SSs are inversely regulated during the pathogenic process. Although the expression of the SPI-1 T3SS at systemic sites has remained controversial, some effector proteins of SPI-1 (e.g., SipA and SopB) are persistently expressed and secreted under favorable conditions for SPI-2 expression during the biogenesis and maturation of the SCV (17).After the SPI-1 T3SS is activated upon host cell contact, the translocators SipB and SipC appear to be inserted into the host cell membrane, where they form a translocation pore, which is connected to the needle complex. A variety of effector proteins encoded within and outside SPI-1 can be translocated into a host cytoplasm and cooperatively induce membrane ruffling (11) and macropinocytosis (16). Among SPI-1 effector proteins, SopE, SopE2, and SopB trigger the actin rearrangement in host cells by activating small GTPases, including Rac1, Cdc42, and RhoG, directly or indirectly (39). A Salmonella serovar Typhimurium mutant carrying null mutations in these effector proteins failed to invade epithelial cells. After bacterial invasion, an activated membrane was subsequently recovered by SptP, another effector protein possessing GTPase-activating protein activity (13).The iacP gene, which is located downstream of sicA- sipBCDA in the SPI-1 locus, was initially identified as a putative acyl carrier protein (ACP) by sequence similarity (26). ACP is an abundant small acidic and highly conserved protein that is essential for various biosynthetic pathways (5). In the process of fatty acid (FA) biosynthesis in Escherichia coli, ACP sequentially delivers the acyl intermediates for FA elongation as a cofactor of FA synthase (20). For the enzymatic activity of ACP, a prosthetic group 4′-phosphopantetheine (4′-PP) that was covalently incorporated into apo-ACP serves as the binding site of acyl groups. It was reported previously that the substitution of serine 36 in Escherichia coli ACP eliminated the attachment site of the 4′-PP and inhibited FA incorporation (27).In addition to lipid biosynthesis, acyl-ACP is required for various bacterial virulence processes: the synthesis of the lipid A moiety of lipopolysaccharide (LPS) (43) and the N-acylhomoserine lactones as signal molecules in quorum sensing (52) and the posttranslational modification of bacterial toxins such as E. coli hemolysin (HlyA) (24). The activation of HlyA requires posttranslational acylation at two internal lysine residues by ACP and the acyl transferase HlyC. The conformation of acylated HlyA is matured into a molten globular form comprised of disordered regions, which is necessary for the hemolytic effects of a toxin to occur (21).As a Salmonella serovar Typhimurium mutant that lacks an entire SPI-1 locus was found to grow as well as the wild type, it is predicted that IacP would be responsible for the modification of other proteins in the T3SS (26). However, it is not known which proteins are targeted by IacP or how the invasion process during SPI-1 activation is affected in the iacP mutant. In this study, we report that IacP promotes SopB, SopA, and SopD secretion during cell entry, thus contributing to the virulence of Salmonella serovar Typhimurium.     

Salmonella enterica Serovar Typhimurium Strains with Regulated Delayed Attenuation In Vivo

