Contribution of Flagellin Pattern Recognition to Intestinal Inflammation during Salmonella enterica Serotype Typhimurium Infection

Salmonella enterica serotype Typhimurium causes acute inflammatory diarrhea in humans. Flagella contribute to intestinal inflammation, but the mechanism remains unclear since most mutations abrogating pattern recognition of flagellin also prevent motility and reduce bacterial invasion. To determine the contribution of flagellin pattern recognition to the generation of innate immune responses, we compared in two animal models a nonmotile, but flagellin-expressing and -secreting serotype Typhimurium strain (flgK mutant) to a nonmotile, non-flagellin-expressing strain (flgK fliC fljB mutant). In vitro, caspase-1 can be activated by cytosolic delivery of flagellin, resulting in release of the interferon gamma inducing factor interleukin-18 (IL-18). Experiments with streptomycin-pretreated caspase-1-deficient mice suggested that induction of gamma interferon expression in the murine cecum early (12 h) after serotype Typhimurium infection was caspase-1 dependent but independent of flagellin pattern recognition. In addition, mRNA levels of the CXC chemokines macrophage inflammatory protein 2 and keratinocyte-derived chemokine were markedly increased early after serotype Typhimurium infection of streptomycin-pretreated wild-type mice regardless of flagellin expression. In contrast, in bovine ligated ileal loops, flagellin pattern recognition contributed to increased mRNA levels of macrophage inflammatory protein 3α and more fluid accumulation at 2 h after infection. Collectively, our data suggest that pattern recognition of flagellin contributes to early innate host responses in the bovine ileal mucosa but not in the murine cecal mucosa.Salmonella enterica serotype Typhimurium is a major cause of gastroenteritis in humans, which is characterized by acute intestinal inflammation and diarrhea (11, 36). One of the serotype Typhimurium virulence factors contributing to intestinal inflammation are flagella. Nonflagellated serotype Typhimurium mutants have been shown to cause less inflammation than their isogenic parents do after infection of bovine ligated ileal loops (59), streptomycin-pretreated mice (65, 74), and chickens (24).Several possible mechanisms by which flagella may contribute to eliciting proinflammatory responses have been proposed. Flagella are surface appendages of serotype Typhimurium that are required for motility and chemotaxis. Motility contributes to serotype Typhimurium invasion of intestinal epithelial cell lines by increasing bacterial contact with host cells (26, 27). The invasion-associated type III secretion system (T3SS-1) is important for inducing intestinal inflammation in animal models (1, 20, 70, 81). Nonmotile serotype Typhimurium mutants may thus cause reduced intestinal inflammation in vivo because the efficiency of T3SS-1-mediated invasion is reduced.In addition to its role in motility and invasion, the proteinaceous monomer of the flagellar filament, flagellin, has been shown to be a potent activator of the innate immune response in tissue culture models. Flagellin is an agonist of Toll-like receptor 5 (TLR5) (21), a pattern recognition receptor (PRR) of the innate immune system expressed on the basolateral surface of intestinal epithelial cells (15, 16) and on the surface of a subset of intestinal dendritic cells (71). In addition, flagellin is delivered into the cytosol of macrophages by the T3SS-1 of serotype Typhimurium (12, 38, 68), where it activates the cytosolic interleukin-1β (IL-1β) converting enzyme-protease activating factor (IPAF), a nucleotide-binding and oligomerization domain-like receptor (NLR) of the innate immune system. Recognition of flagellin by IPAF leads to activation of the inflammasome (i.e., caspase-1), followed by proteolytic activation of IL-1β and IL-18 (12, 38).Although the molecular mechanisms by which flagella influence interaction with host cells have been determined using tissue culture models, it remains unclear which of these mechanisms are operational in vivo. The principal reason for this is that most mutations that prevent the biosynthesis of flagella are associated with a pleiotropic phenotype, including an absence of motility, reduced invasion and reduced stimulation of TLR5 and IPAF. For example, inactivation of the two flagellin genes (fliC and fljB) reduces inflammation in vivo (24, 59, 65, 74), but it is not clear whether the lack of motility or the lack of flagellin pattern recognition exhibited by the fliC fljB mutant is responsible for this observation. Because of the pleiotropic phenotypes of the mutants under study, genetic approaches used in previous reports were not able to distinguish between a reduction in pattern recognition and a reduction in invasiveness as possible causes for reduced inflammatory responses elicited by nonmotile serotype Typhimurium mutants. Therefore, conclusive evidence for a contribution of flagellin pattern recognition to inflammation in vivo is still lacking.Here, we applied a combination of innovative bacterial genetics, mouse genetics, and bovine ligated ileal loop experiments to overcome current limitations and test the hypothesis that flagellin pattern recognition contributes to the initiation of inflammation in vivo.     

