The Mechanisms by Which Salmonella Interferes with Nitric Oxide Production in Macrophages - Essay Example

Name: Course: Tutor: Date The Mechanisms by Which Salmonella Interferes with Nitric Oxide Production in Macrophages. Introduction This paper shows the postulated mechanisms through which Salmonella species manages to survive in macrophages irrespective of the stressful conditions in those cells. Salmonella species cause gastrointestinal and so many life threatening systemic infections in different hosts. It is classified as one of the leading causes (Lahiri et al 1166). The ability of Salmonella species to survive in macrophages and even proliferate in such innate immune cells shows their capacity to cause disease (Gilberthorpe et al 1756). The immune system of hosts infected by different pathogens has reactive mechanisms through which such pathogens are removed from the body. After infection, the immune system responds by producing mechanisms and cells that can fight the pathogen. One of the immune response is the production of macrophages intracellular. Macrophages produce NO (Nitric Oxide) that limits the growth of intracellular pathogens. Salmonella as has been indicated is one of the intracellular pathogens but has the capability to withstand NO produced by the macrophages. Gilberthorpe et al note that Salmonella typhimurium can resist stressful environment created by the macrophages which include; production of reactive Oxygen Species such as superoxide anion (Oˉ2) and production of Reactive Nitrogen Species for example NO (1756). Oxygen reactive species are produced by NAD(P)H Oxidase (Phox) while Reactive Nitrogen Species are produced by inducible Nitric Oxide Synthase (iNOS) (Gilberthorpe et al 1756). One important knowledge is that macrophages produce NO that acts to limit the growth of pathogens within the cells, but the mechanisms through which some pathogenic cells manage to withstand the NO is still unknown. Researchers have proposed several methods which are related but an exact method seems not clear. Some mechanisms proposed indicate that Salmonella species avoid NO toxicity by use of detoxifying protein while some suggest that the pathogens up regulate enzymes involved in the modulation of NO. Some studies have even shown that Salmonella pathogenicity island 2 (SPI-2) acts as an intermediary in the protection of intracellular Salmonella from the toxic RNI (Lahiri et al 1167; Uchiya & Nikai 2004). According to Gilberthorpe et al, Salmonella species interfere with Nitric Oxide production by use of a protein flavohaemoglobin Hmp, which is encoded for by an hmp gene regulated by NsrR (1756). This protein is said to detoxify Nitric oxide and produces a non toxic product to the bacteria N Oˉ3 making the pathogen to continue surviving in the macrophages. In a research titled “NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-c-stimulated J774.2 macrophages” (Gilberthorpe et al 1756), the researchers aim to prove that Salmonella enterica serovar Typhimurium has a genetic regulator that ensures production of the protein flavohaemoglobin Hmp, which results in detoxification of NO produced by the macrophages (Gilberthorpe et al 1756). In another research, researchers try to show that Salmonella species avoid Nitric Oxide toxicity by up regulating arginase II. According to this research Salmonella pathogenicity island (SPI-2) is an intermediary in the protection of Salmonella pathogens from nitrosative stress in macrophages. Up-regulation of arginase II is due to the nature of competition between enzymes that modulate the fate of pathogens in cells. There are two enzymes involved in such modulation, these are iNOS and arginase. Arginase has two forms, arginase I and arginase II. These enzymes use L-arginine as a substrate in the production of Nitric Oxide (Lahiri et al 1166-1167). In activated macrophages, arginase regulates the production of NO so that the cell does not develop tumor mediated cytotoxicity or suffers apoptosis. The enzyme does this by either producing polyamines or depleting L-arginine pool from iNOS. Salmonella up-regulates arginase II which results in reduced L-arginine for iNOS, leading to reduced levels of NO for its survival in the macrophages (Lahiri et al 1166-1167). Some other research suggested that Salmonella Typhimurium involves the induction of an enzyme Cyclooxygenase 2 Expression