Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.     

Salmonella selectively stops traffic

The intracellular pathogen Salmonella replicates in infected host cells within a specialized vacuole referred to as the Salmonella-containing vacuole (SCV). Effector molecules encoded by the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) are essential for Salmonella to survive in the intracellular environment. It was previously shown that SPI-2 allows Salmonella to inhibit the recruitment of NADPH phagocyte oxidase-containing vesicles to SCVs. New research has now revealed that SPI-2 effectors also interfere with the colocalization of inducible nitric oxide synthase (iNOS) to SCVs, thus protecting the pathogen from the antimicrobial actions of reactive nitrogen species.     

In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of na?ve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.     

Implications of Salmonella-induced nitric oxide (NO) for host defense and vaccines: NO, an antimicrobial, antitumor, immunosuppressive and immunoregulatory molecule.

Attenuated Salmonella induce immunosuppressive, microbicidal and tumoricidal macrophages in mice. All three effects are mediated by activated macrophages producing nitric oxide (NO). NO is induced by the innate immune response pathway involving IL-12, NK cells and IFN-gamma in response to infection. NO has beneficial and detrimental effects on the host.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Caloric restriction increases free radicals and inducible nitric oxide synthase expression in mice infected with Salmonella Typhimurium

It is well known that CR (caloric restriction) reduces oxidative damage to proteins, lipids and DNA, although the underlying mechanism is unclear. However, information concerning the effect of CR on the host response to infection is sparse. In this study, 6-month-old mice that were fed AL (ad libitum) or with a CR diet were infected with Salmonella serovar Typhimurium. EPR (electron paramagnetic resonance; also known as ESR (electron spin resonance)) was used to identify FRs (free radicals). These results were subsequently correlated with SOD (superoxide dismutase) catalytic activity, iNOS [inducible NOS (nitric oxide synthase) or NOSII] expression and NO (nitric oxide) content. EPR analysis of liver samples demonstrated that there was a higher quantity of FRs and iron-nitrosyl complex in infected mice provided with a CR diet as compared with those on an AL diet, indicating that CR was beneficial by increasing the host response to Salmonella Typhimurium. Furthermore, in infected mice on the CR diet, NOSII expression was higher, NO content was greater and spleen colonization was lower, compared with mice on the AL diet. No changes in SOD activity were detected, indicating that the NO produced participated more in the formation of iron-nitrosyl complexes than peroxynitrite. These results suggest that CR exerts a protective effect against Salmonella Typhimurium infection by increasing NO production.     

Aminoguanidine renders inducible nitric oxide synthase knockout mice more susceptible to Salmonella typhimurium infection.

Aminoguanidine (AG), a nitric oxide synthase (NOS) inhibitor, has been widely used to study the role of inducible NOS (iNOS) in host defense against infections caused by various pathogens including Salmonella typhimurium. iNOS has been reported to play an important role in host defense against S. typhimurium infection both in vitro and in vivo. In this report we show those AG treatment lead to weight loss in both wild-type and iNOS knockout mice, and rendered them more susceptible to Salmonella infection. These results suggest that AG may have side effects other than the inhibition of iNOS, and that data obtained from studies using AG should be interpreted with caution.     

The O-antigen affects replication of Salmonella enterica serovar Typhimurium in murine macrophage-like J774-A.1 cells through modulation of host cell nitric oxide production

O-antigen-proficient and defined O-antigen-deficient mutants of Salmonella enterica serovar Typhimurium were compared for intracellular replication and induction of nitric oxide (NO) expression in the murine macrophage-like cell line J774-A.1. While O-antigen-proficient bacteria replicated and provoked induction of host cell NO synthesis to expected levels, DeltawaaK, DeltawaaL and DeltawaaKL mutants displayed increased growth yields and induction of significantly lower levels of macrophage NO production. The downregulation of NO production did not involve suppression of inducible nitric oxide synthase (iNOS) expression, yet it depended on bacterial protein synthesis during infection of J774-A.1 cells. In contrast, when inhibitor substances were used to block iNOS activity, the growth yield of the wild type significantly exceeded that of the DeltawaaL mutant bacteria. Inactivation of the Salmonella pathogenicity island 1 (SPI1)-associated bacterial type III secretion system did not affect intracellular replication in the wild type or the DeltawaaL background. However, inactivation of the SPI2-associated type III secretion strongly abrogated bacterial intracellular replication, and the DeltawaaLDeltassaV double mutant lost the ability to suppress NO expression. The results imply that a lack of O-antigen may increase bacterial fitness in J774-A.1 cells through suppression of iNOS activity, and that the O-antigen may protect against NO-independent restriction of bacterial intracellular replication.     

