mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides

It was previously demonstrated that the mig-14 gene of Salmonella enterica serovar Typhimurium is necessary for bacterial proliferation in the liver and spleen of mice following intragastric inoculation and that mig-14 expression, which is induced within macrophages, is under the control of the global regulator PhoP. Here we demonstrate that the mig-14 promoter is induced by growth in minimal medium containing low magnesium or acidic pH, consistent with regulation by PhoP. In addition, mig-14 is strongly induced by polymyxin B, protamine, and the mammalian antimicrobial peptide protegrin-1. While phoP is necessary for the induction of mig-14 in response to protamine and protegrin, mig-14 is still induced by polymyxin B in a phoP background. We also demonstrate that mig-14 is necessary for resistance of S. enterica serovar Typhimurium to both polymyxin B and protegrin-1. Gram-negative resistance to a variety of antimicrobial peptides has been correlated with modifications of lipopolysaccharide structure. However, we show that mig-14 is not required for one of these modifications, the addition of 4-aminoarabinose to lipid A. Additionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of wild-type and mig-14 lipopolysaccharide also shows no detectable differences between the two strains. Therefore, mig-14 contributes to Salmonella resistance to antimicrobial peptides by a mechanism that is not yet fully understood.     

Enhanced resistance to Salmonella enterica serovar Typhimurium infection in mice after coumarin treatment

Coumarin and its derivatives are naturally occurring substances with multiple biological activities. Here we demonstrate that prophylactic peroral administration of coumarin or 7-hydroxycoumarin (7-OHC) enhances resistance to subsequent lethal Salmonella enterica Serovar Typhimurium infection in mice. 7-OHC decreased bacterial load in liver and spleen, and enhanced phagocytosis and bacterial killing by macrophages when applied in vitro and in vivo. 7-OHC treatment induced significant NO release in peritoneal macrophage cultures. The observed protective effect correlated with the induction of Th1-associated cytokines, such as IL-12, IFN-gamma, and TNF-alpha. These data demonstrate a clear immunomodulatory potential of coumarins which might have important therapeutic implications to enhance resistance to infection.     

The two-component sensor kinase KdpD is required for Salmonella typhimurium colonization of Caenorhabditis elegans and survival in macrophages

The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.     

Human genome-wide RNAi screen for host factors that modulate intracellular Salmonella growth

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. While much has been learned about the microbial genes that influence the infectious process through decades of intensive research, relatively little is known about the host factors that affect infection. We performed a genome-wide siRNA screen to identify host genes that Salmonella enterica serovar Typhimurium (S. typhimurium) utilizes to facilitate growth within human epithelial cells. In this screen, with siRNAs targeting every predicted gene in the human genome, we identified 252 new human-host-susceptibility factors (HSFs) for S. typhimurium. We also identified 39 genes whose silencing results in increased intracellular growth of S. typhimurium. The HSFs identified are regulated most centrally by NFκB and associate with each other through an extremely dense network of interactions that center around a group of kinases. Most genes identified were not previously appreciated as playing roles in the intracellular lifecycle of S. enterica. Numerous HSFs identified with interesting characteristics that could play plausible roles in mediating intracellular microbial growth are discussed. Importantly, this study reveals significant overlap between the host network that supports S. typhimurium growth within human epithelial cells and the one that promotes the growth of Mycobacterium tuberculosis within human macrophages. In addition to providing much new information about the molecular mechanisms underlying S. enterica-host cell interplay, all 252 HSFs identified are candidates for new anti-microbial targets for controlling S. enterica infections, and some may provide broad-spectrum anti-microbial activity.     

Impaired macrophage function underscores susceptibility to Salmonella in mice lacking Irgm1 (LRG-47)

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-gamma was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

In vitro and in vivo stability of plasmids in attenuated Salmonella enterica serovar typhimurium used as a carrier of DNA vaccine is associated with its replication origin.

The ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding beta-galactosidase (beta-Gal) and BALB/c mice. We constructed pBRCMVbeta, beta-Gal expression apparatus having a replication origin from low copy pBR322. Comparison of the plasmid stability showed that pBRCMVbeta remained stable in Salmonella even after oral administration, while pUC-based pCMVbeta tended to be lost quickly. However, titers for beta-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVbeta. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology.     

Vasoactive intestinal peptide (VIP) prevents killing of virulent and phoP mutant Salmonella typhimurium by inhibiting IFN-gamma stimulated NADPH oxidative pathways in murine macrophages

Vasoactive intestinal peptide is an immunomodulator with great potential in the treatment of inflammatory pathology. In this study, we have examined the effect of VIP on the growth dynamics of virulent Salmonella enterica. Serovar typhimurium (S. typhimurium) 14028 and 4/74 and an avirulent mutant (14028 phoP) in a murine, macrophage cell line (J774.2). In contrast to standard growth dynamics, in which phoP mutants do not survive in macrophages, we show that VIP (10(-10) M) significantly enhances phoP growth over a 24 h post-infection period even when the cells are co-cultured with IFN-gamma. We examined the effect of VIP on the generation of NADPH-induced reactive oxygen species (ROS) in Salmonella-infected/IFN-gamma cultured J774 cells. VIP inhibited gp91 mRNA levels, gp91 protein and subsequent ROS. The importance of ROS in killing of Salmonella by J774 cells was highlighted by experiments in which ROS production by J774 cells was inhibited using a conventional inhibitor, N-acetyl-L-cysteine captopril (ACC) and in which Salmonella growth significantly increased. Our findings suggest that although VIP inhibits inflammatory pathways in myeloid cells it also promotes the growth of avirulent (phoP) mutants.     

Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.     

