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.     

Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin

Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration. Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear. We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA). Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition [N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l] and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip). We also determined the presence of iNOS using Western blot and immunohistochemistry. LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs. LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs. LPS 455 +/- 27 mg, P > 0.05). Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs. control and P < 0.05 vs. LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs. control and LPS). Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA. Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.     

Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium

Salmonella typhimurium is an intracellular pathogen that can survive and replicate in macrophages. One of the host defense mechanisms that S. typhimurium encounters upon infection is superoxide produced by the phagocytes' NADPH-oxidase. Salmonella has evolved numerous ways of coping with superoxide in the extracellular environment. In addition, Salmonella has to defend itself against superoxide produced as a by-product of aerobic respiration. Over the last decade, research on bacterial mutants has led to the identification of Salmonella strains that differ from their parental strain in susceptibility to superoxide in vitro. However, the consequences of such mutations for bacterial virulence are highly variable, indicating that superoxide sensitivity per se is not a characteristic that renders Salmonella less virulent. By discussing various bacterial mutants classified according to their in vitro sensitivity to superoxide, we will exemplify the complex mechanisms that Salmonella has evolved to cope with superoxide stress.     

IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth

IFN-γ-mediated inducible nitric oxide synthase (iNOS) expression is critical for controlling chlamydial infection through microbicidal nitric oxide (NO) production. Interleukin-17A (IL-17A), as a new proinflammatory cytokine, has been shown to play a protective role in host defense against Chlamydia muridarum (Cm) infection. To define the related mechanism, we investigated, in the present study, the effect of IL-17A on IFN-γ induced iNOS expression and NO production during Cm infection in vitro and in vivo. Our data showed that IL-17A significantly enhanced IFN-γ-induced iNOS expression and NO production and inhibited Cm growth in Cm-infected murine lung epithelial (TC-1) cells. The synergistic effect of IL-17A and IFN-γ on Chlamydia clearance from TC-1 cells correlated with iNOS induction. Since one of the main antimicrobial mechanisms of activated macrophages is the release of NO, we also examined the inhibitory effect of IL-17A and IFN-γ on Cm growth in peritoneal macrophages. IL-17A (10 ng/ml) synergizes with IFN-γ (200 U/ml) in macrophages to inhibit Cm growth. This effect was largely reversed by aminoguanidine (AG), an iNOS inhibitor. Finally, neutralization of IL-17A in Cm infected mice resulted in reduced iNOS expression in the lung and higher Cm growth. Taken together, the results indicate that IL-17A and IFN-γ play a synergistic role in inhibiting chlamydial lung infection, at least partially through enhancing iNOS expression and NO production in epithelial cells and macrophages.     

A single point mutation in 3-deoxy-D-manno-octulosonate-8-phosphate synthase is responsible for temperature sensitivity in a mutant strain of Salmonella typhimurium

Salmonella typhimurium mutants conditionally deficient in 3-deoxy-d-manno-octulosonate-8-phosphate (KDO8P) synthase activity play a central role in our understanding of lipopolysaccharide function in enteric bacteria. The detailed characterization of KDO8P synthase from such a mutant, however, has not been previously reported. To address this issue KDO8P synthase from S. typhimurium AG701 and from a related temperature-sensitive strain (S. typhimurium AG701i50) have been overexpressed in Escherichia coli and purified to homogeneity. The enzyme from the temperature-sensitive strain has a single proline to serine substitution at position 145, leading to an increase in K(m) for both substrates, d-arabinose 5-phosphate and phosphoenolpyruvate. Analytical gel filtration and native polyacrylamide gel electrophoresis indicate that this enzyme also has an altered oligomeric state. These observations are rationalized through an examination of the structure of E. coli KDO8P synthase, which has 93% sequence identity to the enzyme from S. typhimurium.     

Induction of nitric oxide synthase during Japanese encephalitis virus infection: evidence of protective role.

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage-derived factor (MDF) to modulate nitric oxide synthase (NOS) activity in brain and tumor necrosis factor-alpha (TNF-alpha) and the possible antiviral role of NOS during JEV infection were investigated. NOS activity and particularly that of the inducible form of NOS (iNOS) was significantly enhanced in JEV or JEV-induced MDF-treated mice. Following JEV infection, total NOS activity in brain was gradually increased from Day 3 and reached a peak on Day 6. MDF-induced NOS activity and iNOS activity were dose dependent and maximum activity was observed at 1 h after treatment. The response was sensitive to anti-MDF antibody treatment and N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS. Pretreatment of JEV-infected mice with L-NMMA increased the mortality as evident from reduced mean survival time (MST, 11.8 days) compared to placebo treated JEV-infected mice (MST, 17 days). The enhanced level of TNF-alpha observed in the early phase of JEV infection correlated well with the enhanced activity of iNOS. These observations thus provide evidence of the protective role of iNOS during JEV infection and indicate that iNOS may be a key mediator in host innate immune response to infection.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.     

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.     