O-Antigen-Negative Salmonella enterica Serovar Typhimurium Is Attenuated in Intestinal Colonization but Elicits Colitis in Streptomycin-Treated Mice

Lipopolysaccharide (LPS) is a major constituent of the outer membrane and an important virulence factor of Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium). To evaluate the role of LPS in eliciting intestinal inflammation in streptomycin-treated mice, we constructed an O-antigen-deficient serovar Typhimurium strain through deletion of the wbaP gene. The resulting strain was highly susceptible to human complement activity and the antimicrobial peptide mimic polymyxin B. Furthermore, it showed a severe defect in motility and an attenuated phenotype in a competitive mouse infection experiment, where the ΔwbaP strain (SKI12) was directly compared to wild-type Salmonella. Nevertheless, the ΔwbaP strain (SKI12) efficiently invaded HeLa cells in vitro and elicited acute intestinal inflammation in streptomycin-pretreated mice. Our experiments prove that the presence of complete LPS is not essential for in vitro invasion or for triggering acute colitis.Salmonella spp. are a common cause of bacterial food-borne infections. Diseases caused by Salmonella spp. range from gastrointestinal symptoms such as fever, diarrhea, abdominal pain, and nausea to severe systemic infections. Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) is one of the most frequent enteropathogens, causing large numbers of diarrheal infections worldwide by colonizing the gut and triggering mucosal inflammation (33). The type III secretion system 1 (TTSS-1) and TTSS-2 encoded on Salmonella pathogenicity island 1 (SPI1) and SPI2 on the Salmonella genome are employed by the pathogen for mediating bacterial entry into the gut mucosa (SPI1) as well as the intracellular survival followed by systemic spread of the bacteria (SPI2) (9). Acute enteric serovar Typhimurium infection and the mechanisms leading to intestinal inflammation can be analyzed using a well-defined mouse model for Salmonella colitis: streptomycin-pretreated, naïve mice develop a vigorous local inflammation of the large intestine upon intragastric infection with serovar Typhimurium (3).Besides the SPI1- and SPI2-encoded TTSSs, serovar Typhimurium requires numerous additional virulence factors for colonizing the host, resisting host immune defense, and finally, triggering disease. One key virulence factor for serovar Typhimurium is lipopolysaccharide (LPS), a major surface component (42). It contributes to the stability of the outer membrane, serves as a permeability barrier, and protects the bacterium against environmental challenges (34). LPS is composed of three domains. The lipid A part, also known as endotoxin, anchors LPS molecules in the outer membrane with its fatty acid chains. It is connected through the inner core consisting of heptoses and Kdo (3-deoxy-d-manno-octulosonic acid), with the outer core containing hexoses and N-acetylhexoses. Linked to the last glucose of the outer core is the polymeric O-antigen region. This region is composed of 16 to >100 repeats of an oligosaccharide structure containing four to six monosaccharides (27).The endotoxic properties of LPS are mediated by the lipid A moiety, which can be recognized by Toll-like receptor 4 and thus triggers an innate immune response (16, 32). The O antigen, in combination with the inner and outer cores, serves as protection against complement antimicrobial peptides, detergents, and certain antibiotics. Furthermore, the O-antigen region is a key determinant for recognition by the adaptive immune response (40).A number of studies have established an important role for O-antigen side chains in Salmonella virulence. A signature-tagged mutagenesis screening by Morgan and coworkers proved that mutations in genes for enzymes involved in the biosynthesis of O-antigen side chains attenuated bacteria in their ability to colonize chick and calf intestines (25). Interestingly, a mutant in wbaP, the phosphogalactosyltransferase starting O-antigen biosynthesis, was able to colonize calves but showed an attenuated phenotype in chicks (25). Moreover, screening for Salmonella genes required for long-term systemic infection after intraperitoneal injection showed negative selection for mutants in O-antigen biosynthesis (21). Coinfection experiments by Nevola et al. show that mutants lacking O antigen are still able to colonize the murine intestine but are attenuated in competitive infection experiments (30). Furthermore, a recent in vitro study with Salmonella enterica serovar Typhi showed that O-antigen side chains are not necessary for adhesion to and invasion of epithelial cells. However, mutants lacking the complete outer core are severely attenuated (14). In general, the loss of core structures seems more detrimental than the loss of O-antigen side chains. However, it had remained unclear whether the O-antigen side chains are required for triggering intestinal inflammation.We wanted to analyze the role of O-antigen side chains in a well-established mouse model for enteric infections (3) and in an in vitro cellular invasion assay (36). Thus, we deleted the gene encoding the phosphogalactosyltransferase WbaP. This enzyme adds phosphogalactose to undecaprenylphosphate, the first step in O-antigen side chain biosynthesis in the cytoplasm of serovar Typhimurium (35, 43, 44). Streptomycin-pretreated mice were orally infected with the wbaP mutant strain (SKI12), and in line with published work, we found that the ΔwbaP mutant strain (SKI12) was significantly attenuated in a competitive infection assay. In spite of this, the wbaP mutant alone was able to trigger acute colitis. This demonstrates that serovar Typhimurium permits substantial manipulation of the O-antigen structure without losing its ability to trigger mucosal inflammation.     