in Macrophages in its survival in those cells. According to this research, SPI-2 is required for the pathogen to survive in the macrophages. This is because Salmonella enterica activates protein kinase A (PKA) through a pathway that is dependent on the SPI-2. The survival of Salmonella species in the macrophages is due to interference of the processes of macrophages which is up-regulation of interleukin-10 expression. SPI- 2 is involved in the signal transduction pathway that leads to the expression of Cyclooxegenase 2 that in turn leads to the synthesis of prostanoids. These prostanoids when produced at high levels activate the PKA pathway (Uchiya & Nikai 2004). Several other mechanisms may have been revealed but in this paper, only the first two mechanisms described by the first two research papers will be discussed. That is; Salmonella uses a detoxifying protein flavohaemoglobin Hmp to interfere with the NO produced by the macrophages and that Salmonella species up-regulates arginase II to reduce the levels of NO produced in macrophages. These methods used to prove the mechanisms by which Salmonella species interferes with NO production in macrophages will be discussed as well as the results and materials used. The difference or similarity between the mechanisms will be shown and the most appropriate or likely correct mechanism will be shown based on proof from other studies that have been conducted revealing the same results or facts. Paper I “NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-c-stimulated J774.2 macrophages” (Gilberthorpe et al 1756) The aim of the paper Salmonella has been found to be a pathogen that can withstand the oxidative and nitrosative stresses exerted by the macrophages after infection. The process by which it ensures its survival is described by this research paper as by use of a detoxifying protein. This protein is expressed by a gene hmp which is regulated by NsrR. This research paper aimed at showing that the process of Salmonella survival in the macrophages is aided by a regulator NsrR. The main aim was to investigate the roles of NsrR regulons in ensuring that Salmonella Typhimurium survives Nitrosative stress in macrophages (Gilberthorpe et al 1757). S. Typhimurium is said to possess several antioxidative mechanisms that protect it from oxidative stress. These include: hydroperoxidases, superoside mutases and type III secretory system that messes about with Phox vesicles being transported to the phagosomes (Gilberthorpe et al 1757). Nitrosative stress in macrophages is produced by induced Nitric Oxide Syntahses. These syntahses produce Reactive Nitrogen Species and Nitric Oxide that interferes with the respiration of S. Typhimurium. NO is produced by the oxidation of Nitrogen atom obtained from an amino acid L-arginine. Oxidation process is mediated by NAD(P) H that produces Oxygen and other reducing agents (Gilberthorpe et al 1757). On entry of a pathogen into a microorganism’s macrophages, the iNOS is activated by cytokines such as IFN-c, IL-1 and TNFa and the presence of a microorganism in the cells to produce NO. On its activation, Salmonella pathogenicity island 2 secretion system disrupts the localization of iNOS hence avoiding the stress from Reactive Nitrosative species. This process is suspected to be done by the protein previously mentioned flavohaemoglobin Hmp. This protein has two domains, one with binding sites for NAD(P)H. In aerobic conditions, the hmp catalyzes denitrosylase or oxiganase which leads to a conversion of NO stoichiometrically to NOˉ3 that is harmless (Gilberthorpe et al 1757-1758). This study aimed to prove that since flavohaemoglobin Hmp protects S. Typhimurium from nitrosative stress outside macrophages it could be the protein responsible for the microorganism’s protection as well inside the macrophage. This is done by proving that the regulator of the gene responsible for its expression does not change its role while inside the macrophage (Gilberthorpe et al 1756-1758). Problem statement Previous studies about the survival of S. Typhimurium outside macrophages may have acted as the motivation towards this research. Based on the fact that S. Typhimurium was found to be protected from NO by a detoxifying protein, the research aimed at finding out if it could be that the same protein protects the microorganism inside the macrophages. The possible