Recombinant bacterial vaccines must be fully attenuated for animal or human hosts to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Unfortunately, many well-studied means for attenuating Salmonella render strains more susceptible to host defense stresses encountered following oral vaccination than wild-type virulent strains and/or impair their ability to effectively colonize the gut-associated and internal lymphoid tissues. This thus impairs the ability of recombinant vaccines to serve as factories to produce recombinant antigens to induce the desired protective immunity. To address these problems, we designed strains that display features of wild-type virulent strains of Salmonella at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. We recently described one means to achieve this based on a reversible smooth-rough synthesis of lipopolysaccharide O antigen. We report here a second means to achieve regulated delayed attenuation in vivo that is based on the substitution of a tightly regulated araC P_BAD cassette for the promoters of the fur, crp, phoPQ, and rpoS genes such that expression of these genes is dependent on arabinose provided during growth. Thus, following colonization of lymphoid tissues, the Fur, Crp, PhoPQ, and/or RpoS proteins cease to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. Means for achieving regulated delayed attenuation can be combined with other mutations, which together may yield safe efficacious recombinant attenuated Salmonella vaccines.Attenuation of Salmonella vaccine vectors should decrease, if not eliminate, induction of undesirable disease symptoms while the vaccine retains immunogenicity. The attenuated vaccine should be sufficiently invasive and persistent to stimulate both strong primary and lasting memory immune responses and should be designed to minimize consequential adverse events. As even attenuated vaccines may sometimes cause disease (72), the vaccine should be susceptible to clinically useful antibiotics. Achieving a balance between adequate attenuation and safety and maximal immunogenicity in vaccine construction is difficult. Many means to attenuate Salmonella vaccines make them less able to tolerate stresses encountered in the gastrointestinal tract after oral administration, including exposure to acid, bile, increasing osmolarity and iron, and decreasing O₂, and/or reduce invasion of the gut-associated lymphoid tissue (GALT). The doses for recombinant Salmonella vaccines to elicit maximal immune responses in mice are lower for intranasal immunization than they are for oral immunization (37, 55, 58). This may be due, in part, to killing of orally administered vaccines by the acid stress of the stomach (24, 30) quickly followed by exposure to bile in the duodenum. We have determined that these two stresses in succession are more effective in causing bacterial cell death than the sum of killing by each stress alone (M. R. Wilmes-Riesenberg and R. Curtiss, unpublished data). Salmonella possesses a large constellation of genes that confer acid tolerance and resistance to acid stress (1, 17, 20, 21, 51), and inactivation of these genes or their inability to be expressed by induction reduces virulence (76). In this regard, the regulatory proteins RpoS (44), Fur (32), PhoPQ (6, 7), and OmpR (3, 4) are all necessary to confer resistance to acid stress and/or shock in Salmonella enterica serovar Typhimurium. Similarly, many genes are turned on in response to exposure to bile, and some of these gene products transiently repress invasion while bacteria reside in the intestinal lumen (29, 60, 73, 75). The exceedingly low dose of Shigella needed for oral infectivity correlates well with the innate expression of high resistance to acid stresses (74, 75) and the presumed unimportance of bile stress. However, complete lipopolysaccharide (LPS) is of considerable importance as rough mutants of Salmonella lacking LPS O-antigen side chains or portions of the core are avirulent, fail to colonize the intestinal tract, and are deficient in invading cells of the intestinal mucosa (69, 70). This could be due to increased sensitivity to bile or complement and/or an inability to penetrate mucin to enable adherence to intestinal cells prior to invasion. As Salmonella traverses the intestinal tract, there is an increase in osmolarity and a decrease in available oxygen; both of these environmental signals induce the expression of the Salmonella pathogenicity island 1 genes necessary for cell invasion (18, 23, 42), as does the succession of low-pH passage through the stomach followed by the neutral pH of the ileal contents (2). There are also likely stresses to ions, defensins, and other metabolites that might impair the ability of bacterial vaccine vectors, depending on the means of attenuation, to persist in the intestinal tract for sufficient time to enable cell attachment and invasion. In this regard, genes regulated by PhoPQ (25, 26, 61, 73) and PmrAB (77) very much contribute to resistance to bile stress, defensins, and iron stress. Serovar Typhimurium mutants with ΔphoP, ΔphoQ, or ΔphoPQ mutations are all totally avirulent for mice and highly immunogenic in inducing protective immunity to challenge with virulent wild-type strains. This is surprising in that such mutants, although colonizing the GALT to reasonable levels in spite of their increased sensitivity to acid stress, defensins, and bile (61, 73), are found in the mesenteric lymph nodes and spleens of orally immunized mice at much reduced levels (22) compared to titers in numbers of CFU observed after oral administration of either Δaro or Δcya Δcrp attenuated strains (14, 36). These collective results demonstrate that ΔphoPQ mutants are totally avirulent and highly immunogenic but imply that some of the attenuation is due to a reduced ability to colonize lymphoid tissues. RpoS controls expression of the serovar Typhimurium virulence plasmid spv genes (19, 57). The spvRABCD gene cluster controls the growth rate of Salmonella in deep organs and is required for systemic infection and bacteremia in animals and humans (see reference 28 for a review). As expected, Salmonella rpoS mutants have a severely impaired capacity to colonize spleens of infected mice, resulting in avirulence in mice (10, 11, 40). In addition, rpoS mutations reduce the ability of serovar Typhimurium to colonize Peyer''s patches of infected mice (11, 56).Based on the above observations and thoughts, we reasoned that it might be important to have mutations contributing to attenuation or other beneficial vaccine attributes that do not impair the abilities of the vaccine to adjust to and/or withstand a diversity of stresses encountered at any location within the gastrointestinal tract if the vaccine is administered orally or in the respiratory tract if it is administered intranasally. Likewise, there may be a benefit to having a vaccine strain that expresses wild-type abilities not compromised by direct mutations to penetrate through mucin, to attach to cells in the mucosal epithelium, and to be invasive into those cells. To achieve these objectives, we have developed six means using three strategies to achieve regulated delayed attenuation of Salmonella in vivo such that strains at the time of immunization exhibit almost the same abilities as fully virulent wild-type strains to contend with stresses and successfully reach effector lymphoid tissues before displaying attenuation, which precludes onset of any disease symptoms. The first strategy (15) involves a smooth-to-rough phenotypic change in LPS in vivo and makes use of pmi mutants that lack the phosphomannose isomerase needed to interconvert fructose-6-phosphate and mannose-6-phosphate (49). Strains with the Δpmi mutation grown in the presence of mannose synthesize a complete LPS O antigen but lose LPS O-antigen side chains after about seven generations of growth in medium devoid of mannose or in tissues since nonphosphorylated mannose, required for uptake to synthesize O antigen, is unavailable. We report here our second strategy based on regulated delayed expression in vivo of virulence genes. We thus describe four means to be used alone or in combination to provide a regulated delayed attenuation phenotype so that vaccine strains with these mutations have nearly the ability of wild-type Salmonella to colonize lymphoid tissues before exhibiting an attenuated phenotype. Each means confers significant attenuation and improved immunogenicity compared to selected attenuated strains made by direct mutation in virulence genes. Our third strategy (39) uses a system for regulated delayed lysis in vivo to provide both attenuation and biological containment.     

Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.Recognition of microbes is a critical step in the initiation of the host immune response against infection. Indeed, detection of microbe-associated molecular patterns by germ line-encoded receptors such as Toll-like receptors (18) and Nod-like receptors (NLRs) (8) is an early event that leads to inflammatory responses through the production of cytokines and chemokines. Nod1 and Nod2 are cytosolic proteins of the NLR family that detect distinct substructures from bacterial peptidoglycan (8). Whereas Nod2 detects muramyl dipeptide (12, 16), a motif common to gram-negative and gram-positive bacteria, Nod1 senses meso-diaminopimelic acid-containing peptidoglycan (3, 11), which is more commonly found in gram-negative bacteria. In macrophages and dendritic cells (DC), triggering of Nod1 and Nod2 induces proinflammatory cytokines and costimulatory molecules (21). In addition, synergistic effects of Nod ligands with Toll-like receptor ligands in myeloid cells have been reported (9, 29). Nod1 has been shown to regulate the colonization of mice by Helicobacter pylori (31), and Nod2 affects the pathogenicity of Listeria monocytogenes (19) and Mycobacterium tuberculosis (6) in mice models. However, no studies have been conducted on the impact of Nod1 and Nod2 on the in vivo infection process of the specific enteric pathogen, Salmonella enterica serovar Typhimurium.Salmonella enterica is a gram-negative bacterium of the Enterobacteriaceae family. S. enterica serovar Typhimurium is a mouse pathogen that provokes a typhoid-like syndrome in orally infected mice, with colonization of the deeper organs, including the liver and spleen (5). S. Typhimurium is capable of entering intestinal epithelial cells using a unique mechanism involving a type 3 secretion system, Salmonella pathogenicity island 1 (Spi1) (10), and resides in a vacuole within infected cells via a mechanism dependent on a second type 3 secretion system, Spi2 (27). Hence, the bacteria are able to avoid killing and spread throughout the infected host by invading immune cells. The intracellular lifestyle of Salmonella is in line with a possible implication of Nod proteins during the course of the infection. However, to date, no study has been conducted in vivo to determine the role of Nod1 and Nod2 after oral infection with S. Typhimurium.Spi1 is critical for the invasiveness of the bacteria in epithelial cells and is thought to be responsible for the main route of entry of the bacteria through Peyer''s patches (13, 30). Strikingly, bacteria deficient for Spi2 are completely avirulent whereas Spi1 mutants are still capable of inducing the disease (26). Recently, myeloid cells from the intestinal lamina propria have been shown to sample the luminal contents of the gut, including intact bacteria. This mechanism is crucial for gut homeostasis but provides a portal of entry for S. Typhimurium and explains the persistent virulence of Spi1-deficient bacteria (4, 24, 30).In the present study we show that Nod1 deficiency leads to increased susceptibility to Spi1 deficient-S. Typhimurium but not the wild-type (WT) strain, suggesting a critical role for Nod1 in myeloid cells from the intestinal lamina propria for defense against S. Typhimurium infection in vivo. Accordingly, using bone marrow-chimeric mice, we have been able to locate the defect in in vivo hematopoietic cells. Indeed, Nod1 deficient animals show increased S. Typhimurium in the lamina propria DC subpopulation and an impaired cellular response after infection with Spi1-deficient bacteria. Additionally, we observed an impaired response of Nod1-deficient DCs toward the bacteria. Taken together, our findings uncover a surprising role of Nod1 in lamina propria DCs in the control of S. Typhimurium infection in vivo.     