Activation of mouse peritoneal macrophages during infection with Salmonella typhimurium does not result in enhanced intracellular killing

Our study was performed to investigate whether macrophages become activated during an infection with Salmonella typhimurium and, if so, whether these activated macrophages kill S. typhimurium faster than resident macrophages. Mice received i.v. injections with a sublethal number of S. typhimurium; on about day 12 of the infection the numbers of bacteria in the liver and the spleen were maximal. During the infection, activation of peritoneal macrophages could be demonstrated on the basis of three criteria, i.e., the ability to inhibit the proliferation of Toxoplasma gondii, an enhanced production of H2O2 and an increased expression of Ia Ag. The rate of in vitro intracellular killing of S. typhimurium by these activated macrophages was not increased compared to that for resident macrophages. To determine the growth of S. typhimurium in activated mice a nalidixic acid-resistant mutant strain, called S. typhimurium 510R, was used. The net growth rates of the mutant S. typhimurium 510R in the spleen of S. typhimurium 510-activated and normal mice were similar. However, in the liver of S. typhimurium 510-activated mice the number of S. typhimurium 510R did not change during 3 to 48 h after injection. The role of specific antibodies during the initial phase of the infection was negligible, because only low levels of antibodies were detected during the first 15 days of infection and the growth rates of S. typhimurium 510 in the spleen and liver of mice with high titers of antibodies were not significantly different from the rates in normal mice. The results of this study demonstrate that although macrophages become activated during an infection with S. typhimurium, these cells do not display an enhanced bactericidal activity in vitro and in vivo no significant effect on the growth rate of S. typhimurium in the spleen and a bacteriostatic effect in the liver is found. Hence macrophage activation is probably not very important in the host defense against S. typhimurium.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

PIR-B-deficient mice are susceptible to Salmonella infection

Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection, PIR-B-deficient (PIR-B(-/-)) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica Typhimurium (WB335). PIR-B(-/-) mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns of inflammatory lesions were found in their livers at 7-14 days postinfection: diffuse spreading along the sinusoids in PIR-B(-/-) mice vs nodular restricted localization in WT mice. PIR-B(-/-) mice have more inflammatory cells in the liver but fewer B cells and CD8(+) T cells in the spleen than WT mice at 14 days postinfection. PIR-B(-/-) bone marrow-derived macrophages (BMMphi) failed to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared with WT BMMphi. PIR-B(-/-) BMMphi also produced more nitrite and TNF-alpha upon exposure to Salmonella than WT BMMphi did. These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial infection.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria

The genotype of a mouse influences whether or not it will survive infection with the agent of murine typhoid, Salmonella typhimurium. The best-characterized murine salmonella response gene is a Chromosome 1 locus designated Ity. Inbred strains of mice that express the Itys allele are unable to contain the net growth of Salmonella typhimurium within their spleens and livers, and usually die early in the infection. By contrast, mice homozygous or heterozygous for the Ityr allele are able to control the net multiplication of Salmonella typhimurium within these organs. The Ity gene also appears to regulate the extent of replication within murine reticuloendothelial cell tissues of the obligate intracellular parasite Leishmania donovani, as well as the facultative intracellular bacteria Mycobacterium bovis and Mycobacterium lepraemurium. Previous studies from our laboratory strongly suggested that Ityr mice are more resistant to S. typhimurium infection than are Itys mice, because resident Ityr macrophages kill salmonellae more efficiently than do Itys macrophages. In this study, we used an in vitro macrophage assay to assess the specificity of the enhanced killing capacity of Ityr macrophages. We found that Ityr macrophages were better able than Itys macrophages to kill both intracellular bacteria (Salmonella typhi) and extracellular bacteria (Escherichia coli, Staphylococcus aureus, Corynebacterium diphtheriae). Thus, the diversity of organisms affected by Ity expression suggests that the product of this gene may play a key regulatory role in the initial interaction of mice with a variety of microbial agents.     

The alternative sigma factor sigma is required for resistance of Salmonella enterica serovar Typhimurium to anti-microbial peptides

The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters a variety of anti-microbial peptides during the course of infection. We report here that the extracytoplasmic sigma factor sigma(E) (RpoE) is required for Salmonella resistance to killing by the bactericidal/permeability-increasing protein (BPI)-derived peptide P2 and the murine alpha-defensin cryptdin-4 (Crp4). Moreover, sigma(E)-deficient S. Typhimurium is attenuated for virulence after oral infection of immunocompromised gp91phox(-/-) mice that lack a functional NADPH phagocyte oxidase, suggesting that sigma(E) plays an important role in resistance to non-oxidative mucosal host defences such as anti-microbial peptides. Although both P2 and Crp4 target the cell envelope, bacterial killing by these peptides appears to occur by distinct mechanisms. Formate enhances bacterial resistance to P2, as previously demonstrated, but not to Crp4. Both sigma(E) and cytoplasmic membrane-associated formate dehydrogenase are required for the protective effect of formate against P2. In contrast to P2, Crp4 does not inhibit bacterial respiration at lethal concentrations. However, both peptides induce expression of rpoE, suggesting that they trigger a common mechanism for sensing extracytoplasmic stress.     

CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154-/-) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154-/- mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154-/- mice exhibited a defect both in the production of IL-12, IFN-gamma, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154-/- animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in > or =90% protection. The lack of protective immunity in CD154-/- mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154-/- mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.     

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.     

Role of peritoneal macrophages in the development of an experimental Salmonella infection

The effect produced on the course of Salmonella infection in mice by the removal of peritoneal macrophages with agarose has been studied. Peritoneal macrophages have been shown to control the multiplication of faintly virulent and virulent S. typhimurium strains in the spleen of mice. In immune mice the elimination of the virulent strain of the causative agent of superinfection may occur without the control of peritoneal macrophages.