Laboratory-Acquired Infection with Salmonella enterica Serovar Typhimurium Exposed by Whole-Genome Sequencing

Despite advances in laboratory design, professional training, and workplace biosafety guidelines, laboratory-acquired infections continue to occur. Effective tools are required to investigate cases and prevent future illness. Here, we demonstrate the value of whole-genome sequencing as a tool for the identification and source attribution of laboratory-acquired salmonellosis.     

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.     

Increasing Incidence and Comparison of Nalidixic Acid-Resistant Salmonella enterica subsp. enterica Serotype Typhimurium Isolates from Humans and Animals

We determined the resistance to quinolone of 309 Salmonella enterica subsp. enterica serotype Typhimurium strains isolated from humans and animals (cattle, pigs, or poultry) in 1995 or 1996. Nalidixic acid resistance increased from 8.5% in 1995 to 18.6% in 1996. The highest resistance levels correlated with a mutation at Ser-83 (or Asp-82). All strains remained ciprofloxacin susceptible. Human and animal isolates were compared by pulsed-field gel electrophoresis, and the banding patterns of the human isolates most closely matched those of the bovine isolates.     

Increased Susceptibility of Melanin-Concentrating Hormone-Deficient Mice to Infection with Salmonella enterica Serovar Typhimurium

Melanin-concentrating hormone (MCH) was initially identified in mammals as a hypothalamic neuropeptide regulating appetite and energy balance. However, the wide distribution of MCH receptors in peripheral tissues suggests additional functions for MCH which remain largely unknown. We have previously reported that mice lacking MCH develop attenuated intestinal inflammation when exposed to Clostridium difficile toxin A. To further characterize the role of MCH in host defense mechanisms against intestinal pathogens, Salmonella enterocolitis (using Salmonella enterica serovar Typhimurium) was induced in MCH-deficient mice and their wild-type littermates. In the absence of MCH, infected mice had increased mortality associated with higher bacterial loads in blood, liver, and spleen. Moreover, the knockout mice developed more-severe intestinal inflammation, based on epithelial damage, immune cell infiltrates, and local and systemic cytokine levels. Paradoxically, these enhanced inflammatory responses in the MCH knockout mice were associated with disproportionally lower levels of macrophages infiltrating the intestine. Hence, we investigated potential direct effects of MCH on monocyte/macrophage functions critical for defense against intestinal pathogens. Using RAW 264.7 mouse monocytic cells, which express endogenous MCH receptor, we found that treatment with MCH enhanced the phagocytic capacity of these cells. Taken together, these findings reveal a previously unappreciated role for MCH in host-bacterial interactions.     

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.     