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

The secretion of bacterial toxin proteins is achieved by dedicated machineries called secretion systems. The type VI secretion system (T6SS) is a widespread versatile machine used for the delivery of protein toxins to both prokaryotic and eukaryotic cells. In Salmonella enterica serovar Typhimurium, the expression of the T6SS genes is activated during macrophage or mouse infection. Here, we show that the T6SS gene cluster is silenced by the histone-like nucleoid structuring H-NS protein using a combination of reporter fusions, electrophoretic mobility shift assays, DNase footprinting, and fluorescence microscopy. We further demonstrate that derepression of the S. Typhimurium T6SS genes induces T6SS-dependent intoxication of competing bacteria. Our results suggest that relieving T6SS H-NS silencing may be used as a sense-and-kill mechanism that will help S. Typhimurium to homogenize and synchronize the microbial population to gain efficiency during infection.     

Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 10(4) to 10(7) bacteria in C3H/HeN and 10(1) to 10(4) bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.     

Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nal^r strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.     

Toll-Like Receptor 5-Dependent Regulation of Inflammation in Systemic Salmonella enterica Serovar Typhimurium Infection

Salmonella enterica, a gram-negative pathogen, causes a spectrum of human infections including enterocolitis and typhoid fever. We previously showed that Salmonella flagellin played a role in suppressing intestinal mucosal inflammation in a murine model of acute enterocolitis. In this study, we examined the role of flagellin in the typhoid-like systemic murine Salmonella infection by measuring bacterial proliferation, inflammation, leukocyte recruitment, and cellular apoptosis in Peyer''s patches (PPs), mesenteric lymph node (MLN), and spleen. We found that relative to an isogenic wild-type (WT) strain, aflagellate Salmonella exhibited increased proliferation at 4 days postinfection in PPs and MLN but not spleen. The aflagellate mutant also elicited increased local and systemic secretion of inflammatory cytokines such as interleukin-1β, gamma interferon, and tumor necrosis factor alpha and enhanced surface expression of ICAM-1 on macrophages and dendritic cells (DCs). Furthermore, the recruitment of macrophages and DCs in PPs and MLN, but not spleen, was enhanced upon infection with aflagellate Salmonella. The relative differences between WT and aflagellate Salmonella were highly attenuated in Toll-like receptor 5-deficient (TLR5^−/−) mice, indicating involvement of TLR5-dependent signaling. Interestingly, infection with the aflagellate mutant also resulted in decreased levels of T-cell apoptosis in PPs relative to infection with WT Salmonella. We postulate that the initial lack of detection of the aflagellate mutant in the mucosa permits increased proliferation within the host and enhances inflammatory signaling in nonepithelial cell types, which subsequently promotes leukocyte recruitment. In contrast, lack of difference in any disease parameter measured in the spleen likely reflects that Salmonella expression of flagellin is downregulated in this organ. Thus, the characteristic inflammatory pathology of Salmonella infection occurs only in PPs and to a lesser extent in MLN during the initial phases of infection and these early responses are dependent on TLR5.Salmonella enterica is a food- and waterborne gram-negative pathogen that causes a range of infectious diseases in a variety of hosts. In humans, infection with Salmonella enterica serovar Typhi typically causes severe systemic illness while infection with serovar Typhimurium is commonly associated with self-limiting gastroenteritis. S. enterica serovar Typhimurium infections are a common cause of food poisoning in industrialized countries. While the infection is usually confined to the intestinal tract, it can sometimes result in severe complications and is a particular threat to immunocompromised persons. In contrast, systemic infections by S. enterica serovar Typhi, often referred to as “typhoid fever,” commonly cause severe, sometimes lethal, illness, making this bacterium a major menace in the developing world. Importantly, in contrast to Salmonella-induced gastroenteritis, systemic infections with S. enterica serovar Typhi are not typically associated with significant intestinal injury during early stages of disease.The mechanisms of Salmonella infection in mice have been extensively studied. The most efficient route of systemic infection appears to be that, following ingestion and gastric passage, Salmonella reaches the small intestine lumen, where it crosses the epithelium overlaying mucosal lymphoid aggregates, or Peyer''s patches (PPs). The epithelium overlaying