reason for such kind of hypothesis was the classification and the role of the regulator of the gene responsible for flavohaemoglobin Hmp expression. NsrR was found to be a regulator of hmp and a member of the Rrf2family. IscR also belongs to the family of transcriptional regulators and regulates genes involved in cluster biogenesis of Fe-S. NsrR was therefore suspected to be “an NO sensitive [Fe-S] cluster” (Gilberthorpe et al 1757). This hypothesis is supported by previous research that revealed the role of NsrR in Bacillus subtilis. Salmonella hmp transcription however is mediated by another regulator known as RamA (Gilberthorpe et al 1757). Objectives Since the mechanism through which Salmonella avoids NO effects aerobically had been obtained, the research had these objectives todetermine the mechanism used in anaerobic conditions. To determine the role of NsrR both invitro and invivo, to determine NsrR regulons, to determine the specific regulon involved in survival of S. Typhimurium in macrophages, to determine other agents or factors that help Salmonella Typhimurium survive in macrophages apart from flavohaemoglobin hmp, to find out the real components involved in Salmonella typhimurium protection from NO and to determine the process through which NO is interfered with in the macrophages. Methods and materials In order to achieve the objectives these methods were used to carry out the research. Western blotting, Mutagenesis, cloning, macrophage culture and infection, GSNO synthesis, fluorescence microscopy, assay of intracellular Salmonella viability, minimum bactericidal concentration test, assays of nitrite and nitrate accumulation in tissue culture supernatants, Quantitative real-time PCR, (qRT-PCR Reverse transcriptase PCR (RT-PCR) and determination of inhibition of respiration by NO (Gilberthorpe et al 1759-1760). The λ red system was used in mutagenesis. This research aimed at finding out the roles of NsrR both invivo and onvitro therefore a mutant of NsrR was produced and the roles studied. Western blotting was necessary for determination of the changes in expression of the genes and cloning and PCR processes were important for production of similar strains except for qRT-PCR which was used to test the influence of nsrR mutation on four genes regulated by nsrR. J774.2 macrophages were used; to be infected with S. Typhimurium for the research. “Wild type S. enterica Serovar Typhimurium ATCC 14028s or SL1344 and their isogenic derivatives were used for the research. These bacteria were cultured at 37 uC with antibacterial chloramphenical or Kanamycin. The sensitivity of the strains was assessed to ensure that it does not affect the research results which were found to be the same in all strains. The sensitivity assessment was conducted by omitting antibiotics from the media. Cells were grown in aerobic and anaerobic cultures for different processes for example western blotting. The “cells were stored as pellets at 220 uC” (Gilberthorpe et al 1758). Results In determination of the role of NsrR invivo and invitro, the S. Tymphimurium nsrR mutants were found to synthesize hmp both in aerobic conditions and anaerobic conditions. NsrR was found to regulate four other genes apart from hmp which included hcp-hcr (nipAB), ytfE (nipC), tehB and ygbA. ytfE function was not clearly established, ygbA was found to have no function while hcp-hcr (nipAB) were upregulated by nsrR. Hmp gene was the one found to be responsible for GSNO resistance. Hmp was found to be expressed in an nsrR mutant and so its resistance to nitrosative stress was also tested. This revealed the growth of the wild type S. Typhimurium was more affected that the mutant in the presence of GSNO (Gilberthorpe et al 11760-1761). Other findings included, that protection of S. Typhimurium from NO inhibition is also made possible by over expression of genes apart from hmp, that “nsrR, hmp and fur mutations attenuate survival in J774.2 macrophages” (Gilberthorpe et al 1763), that tissue culture supernatants of stimulated macrophages accumulated more NO2 as compared to the non stimulated ones showing that stimulated macrophages produce more NO to fight pathogens, that wild type S. Typhimurium converts NO to NOˉ3, and that sensitivity to oxidative stress was enhanced by nsrR mutation even in the absence of hmp (Gilberthorpe et al 1760-1766). The results indicated that