Induction of CD8+ T lymphocytes by Salmonella typhimurium is independent of Salmonella pathogenicity island 1-mediated host cell death

Salmonella are intracellular bacterial pathogens that reside and replicate inside macrophages, and attenuated strains of Salmonella typhimurium can be used to deliver heterologous Ags for MHC class I and/or MHC class II-restricted presentation. Recently, it was shown that invasion of macrophages by S. typhimurium may result in the death of host macrophages via a mechanism harboring features of apoptotic and necrotic cell death. However, it is unknown whether this bacterial-induced host cell death affects immunity. In addition, it has been hypothesized that macrophage death following infection with S. typhimurium and subsequent uptake of apoptotic cells by APC are fundamental to the induction of CTL responses. In this study we investigated the in vivo induction of Ag-specific CD8+ T lymphocyte responses and compared CD8+ T lymphocyte responses elicited with S. typhimurium strains carrying a mutation in their invA gene, and therefore an inability to induce Salmonella pathogenicity island 1 (SPI-1)-mediated macrophage death, with responses elicited by an attenuated deltaaroAD strain. Ag-specific CD8+ T lymphocyte responses were analyzed using IFN-gamma ELISPOT, tetramer binding, and in vivo and in vitro CTL assays. Our results showed that deltaaroAD and deltaaroADdeltainvA induced comparable levels of Ag-specific CD8+ T lymphocyte responses as well as protective, Ag-specific B and CD4+ T lymphocyte immunity. Furthermore, experiments in macrophage-depleted mice showed that CD8+ T lymphocyte responses were effectively induced in the absence of macrophages. Together, our results imply that in this infection model, SPI-1-mediated cell death does not affect the immunological defense response and is not important for the induction of CD8+ T lymphocyte responses.     

A mouse model for S. typhimurium-induced enterocolitis

Salmonella typhimurium has emerged as a model pathogen that manipulates host cells in a complex fashion, thus causing disease. In humans, S. typhimurium causes acute intestinal inflammation. Intriguingly, type III secreted virulence proteins have a central role in this process. At the cellular level, the functions of these factors are well characterized; at present, animal models are required for elucidating how these factors trigger inflammatory disease in vivo. Calf infection models have been employed successfully and, recently, a mouse model was identified: in streptomycin-pretreated mice, S. typhimurium causes acute colitis. This mouse model provides a new avenue for research into acute intestinal inflammation because it enables the manipulation and dissection of both the bacterial and host contributions to the disease in unsurpassed detail.     

Host resistance to mucosal gram-negative infection. Susceptibility of lipopolysaccharide nonresponder mice

The effect of Lps on the resistance of mice to gram-negative infection was compared in two genetically different backgrounds; C3H and C57BL. To mimic the natural sequence of pathogenetic events, infection was induced via a mucosal surface (intravesically), with Escherichia coli which remained at the mucosal site and Salmonella typhimurium which invaded to e.g., livers and spleens. Susceptibility was assessed as the bacterial persistence in kidneys, bladders, livers, and spleens at various times after infection. The initial clearance of both bacterial species from the mucosal site was significantly impaired in Lpsd mice both in the C3H and C57BL backgrounds. In the C57BL mice, additional unknown determinants conferred increased resistance to mucosal infection compared to the C3H mouse. For S. typhimurium, these resistance factors and alleles at the Lps locus dominated over Ity as determinants of resistance to mucosal infection. The Itys genotype conferred a significant increase in the susceptibility only to systemic infection, especially in the Lpsd, Itys mice. These results demonstrate an important difference between the genetic determinants of host resistance at mucosal and systemic sites, and emphasize the role of LPS induced host defense mechanisms for bacterial clearance from mucosal surfaces.     

Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production

To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth. J774-A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

Pathogen-specific TLR2 protein activation programs macrophages to induce Wnt-beta-catenin signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus nonpathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-β-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-β-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-β-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-β-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-β-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-β-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.     

大鼠肢体缺血/再灌注后肺损伤及一氧化氮合酶的变化与意义

目的：研究大鼠肢体缺血／再灌注后急性肺损伤时,内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（i-NOS）的表达及其在急性肺损伤发生中的作用。方法：雄性Wistar大鼠于后肢根部阻断血流后松解（4h／4h）,分别给予L-Arg和氨基胍（AG）预先干预,分为control、IR、L-Arg和AG组,免疫组织化学方法检测肺组织中iNOS和eNOS的表达,同时检测肺组织中MDA、MPO、W／D和NO2^-／NO3^-值,肺组织形态学观察以评价肺损伤的程度。结果：与control组比较,I／R组eNOS表达降低,iNOS表达增强,MDA、MPO、W／D和NO2^-／NO3^-值增加。肺组织充血、炎细胞浸润,肺泡腔渗液;与I／R组比较,L-Arg组eNOS、iNOS表达无明显变化,NO2^-／NO3^-增加。MDA、MPO、W／D降低,肺组织损伤有减轻趋势,AG组eNOS表达无明显变化,iNOS活性降低,NO2^-／NO3^-减少,MDA、MPO、W／D增加,肺组织损伤有加重趋势。结论：肢体缺血／再灌注急性肺损伤过程中,iNOS表达增加,NO生成增多,在肺损伤发生中有一定的保护作用。     

Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment

In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles. Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system. Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA. The induction of eNOS mRNA was followed by an increase in eNOS protein. Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels. Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection.     

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in th...