PPs—the follicle-associated epithelium—contains specialized enterocytes, referred to as “microfold” cells (or M cells), that are adapted for uptake of particulate antigens and are exploited by Salmonella to breach the epithelial barrier (31). PPs are distributed all along the small intestine, predominantly in the distal ileum. Histologically, they are composed of a subepithelial dome region containing dendritic cells (DCs) and discrete B-cell-rich germinal centers with a surrounding T-cell region. Salmonella initially enters the PPs and replicates intracellularly within DCs. This provokes inflammatory processes resulting in recruitment and accumulation of monocytes/macrophages, neutrophils, and additional DCs (22, 31). Neutrophils and monocytes predominately accumulate in the subepithelial dome, whereas monocytes alone are found in the T-cell area (25). DCs present in the subepithelial dome region migrate to the T-cell region following initial invasion (31). Free bacteria or infected DCs then egress via the lymphatics to reach the mesenteric lymph node (MLN) and ultimately to systemic reticuloendothelial tissues of the spleen and liver.As with most pathogens, Salmonella possesses a range of virulence factors that are necessary to invade and survive in the hostile environment encountered within the host. One of these virulence factors is the flagella responsible for the bacterial motility and chemotaxis, which allows the organism to move in response to environmental signals (23). The long helical filament of flagella is mainly composed of monomers of two distinct but related bacterial flagellin proteins, FliC and FljB (18). It is now well known that flagellin monomers are recognized by the innate immune system via extracellular Toll-like receptor 5 (TLR5), a member of the TLR family (21, 37). TLR5 is expressed on the basolateral surface of intestinal epithelial cell lines and human colon (8, 24, 37) and on CD11c⁺ lamina propria DCs (33). TLR5 is able to sense flagellin from a variety of flagellated pathogens, including enteropathogenic Escherichia coli and Salmonella. Additionally, flagellin released or shed from intracellular bacteria can be recognized by the cytoplasmic pattern recognition receptors IPAF (IL-1β-converting enzyme protease activating factor) and/or NAIP5 (neuronal apoptosis inhibitor protein 5), expressed inside antigen-presenting cells such as macrophages (7, 13, 17, 20, 29). Flagellin has been shown to potently induce inflammation via the NF-κB and mitogen-activated protein kinase pathways and stimulate the production of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-18 (13, 26, 41), and thus represents a major proinflammatory determinant of Salmonella. We have shown that in in vitro epithelial systems, aflagellate Salmonella (FliC⁻ FljB⁻) was virtually devoid of the potent proinflammatory signaling mediated by isogenic wild-type (WT) Salmonella (41). Additionally, we have demonstrated in an acute model of murine enterocolitis that aflagellate Salmonella, while showing suppressed inflammation and epithelial apoptosis at very early stages (6 to 24 h post oral infection), stimulated markedly increased mucosal inflammation and apoptosis at 48 h, suggesting a time-dependent attenuating effect of flagellin on both cellular inflammation and apoptosis (39). Interestingly flagellin is rather involved in the early innate response in the bovine ileal loop model (40). In contrast to studies of Salmonella enterocolitis in which many bacteria directly invade throughout the gut epithelium, the role of flagellin in systemic infection, in which infection occurs via the PPs, is not known.Salmonella has been shown to induce several types of programmed cell death (6, 11), a fundamental mechanism necessary to eliminate infected, damaged, or superfluous cells. Apoptotic programmed cell death is characterized by the condensation of the nucleus and the cytoplasm and the fragmentation of the cell, leading to the formation of apoptotic bodies eliminated by macrophages, thus preventing release of noxious cellular contents (6). However, any type of cell death can also become deleterious and cause pathological tissue damage when it is prolonged or amplified, as often occurs during infection with a pathogen such as Shigella (42). Salmonella is able to induce apoptosis in several cell types, including both epithelial cells and leukocytes (16, 22, 30).In this study, we examine the role of Salmonella flagellin in a typhoid fever model in C57BL/6 mice. We report that a mutant lacking flagellin is able to replicate more efficiently in PPs and to a lesser extent in the MLN, leading to an increase in inflammatory mediator expression and leukocyte recruitment in these tissues. We also observed that this effect does not occur in the spleen. Furthermore, the aflagellate mutant decreases T-cell apoptosis at day 4 postinfection via TLR5-independent mechanisms.     