Adaptive acid tolerance response in Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Typhi

The survival of bacteria in various environments depends on a number of protective responses including acid tolerance response (ATR). In this study, ATR phenomenon was compared in Salmonella enterica serovar Typhi 6 and Salmonella enterica serovar Typhimurium 98 under different culture conditions. Survival of the adapted culture (pre-acid shocked to pH 5.5) was significantly better (p < 0.05) as compared to control, unadapted culture after acid shock at pH 3.3. However, the ATR varied with the serovar, incubation temperature and the growth medium used (all p-values < 0.05). S. Typhi 6 failed to grow in pH 3.3 at 45 degrees C. The addition of tetracycline or chloramphenicol (1.0 microg ml(-1)) to adapted cultures during or after acid shock (pH 3.3) had no effect on ATR expression. In S. Typhimurium 98, growth was increased by 10% or greater in adapted culture (when grown at pH 3.3) as compared to growth observed with an unadapted culture (when grown at pH 7.3) on transfer to fresh growth medium at pH 7.3. A poor ATR observed in non-growing S. Typhimurium 98 suspensions clearly showed that ATR is an energy-consuming process. Storage of S. Typhimurium 98 cultures in pH 4.5 nutrient broth at 4 degrees C demonstrated that prolonged exposure to acidic conditions is more detrimental in comparison to the cultures stored at pH 7.3 at this temperature.     

The Ferric Enterobactin Transporter Fep Is Required for Persistent Salmonella enterica Serovar Typhimurium Infection

Most bacterial pathogens require iron to grow and colonize host tissues. The Gram-negative bacterium Salmonella enterica serovar Typhimurium causes a natural systemic infection of mice that models acute and chronic human typhoid fever. S. Typhimurium resides in tissues within cells of the monocyte lineage, which limit pathogen access to iron, a mechanism of nutritional immunity. The primary ferric iron import system encoded by Salmonella is the siderophore ABC transporter FepBDGC. The Fep system has a known role in acute infection, but it is unclear whether ferric iron uptake or the ferric iron binding siderophores enterobactin and salmochelin are required for persistent infection. We defined the role of the Fep iron transporter and siderophores in the replication of Salmonella in macrophages and in mice that develop acute followed by persistent infections. Replication of wild-type and iron transporter mutant Salmonella strains was quantified in cultured macrophages, fecal pellets, and host tissues in mixed- and single-infection experiments. We show that deletion of fepB attenuated Salmonella replication and colonization within macrophages and mice. Additionally, the genes required to produce and transport enterobactin and salmochelin across the outer membrane receptors, fepA and iroN, are needed for colonization of all tissues examined. However, salmochelin appears to be more important than enterobactin in the colonization of the spleen and liver, both sites of dissemination. Thus, the FepBDGC ferric iron transporter and the siderophores enterobactin and salmochelin are required by Salmonella to evade nutritional immunity in macrophages and cause persistent infection in mice.     

Coinfection with an Intestinal Helminth Impairs Host Innate Immunity against Salmonella enterica Serovar Typhimurium and Exacerbates Intestinal Inflammation in Mice

Salmonella enterica serovar Typhimurium is a Gram-negative food-borne pathogen that is a major cause of acute gastroenteritis in humans. The ability of the host to control such bacterial pathogens may be influenced by host immune status and by concurrent infections. Helminth parasites are of particular interest in this context because of their ability to modulate host immune responses and because their geographic distribution coincides with those parts of the world where infectious gastroenteritis is most problematic. To test the hypothesis that helminth infection may negatively regulate host mucosal innate immunity against bacterial enteropathogens, a murine coinfection model was established by using the intestinal nematode Heligmosomoides polygyrus and S. Typhimurium. We found that mice coinfected with S. Typhimurium and H. polygyrus developed more severe intestinal inflammation than animals infected with S. Typhimurium alone. The enhanced susceptibility to Salmonella-induced intestinal injury in coinfected mice was found to be associated with diminished neutrophil recruitment to the site of bacterial infection that correlated with decreased expression of the chemoattractants CXCL2/macrophage inflammatory protein 2 (MIP-2) and CXCL1/keratinocyte-derived chemokine (KC), poor control of bacterial replication, and exacerbated intestinal inflammation. The mechanism of helminth-induced inhibition of MIP-2 and KC expression involved interleukin-10 (IL-10) and, to a lesser extent, IL-4 and IL-13. Ly6G antibody-mediated depletion of neutrophils reproduced the adverse effects of H. polygyrus on Salmonella infection. Our results suggest that impaired neutrophil recruitment is an important contributor to the enhanced severity of Salmonella enterocolitis associated with helminth coinfection.     

