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.     

Interplay between MgtC and PagC in Salmonella enterica serovar Typhimurium

In Salmonella enterica serovar Typhimurium, MgtC and PagC are positively regulated by the PhoP-PhoQ two-component system, which is activated under magnesium deprivation. Both MgtC and PagC are of unknown function but have been involved in intramacrophage survival. We have found that the amount of PagC is lowered in a DeltamgtC mutant strain grown in magnesium depleted medium. However, the effect of MgtC on PagC does not account for the growth defect of a DeltamgtC mutant in macrophages since, in contrast to previous reports, our results indicate that PagC does not contribute to intramacrophage survival. In addition, a pagC null mutant is only poorly attenuated in Nramp1-negative or Nramp1-positive mice. On the other hand, a mgtC null mutant is significantly more attenuated with Nramp1-positive than Nramp1-negative mice, suggesting that a functional Nramp1 (Slc11a1) further limits the multiplication of this mutant within the host.     

Interaction between Host Cells and Septicemic Salmonella enterica Serovar Typhimurium Isolates from Pigs

Salmonella enterica serovar Typhimurium is an important pathogen in swine and is also a frequently reported zoonotic agent. The objective of this study was to characterize isolates of S. enterica serovar Typhimurium associated with septicemia in swine and to compare them to isolates recovered from clinically healthy pigs. We were particularly interested in comparing the two groups of isolates for their ability to adhere to and invade host cells, to be phagocytized and survive in monocyte cells, to induce apoptosis, and to adhere to intestinal mucus. Their surface properties were also evaluated by interactions with solvents. The isolates recovered from diseased animals were shown to invade intestinal epithelial cell lines at a higher rate (P = 0.003) than isolates from healthy pigs. Septicemic isolates were phagocytized by human monocytes at a higher rate than isolates from healthy pigs (P = 0.009). The mean percentages of phagocytosis were significantly lower for human monocytes than for porcine monocytes (P = 0.02 and P = 0.008, respectively) for isolates from both diseased and healthy animals. Healthy animal isolates were phagocytized more by porcine monocytes at 15 min (P = 0.02) than septicemic isolates. No difference between isolates from septicemic pigs and isolates from healthy pigs was detected for other tested parameters. These results suggest that septicemic isolates have a particular pattern of invasion.In pigs, Salmonella strains are related to significant animal infections associated with clinical signs and economic losses but are mainly associated with a carrier state, becoming a reservoir for human infections (3). Infection and/or silent carriage of Salmonella in pigs is an important public health concern. Multiresistance to antibiotics is often associated with Salmonella enterica serovar Typhimurium (14). In order to develop control measures, it is important to characterize these isolates and better understand the pathogenesis of infection.The pattern of infection by Salmonella is oral ingestion of the bacteria followed by passage through the mucus which covers the epithelial cells to invade mucous membranes and cause disease (35). The intestinal mucus can then serve as the initial binding site for bacteria. Initial adhesion is mainly a physicochemical process founded on nonspecific interactions (van der Waals and coulombic interactions) (17, 33). This type of adhesion can be reversible or not, and surface properties of some bacteria have been show to influence nonspecific interactions with host cells. The first steps of infection are adhesion on the surface, firm attachment, and penetration into intestinal epithelial cells. The invasion of intestinal epithelial cells is believed to be a very important step related to the virulence of Salmonella strains associated with infections (5).After invasion of the epithelial cells, the bacteria reach the subepithelial lymph tissue and the lamina propria, where Salmonella cells meet host immune cells (28). The overall phagocytic process can be divided into at least two main parts. First, bacteria must adhere to the phagocyte surface in a process called adherence. The second step of phagocytosis involves internalization or ingestion of the adherent particle. Following initial adhesion to phagocytes, special bacterial cell surface structures recognize receptors on the target cell surface (17, 33). The virulence genes of S. enterica serovar Typhimurium located on SPI-1, which encode a type III protein export machinery, are necessary for invasion of either nonphagocytic (5) or phagocytic (28) cells. Salmonella is able to induce cell death in macrophages in two different ways (32). Rapid activation of programmed macrophage cell death depends on SipB and SPI-1, whereas delayed induction of apoptosis in infected macrophages is SPI-1 independent (32). The results reported by van der Velden et al. indicate that ompR and a functional SPI-2-encoded type III protein secretion apparatus are required for delayed induction of apoptosis (32). The survival in phagocytes is an important step to induce septicemia in pigs, causing clinical signs similar to those in humans (16, 31); therefore, this animal model may be used to study human salmonellosis. Salmonella is able to survive and replicate in phagocytic cells, and this is an essential component of the virulence of these bacteria (1). S. enterica serovar Typhimurium cells that have invaded the macrophage by phagocytosis are able to replicate intracellularly (28) and induce apoptosis (32). In the past 10 to 15 years, an increased number of cases of clinical salmonellosis associated with Salmonella serovar Typhimurium were observed in pigs (11).The aim of this study was to characterize isolates of S. enterica serovar Typhimurium associated with septicemia in swine and to compare them to isolates recovered from clinically healthy pigs. We were particularly interested in comparing the two groups of isolates in regard to their abilities to adhere to host cells, invade host cells, be phagocytized, survive in cells, generate apoptosis, adhere to intestinal mucus, and adhere to solvents.     

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 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.     

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.     

Design of Glycoconjugate Vaccines against Invasive African Salmonella enterica Serovar Typhimurium

Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S. Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM₁₉₇). The OAg-CRM₁₉₇ conjugates varied in (i) OAg source, with three S. Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM₁₉₇ ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S. Typhimurium glycoconjugate by challenge with live Salmonella. We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM₁₉₇ ratio. In addition, we found that bactericidal activity can be influenced by S. Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S. Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S. Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.     

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.     

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways.     

Salmonella enterica Serovar Typhimurium Response Involved in Attenuation of Pathogen Intracellular Proliferation

Salmonella enterica serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate. S. enterica serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes phoQ, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate. phoP- and phoQ-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named igaA for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), spiA, and rpoE, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.