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.     

Characterization of the Murine T-Lymphocyte Response to Salmonella enterica Serovar Typhimurium Infection

Infection of mice with Salmonella enterica serotype Typhimurium induces a strong Th1 cell response that is central for the control of infection. We infected mice of a resistant background with a virulent strain of S. enterica serovar Typhimurium and analyzed the kinetics and magnitude of the T-cell response. After infection, the majority of CD4(+) and CD8(+) splenocytes acquired an activated phenotype, as indicated by expression levels of CD44 and CD62L. In addition, after 3 to 4 weeks of infection, more than 20% of the CD4(+) and more than 30% of the CD8(+) T cells produced gamma interferon (IFN-gamma) in response to short-term polyclonal stimulation. In contrast, we detected only a moderate (two- to threefold) expansion of both T-cell populations, and BrdU incorporation revealed that there was either no or only a limited increase in the in vivo proliferation of CD4(+) and CD8(+) T cells, respectively. Our results indicate that although an unexpectedly large population of both CD4(+) and CD8(+) T cells is activated and acquires the potential to secrete IFN-gamma, this activation is not paralleled by substantial expansion of these T-cell populations.     

Characterization of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated in Japan

A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.     

Genetic Analysis of Non-Hydrogen Sulfide-Producing Salmonella enterica Serovar Typhimurium and S. enterica Serovar Infantis Isolates in Japan

Whole-genome sequencing of non-H₂S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 β-lactamase. The lack of production of H₂S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.