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.     

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.     

Effect of Human Immunodeficiency Virus Infection on Plasma Bactericidal Activity against Salmonella enterica Serovar Typhimurium

Individuals with human immunodeficiency virus (HIV) infection have increased susceptibility to invasive disease caused by Salmonella enterica serovar Typhimurium. Studies from Africa have suggested that this susceptibility is related in part to the development of a high level of lipopolysaccharide (LPS)-specific IgG that is able to inhibit the killing of S. Typhimurium by bactericidal antibodies in healthy individuals. To explore this issue further, we examined the bactericidal activity against S. Typhimurium using serum and plasma samples from healthy controls and various clinical subgroups of HIV-infected adults in the United States. We found that the bactericidal activity in the samples from HIV-positive elite controllers was comparable to that from healthy individuals, whereas it was significantly reduced in HIV-positive viremic controllers and untreated chronic progressors. As demonstrated previously for healthy controls, the bactericidal activity of the plasma from the elite controllers was inhibited by preincubation with S. Typhimurium LPS, suggesting that it was mediated by anti-LPS antibodies. S. Typhimurium LPS-specific IgG was significantly reduced in all subgroups of HIV-infected individuals. Interestingly, and in contrast to the healthy controls, plasma from all HIV-positive subgroups inhibited in vitro killing of S. Typhimurium by plasma from a healthy individual. Our results, together with the findings from Africa, suggest that multiple mechanisms may be involved in the HIV-induced dysregulation of humoral immunity to S. Typhimurium.     

Salmonella enterica Serotype Dublin Infection: an Emerging Infectious Disease for the Northeastern United States

Salmonella enterica subspecies enterica serotype Dublin (S. enterica Dublin) emerged for the first time in New York, Pennsylvania, and Ohio in 1988. Since that time this host-adapted serotype has spread throughout the veal- and dairy beef-raising operations in the region; very few dairy farms have experienced clinical S. enterica Dublin infections. This study details the epidemiology of the outbreaks in cattle. During the period 1988 through 1995, nine New York and four Pennsylvania counties have been affected; 13 different locations were involved in New York, and 10 were involved in Pennsylvania. The morbidity and mortality and seasonal distribution of outbreaks, which totaled 35, is described. The antimicrobial susceptibility pattern of isolates revealed that many of the strains were resistant to a number of commonly used drugs. Clinical case details and pathology information are provided, with a caution to clinicians and microbiologists presented with suspect animals, i.e., most cases occurred in older calves, which is atypical for salmonellosis for this region (calves were 8 or more weeks old) and presented as pneumonia and septicemia rather than the primarily diarrheal syndrome that is more typically recognized for the region. The epidemiology of cases is analyzed through cluster analysis of bacterial isolates and their fatty acid methyl ester profiles; at least six clones appeared in the region during the study period. Results of the epidemiology analysis are used to support a hypothesis regarding the source of S. enterica Dublin for the region and its manner of dissemination.     

Prolonged Impact of Antibiotics on Intestinal Microbial Ecology and Susceptibility to Enteric Salmonella Infection