Hmp is important in protection of Salmonella Typhimurium from nitrosative stress both aerobically and anaerobically. They also showed that NsrR regulation of Hmp levels is important in protection of Salmonella from oxidative stress in macrophages (Gilberthorpe et al 1756). Paper II “Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection” Aim of the paper iNOS and arginase have been determined to be the enzymes responsible for modulating the destiny of pathogens that infect macrophages. These enzymes compete for L-arginine which is required for the formation of NO in a macrophage. Arginase prevents cell damage by NO while iNOS forms NO to limit the survival of pathogens in macrophages. Due to the competition for L-arginine, Lahiri et al did a research to prove that S. Typhimurium uses this mechanism to survive in the macrophages (1166). According to Lahiri et al (1166), iNOS inhibition results in reduced bacterial survival which means more NO is produced by the iNOS to fight the pathogen. Arginase plays the role of preventing self cell death and development of tumor mediated cytotoxicity from accumulation of NO by addition of polyamines or reduction of NO formed in the cell. S. Typhimurium therefore up-regulates arginase enzyme which reduces the number of L-arginine available for iNOS for formation of NO, therefore reduced NO for their survival (Lahiri et al 1166-1167). Problem statement The research hypothesizes that the NO production by macrophages is modulated by two enzymes one of which is upregulated by S. Typhimurium. These two enzymes are: iNOS and arginase. According to this paper, arginase appears in two forms depending on the type of cell in which it is. There is arginase I and arginase II. Based on research about the role of arginine in macrophages, this research sort to prove that, S. Typhimurium interferes with the production of NO by up-regulation of arginase II. Arginase II has been found to be responsible for protection of host cells from tumor mediated cytotoxicity and apoptosis and iNOS inhibition has been found to lead to increased intracellular proliferation and bacterial burden in macrophages. This knowledge proves that for the S.Typhimurium to survive in macrophages iNOS must not be inhibited and arginase II must be up-regulated. This is the first study to prove that S. Typhimurium upregulates arginase II in macrophages (Lahiri et al 1167). Objectives of the paper With the aim to show the mechanism through which Salmonella avoids NO or survives in macrophages, the research methods sort to determine the effect of Salmonella on argianse enzyme, to find out the effect of inhibiting arginase activity on the macrophage and Salmonella, to find out the effect of other arginase activities on the macrophage and Salmonella and to find out the specific isoform of arginase involved in NO modulation in macrophages (Lahiriet al 1166-1171). Methods The methods used included: Measurement of activity of the enzyme arginase, measurement of bacterial growth during inhibition or arginase activity and stimulation of arginase activity, western blot analysis to determine the arginase I and arginase II, Semiquantitative reverse transcription-PCR (RT-PCR), intracellular measurement of NO, assay of serum and cell supernatant to determine Nitrite accumulation, flouresent microscopy, “Infection and in vivo treatment with Nu-hydroxynor-L-arginine (nor-NOHA) and/or L-arginine” (Lahiri et al 1168) and nitrotyrosine localization by flouresent microscopy (Lahiri et al 1167-1168). Murine macrophage of RAW264.7 cell line were used in the research with “Salmonella enterica serovar Typhimurium strain 12023 (WT)” (Lahiri et al 1167). Culture of the S Typhimurium was grown at 37°C with Carbenicillin used as the antibacterial for other unwanted bacteria. The research also prepared heat killed bacteria to determine the difference in reaction of macrophages by heating bacterial culture for 20 minutes at 80 °C. The research analyzed invitro data by use of paired t-test where 0.05 was the P value considered significant (Lahiri et al 1168). Results The research found out that arginase activity was stimulated by salmonella after infection, that polyamines produced by arginase II had no effect on Salmonella species, that the specific arginase isoform stimulated by Salmonella infection was arginase II, and that inhibition of the enzyme arginase