Oxygen-dependent anti-Salmonella activity of macrophages

Numerous observations have established a crucial role for phagocytic cells in host resistance to Salmonella. Activated macrophages rely on a complex array of oxygen-dependent antimicrobial molecules to inhibit or kill intracellular Salmonella. An initial oxidative bactericidal phase, which is dependent on the respiratory burst phagocyte oxidase (phox) is succeeded by a prolonged nitrosative bacteriostatic phase, which is dependent on inducible nitric oxide synthase (iNOS). The sequential contribution of phox and iNOS to anti-Salmonella innate immunity has been demonstrated both in vitro and in vivo. The temporal progression from the predominant production of reactive oxygen species to the production of nitrogen oxides could optimize the initial reduction in microbial burden while minimizing the immunopathological consequences of the host inflammatory response.     

The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion

The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post-invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response.     

Protective efficacy of probiotic alone or in conjunction with a prebiotic in Salmonella-induced liver damage

In view of the increasing interest in the bioecological and nutritional control of diseases, use of probiotics alone or in combination with prebiotics (synbiotics) appears as a therapeutic option for various diseases. In this study, an attempt was made to explore the protective potential of Lactobacillus acidophilus as a probiotic, inulin as a prebiotic and both L. acidophilus and inulin as synbiotic against Salmonella -induced liver damage in a murine model. The probiotic, prebiotic and synbiotic supplementation resulted in decreased bacterial translocation in the liver of mice challenged with Salmonella typhimurium and decreased levels of serum aminotransferases, suggesting their protective role against Salmonella infection. Mice supplemented with these preparations before Salmonella challenge also revealed decreased levels of lipid peroxidation, increased levels of superoxide dismutase and glutathione, along with reduced levels of nitric oxide. Thus, bacteriological and biochemical alterations correlated well with the histological evidence. Protection afforded by supplementation with the probiotic alone was found to be more effective. None of the observations was suggestive of the synergistic effect in the synbiotic-supplemented animals. Thus, it is indicated that the probiotic and the prebiotic used in the present study may act by different mechanisms involved in affording protection against Salmonella -induced liver damage.     

The mutagenic activity of the S-nitrosoglutathione/glutathione system in Salmonella typhimurium TA1535

S-nitrosoglutathine (GSNO) and reduced glutathione (GSH) were tested for mutagenicity against strain Salmonella typhimurium TA1535 in the Ames Standard plate incorporation assay. Neither GSNO not GSH were mutagenic when tested alone. In combination, the GSNO/GSH system induced a positive mutagenic response that ranged from 3 to 20 x over background at concentrations of 10 to 50 micromol (micromol)per plate, respectively. This mutagenic response can be attributable to the generation nitric oxide, among the many other reactive products generated by the reaction of GSNO with GSH.     

The effect of nitric oxide combined with fluoroquinolones against Salmonella enterica serovar Typhimurium in vitro

Two regulons, soxRS and marRAB, are associated with resistance to quinolones or multiple antibiotic in Salmonella enterica serovar Typhimurium. These regulons are activated by nitric oxide and redox-cycling drugs, such as paraquat and cause on activation of the acrAB-encoded efflux pump. In this study, we investigated the effect of nitric oxide (NO) alone and in combination with ofloxacin, ciprofloxacin, and pefloxacin against S. typhimurium clinical isolates and mutant strains in vitro. We did not observe synergistic effect against clinical isolates and SH5014 (parent strain of acr mutant), while we found synergistic effect against PP120 (soxRS mutant) and SH7616 (an acr mutant) S. typhimurium for all quinolones. Our results suggest that the efficiencies of some antibiotics, including ofloxacin, ciprofloxacin, and pefloxacin are decreased via activation of soxRS and marRAB regulons by NO in S. enterica serovar Typhimurium. Further studies are warranted to establish the interaction of NO with the genes of Salmonella and, with multiple antibiotic resistance.     