The impact of antibiotics on the host''s protective microbiota and the resulting increased susceptibility to mucosal infection are poorly understood. In this study, antibiotic regimens commonly applied to murine enteritis models are used to examine the impact of antibiotics on the intestinal microbiota, the time course of recovery of the biota, and the resulting susceptibility to enteric Salmonella infection. Molecular analysis of the microbiota showed that antibiotic treatment has an impact on the colonization of the murine gut that is site and antibiotic dependent. While combinations of antibiotics were able to eliminate culturable bacteria, none of the antibiotic treatments were effective at sterilizing the intestinal tract. Recovery of total bacterial numbers occurs within 1 week after antibiotic withdrawal, but alterations in specific bacterial groups persist for several weeks. Increased Salmonella translocation associated with antibiotic pretreatment corrects rapidly in association with the recovery of the most dominant bacterial group, which parallels the recovery of total bacterial numbers. However, susceptibility to intestinal colonization and mucosal inflammation persists when mice are infected several weeks after withdrawal of antibiotics, correlating with subtle alterations in the intestinal microbiome involving alterations of specific bacterial groups. These results show that the colonizing microbiotas are integral to mucosal host protection, that specific features of the microbiome impact different aspects of enteric Salmonella pathogenesis, and that antibiotics can have prolonged deleterious effects on intestinal colonization resistance.The mammalian host is colonized by trillions of microbes that live in a predominantly symbiotic relationship with their host (18, 43). The majority of these microbes inhabit the gastrointestinal (GI) tract (12, 17), and a large percentage of these microbes cannot be cultured by currently available methods, necessitating the use of molecular approaches for the identification and quantification of these organisms (40). The recent application of 16S rRNA gene sequences for the study of complex microbial ecosystems has greatly advanced the understanding of intestinal microbial ecology (2, 40). Current analyses of the intestinal microbiota suggest that the gut is colonized by more than 1,000 different bacterial species (12). The intestinal microbiotas are involved in mucosal and immunological growth and development, nutrition, and mucosal protection (14, 29, 48) and have been implicated in pathophysiology as well.The importance of an intact biota for mucosal protection from bacterial infection has been demonstrated with animal models and the human host. Germfree animals have stunted mucosal and immune development and are highly susceptible to enteric infection (14). Recently, associations between the ability of an enteric pathogen to disrupt the microbial ecology of the gut and the ability of the pathogen to cause enteritis have been shown (4, 25, 39). In humans, treatment with broad-spectrum oral antibiotics may result in the development of Clostridium difficile infections, a common colonizer of the human gut whose growth is held in check by the normal biota but which overgrows the biota upon antibiotic use (6, 21). Many mouse models of enteritis employ the use of antibiotics to eliminate and/or perturb the indigenous biota to allow consistent enteric infection by a variety of pathogens including Salmonella enterica (5, 8, 30, 36), Vibrio cholerae (26), Escherichia coli (46, 47), and Enterococcus faecalis (45) and have demonstrated the importance of colonization resistance by an intact microbiota.The effects of antibiotics on the intestinal microbiota have often focused on analyses of culturable bacterial species (27, 46, 47). More-recent studies using antibiotics to sterilize the gut have used culture techniques to suggest the loss of all colonizing bacteria (31). Because a large percentage of the microbiota cannot be cultured, there is a limited understanding of the impact of antibiotics on intestinal microbial ecology and the relationship between perturbation of the microbiota and susceptibility to enteric infection. We hypothesized that the ability of Salmonella to colonize the murine intestinal tract and the severity of enteritis and systemic spread would be correlated with the extent of the disruption of the protective microbiota.In this study, we treated mice with three different regimens of antibiotics that are commonly used to disrupt the microbiota in mouse models of enteric infection and inflammation and evaluated their effect on intestinal microbial colonization by several dominant bacterial groups (35). Using the same regimens, mice were challenged with oral Salmonella infection to investigate the role of antibiotic-induced biota disruption in host susceptibility to infection. We found that antibiotics varied in their abilities to reduce total bacterial numbers in the gut, but none of the regimens tested completely eliminated the microbiota. There was also variation in the impact of antibiotic on specific dominant bacterial species in the microbiota. Unexpectedly, all the antibiotic regimens used to perturb the microbial ecosystem enhanced Salmonella colonization of the gut, mucosal inflammation, and invasion irrespective of the antibiotic regimen or its relative ability to eliminate colonizing bacteria. These findings were confirmed by antibiotic recovery experiments. Analysis of the recovery of the intestinal biome after withdrawal of antibiotics demonstrated a rapid recovery of total bacterial numbers but persistent changes in the biome composition over several weeks. The enhanced ability of Salmonella to translocate the intestinal tract diminished rapidly after antibiotic withdrawal. However, even after the biome had recovered in many aspects, including total numbers, the ratio of aerobes/anaerobes, and the abundance of several dominant bacterial groups, the mice retained their susceptibility to Salmonella colonization and enteritis. These results suggest that total numbers of bacteria comprising the microbiota contribute to limit pathogen invasion but that complete colonization resistance depends on the correct complex balance of bacterial diversity and quantity and that the use of antibiotics can have lasting deleterious effects on the capacity of the intestinal microbiome to resist infection.