Modulation of the Arginase Pathway in the Context of Microbial Pathogenesis: A Metabolic Enzyme Moonlighting as an Immune Modulator

Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology.     

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.     

Microplate assay for detection of bacterial contamination in tissue culture media

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.     

Development of micro-shock wave assisted dry particle and fluid jet delivery system

Small quantity of energetic material coated on the inner wall of a polymer tube is proposed as a new method to generate micro-shock waves in the laboratory. These micro-shock waves have been harnessed to develop a novel method of delivering dry particle and liquid jet into the target. We have generated micro-shock waves with the help of reactive explosive compound [high melting explosive (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and traces of aluminium] coated polymer tube, utilising ??9?J of energy. The detonation process is initiated electrically from one end of the tube, while the micro-shock wave followed by the products of detonation escape from the open end of the polymer tube. The energy available at the open end of the polymer tube is used to accelerate tungsten micro-particles coated on the other side of the diaphragm or force a liquid jet out of a small cavity filled with the liquid. The micro-particles deposited on a thin metal diaphragm (typically 100-??m thick) were accelerated to high velocity using micro-shock waves to penetrate the target. Tungsten particles of 0.7???m diameter have been successfully delivered into agarose gel targets of various strengths (0.6?C1.0?%). The device has been tested by delivering micro-particles into potato tuber and Arachis hypogaea Linnaeus (ground nut) stem tissue. Along similar lines, liquid jets of diameter ??200?C250???m (methylene blue, water and oils) have been successfully delivered into agarose gel targets of various strengths. Successful vaccination against murine salmonellosis was demonstrated as a biological application of this device. The penetration depths achieved in the experimental targets are very encouraging to develop a future device for biological and biomedical applications.     

A Safe Vaccine (DV-STM-07) against Salmonella Infection Prevents Abortion and Confers Protective Immunity to the Pregnant and New Born Mice

Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before the onset of pregnancy and followed the immune reaction against challenge with virulent S. Typhimurium in pregnant mice. Here we demonstrate that DV-STM-07 vaccine gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. These results suggest that our vaccine DV-STM-07 can be very useful in preventing abortion in the pregnant individuals and confer immunity to the new born. Since there are no such vaccine candidates which can be given to the new born and to the pregnant women, this vaccine holds a very bright future to combat Salmonella induced pregnancy loss.     

Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.     

Curcumin Increases the Pathogenicity of Salmonella enterica Serovar Typhimurium in Murine Model

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer''s patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin''s mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.     

Intracellular activities of Salmonella enterica in murine dendritic cells

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.     

Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further (35)S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of 'S' phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of 'S' phase cells and IL-6 synthesis