Slc11a1 limits intracellular growth of Salmonella enterica sv. Typhimurium by promoting macrophage immune effector functions and impairing bacterial iron acquisition

The natural resistance-associated macrophage protein 1, Slc11a1, is a phagolysosomal transporter for protons and divalent ions including iron that confers host protection against diverse intracellular pathogens including Salmonella . We investigated and compared the regulation of iron homeostasis and immune function in RAW264.7 murine phagocytes stably transfected with non-functional Slc11a1 and functional Slc11a1 controls in response to an infection with Salmonella enterica serovar Typhimurium. We report that macrophages lacking functional Slc11a1 displayed an increased expression of transferrin receptor 1, resulting in enhanced acquisition of transferrin-bound iron. In contrast, cellular iron release mediated via ferroportin 1 was significantly lower in Salmonella -infected Slc11a1-negative macrophages in comparison with phagocytes bearing Slc11a1. Lack of Slc11a1 led to intracellular persistence of S. enterica serovar Typhimurium within macrophages, which was paralleled by a reduced formation of nitric oxide, tumour necrosis factor-alpha and interleukin-6 in Slc11a1-negative macrophages following Salmonella infection, whereas interleukin-10 production was increased. Moreover, Slc11a1-negative phagocytes exhibited higher cellular iron content, resulting in increased iron acquisition by intracellular Salmonella . Our observations indicate a bifunctional role for Slc11a1 within phagocytes. Slc11a restricts iron availability, which first augments pro-inflammatory macrophage effector functions and second concomitantly limits microbial iron access.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

Heterologous expression of bacterial nitric oxide synthase gene: a potential biological method to control biofilm development in the environment

The nitric oxide synthase gene from Bacillus subtilis was heterologously expressed in Pseudomonas putida using a broad-host expression vector. Both the performance of the nitric oxide-specific fluorescent probe and the quantitative assessment of the nitric oxide end products demonstrated the generation of nitric oxide. The endogenous nitric oxide increased the motility of P.?putida and decreased the capacity of P. putida and other multispecies bacteria to develop biofilms. On the practical side, endogenous nitric oxide offers an advantage in generating continuous, controllable levels of nitric oxide, which suggests a new method to use nitric oxide in the control of biofilms.     

一氧化氮对家榆种子老化的影响及其作用机制研究

以家榆种子为试材,采用种子活力检测技术、激光共聚焦显微镜技术、蛋白质S-亚硝基化检测技术,结合多种相关抑制剂的使用,研究了NO对种子老化的影响及其作用机制。结果表明:(1)外源NO可显著提升老化处理后种子的活力,NO清除剂cPTIO可降低老化处理后种子的活力,且此影响可被NO供体硝普钠所恢复。(2)硝酸还原酶底物亚硝酸钠、类一氧化氮合酶底物L-精氨酸(L-Arg)均可提高老化处理后种子的活力,2种酶的抑制剂可降低种子活力,且此影响可被NO供体硝普钠所恢复,即硝酸还原酶与类一氧化氮合酶可参与种子老化过程中NO的产生。(3)种子老化过程中NO首先在子叶中合成,随后在胚根尖部、生长点与下胚轴等部位出现,蛋白质S-亚硝基化水平与NO在种子中产生的时间特点一致。研究认为,NO可提高种子抗老化能力,种子内NO可通过硝酸还原酶途径和类一氧化氮合酶途径产生,且与种子蛋白质S-亚硝基化水平相关。     

Immunity to systemic Salmonella infections

Salmonella infections are a serious public health problem in developing countries and represent a constant concern for the food industry. The severity and the outcome of a systemic Salmonella infection depends on the "virulence" of the bacteria, on the infectious dose as well as on the genetic makeup and immunological status of the host. The control of bacterial growth in the reticuloendothelial system (RES) in the early phases of a Salmonella infection relies on the NADPH oxidase-dependent anti-microbial functions of resident phagocytes and is controlled by the innate resistance gene Nramp1. This early phase is followed by the suppression of Salmonella growth in the RES due to the onset of an adaptive host response. This response relies on the concerted action of a number of cytokines (TNFalpha, IFNgamma, IL12, IL18, and IL15), on the recruitment of inflammatory phagocytes in the tissues and on the activation of the recruited cells. Phagocytes control bacterial growth in this phase of the infection by producing reactive nitrogen intermediates (RNI) generated via the inducible nitric oxide synthase (iNOS). Clearance of the bacteria from the RES at a later stage of the infection requires the CD28-dependent activation of CD4+ TCR-alphabeta T-cells and is controlled by MHC class II genes. Resistance to re-infection with virulent Salmonella micro-organisms requires the presence of Th1 type immunological memory and anti-Salmonella antibodies. Thus, the development of protective immunity to Salmonella infections relies on the cross-talk between the humoral and cellular branches of the immune system.