reduced the burden of NO to the bacteria in macrophages. Other findings that supported the research included: that nitrite response in macrophages is suppressed by arginase II, that nor-NOHA treatment causes increased formation of peroxynitrite, decreased burden on bacteria with no effect on WT induced iNOS protein expression and that upregulation of arginase II happens in the spleen with no expression s in the liver (Lahiri et al 1168-1171). The use of heat killed bacteria that produced a similar response in macrophages indicated that up-regulation of arginase II did not require active prokaryotic protein synthesis by salmonella ((Lahiri et al 1171). The results indicate the L-arginine availability affects the survival of S. Typhimurium in macrophages. If L- arginine concentration is reduced, S.Typhimurium survival is enhanced. The up-regulation of arginase II in the spleen and its absence in the liver reveal that arginase isoform II is only produced in certain cells ((Lahiri et al 1171). Conclusion The first paper proves that the mechanism is regulated by NsrR and NsrR expression is unchanged invivo or invitro. It therefore reveals that NsrR is responsible for its survival invitro and invivo and that the protein expressed is the one that detoxifies NO. The presence of two domains in the Hmp, one with the capability of electron transfer from NAD(P)H to O2 therefore the formation of NOˉ3 makes Hmp protein detoxify NO in macrophages. The second research paper shows that the mechanism through which Salmonella survives in macrophages is through stimulation of arginase II. Both researches acknowledge that, Salmonella pathogenicity island 2 is involved in the avoiding effects of NO and that interference with iNOS activity leads to its survival. The first paper explains that S. Typhimurium localizes iNOS while the second research paper notes that iNOS is depleted of its substrate L-arginine leading to reduced NO production. The difference in the mechanisms is that interference in the first paper is about detoxification of NO while interference of the iNOS in the second is by reducing the availability of substrate. Most research papers have indicated that Salmonella pathogenicity island 2 is involved in the survival of Salmonella species in macrophage. One such research is the proof that S. Typhimurium uses a detoxification protein to survive outside the cell (Liu 2006) and that the regulator of the gene of the detoxification protein is NsrR. The second research only concerns arginase activity in the spleen where arginase II activity is up-regulated (Lahiri 1173). The most likely correct mechanism therefore is the use of detoxification protein flavohaemoglobin Hmp of the research titled; “NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-c-stimulated J774.2 macrophages” (Gilberthorpe et al 1756.) Works Cited Gilberthorpe, Nicola J., Lee, Margaret E., Stevanin, Tania M. Read, Robert C. and Poole, Robert K. NsrR: A key Regulator Circumventing Salmonella enterica serovar Typhimurium Oxidative and Nitrosative Stress In vitro and in IFN-c-stimulated J774.2 Macrophages: The NsrR Regulon of Salmonella. Microbiology (2007), 153, 1756–1771. Uchiya, Kei-ichi and Nikai, Toshiaki. Salmonella enterica Serovar Typhimurium Infection Induces Cyclooxygenase 2 Expression in Macrophages: Involvement of Salmonella Pathogenicity Island 2. Department of Microbiology, Faculty of Pharmacy, Meijo University, Tenpaku-ku, Nagoya, Japan. 2 September 2004. Retrieved on 2nd April 2009 from:http://iai.asm.org/cgi/content/full/72/12/6860?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=postinfection&searchid=1&FIRSTINDEX=570&resourcetype=HWFIG Liu, L., Vazquez-Torres, A., Crouch, ML. Stamler, JS., and Fang, FC. Maintenance of Nitric Oxide and Redox Homeostasis by the Salmonella Flavohemoglobin hmp. Nitric Oxide Research .281: 28039-47, 2006. Retrieved on 2nd April 2009 from: http://www.nitricoxideresearch.com/showabstract.php?pmid=16873371 Lahiri, Amit ., Das, Priyanka., and Chakravortty, Dipshikha. Arginase Modulates Salmonella Induced Nitric Oxide Production in RAW264.7 Macrophages and is Required for Salmonella Pathogenesis in Mice Model of Infection. Centre for Infectious Disease Research and Biosafety Laboratories, Department of Microbiology and Cell Biology, Indian Institute of Science, CV Raman Avenue, Bangalore 560012, 26 June 2008. Read More