Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN- ) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN- -induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN- -treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN- -induced NO production, and they highlight the critical role of nirC as a virulence gene.

The biology and pathogenesis of hepatitis viruses

Viral hepatitis is caused mainly by infection with one of the five hepatitis viruses, which use the liver as their primary site of replication. Each of these, known as hepatitis A through E viruses (HAV to HEV), belong to different virus families, have unique morphology, genomic organization and replication strategy. These viruses cause similar clinical manifestations during the acute phase of infection but vary in their ability to cause chronic infection. While HAV and HEV cause only acute disease with no chronic sequelae, HBV, HCV and HDV cause varying degrees of chronicity and liver injury, which can progress to cirrhosis and liver cancers. Though specific serological tests are available for the known hepatitis viruses, nearly 20% of all hepatitis cases show no markers. Antiviral therapy is also recommended for some hepatitis viruses and a preventive vaccine is available only for hepatitis B. More research and public awareness programmes are needed to control the disease. This review will provide an overview of the hepatitis viruses and the disease they cause.

Japanese encephalitis: pathogenesis, prophylactics and therapeutics

Japanese encephalitis (JE) is one of the most dreaded mosquito-borne viral encephalitis known to afflict humans. The Japanese encephalitis virus (JEV) is a neurotropic flavivirus that affects the CNS, causing extensive damage that may lead to fatality in about one third of bpatients. Half of the survivors suffer from severe neuropshychiatric sequelae. With nearly 3 billion people living under the current JE-endemic region, recurring incidents of epidemic are being reported at regular intervals. With no established antiviral therapies against JE available, vaccination has been the only way of preventing JE. Two types of JE vaccines are currently in vogue although the safety of administering them is questionable, in certain individuals. Thus, there is a need to develop a safe, affordable and potent JE vaccine and this review addresses the current efforts in this direction. This review also focuses on the pathophysiology of JE and efforts towards a possible breakthrough in anti-JEV therapy.

An approach for studying the mediators of pathogenesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis is an example of an intracellular pathogen that mediates the disease state through complex interactions with the host's immune system. Not only does this organism replicate in the hostile environment prevailing within the infected macrophage, but it has also developed intricate mechanisms to inhibit several defence mechanisms of the host's immune system. It is postulated here that the mediators of these interactions with the host are products of differentially expressed genes in the pathogen, B and T fell responses of the host are hence to be used as tools to identify such gene products from an expression library of the Mycobacterium tuberculosis genome. The various pathways of generating a productive immune response that may be targeted by the pathogen are discussed.

Role of TGF-beta beta and BMP7 in the pathogenesis of oral submucous fibrosis

To understand the molecular pathogenesis of oral submucous fibrosis (OSF), which is a chronic inflammatory disease, gene expression profiling was performed in 10 OSF tissues against 8 pooled normal tissues using oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (P < a parts per thousand currency sign 0.05 and fold change >= a parts per thousand yen 1.5). Among these, 2884 are upregulated and 2404 are downregulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed upregulation of transforming growth factor-beta beta 1 (TGF-beta beta 1), TGFBIp, THBS1, SPP1, and TIG1 and downregulation of bone morphogenic protein 7 (BMP7) in OSF tissues. Furthermore, activation of TGF-beta beta pathway was evident in OSF as demonstrated by pSMAD2 strong immunoreactivity. Treatment of keratinocytes and oral fibroblasts by TGF-beta beta confirmed the regulation of few genes identified in microarray including upregulation of connective tissue growth factor, TGM2, THBS1, and downregulation of BMP7, which is a known negative modulator of fibrosis. Taken together, these data suggest activation of TGF-beta beta signaling and suppression of BMP7 expression in the manifestation of OSF.

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.

Circulating miRNA profile in HCV infected serum: novel insight into pathogenesis

Changes in circulating miRNA profiles have been associated with different diseases. Here we demonstrate the circulating miRNA profile in serum of HCV infected individuals using a microRNA array that profiles the expression of 940 miRNAs. Serum samples from two HCV genotype -1 and two HCV genotype -3 infected individuals were compared with healthy controls. Expression levels of miR-134, miR-198, miR-320c and miR-483-5p that were commonly upregulated in case of both genotypes were validated in 36 individual patient serum samples. Serum miR-134, miR-320c and miR-483-5p were significantly upregulated during HCV infection. miR-320c and miR-483-5p were also upregulated in HCV-JFH1 infected cells and cell culture supernatant. Pathway analysis of putative target genes of these miRNAs indicated involvement of PI3K-Akt, NFKB and MAPK signaling pathways. Results revealed novel insights on the role of circulating miRNAs in mediating pathogenesis in HCV-infected cells.

Quorum sensing and pathogenesis: role of small signalling molecules in bacterial persistence

The pathogenesis of Mycobacterium tuberculosis is associated with its ability to survive inside the human host and the bacteria use a variety of mechanism to evade the host's defence. A clearer understanding of the host pathogen interaction is needed to follow the pathogenicity and virulence. Recent advances in the study of inter and intra-cellular communication in bacteria had prompted us to study the role of quorum sensing in bacterial survival and pathogenicity. The cell cell communication in bacteria (quorum sensing) is mediated through the exchange of small molecules called as autoinducers that allow bacteria to modulate their gene expression in response to change in cell-population density. It is a coordinated response that confers multicellularity to a bacterial population in response to stress from external environment. Quorum sensing molecules are the global regulators and regulate a wide range of physiological processes including biofilm formation, motility, cell differentiation, long-term survival and many others. Many bacterial pathogens require quorum sensing to produce the virulence factors in response to host pathogen interaction. Here, we summarize our current understanding on small molecule signalling and their role in the bacterial persistence. New discoveries in these areas have enriched our knowledge on intracellular signalling and their role in the long-term survival of mycobacteria under nutrient starvation.

Role of Areca Nut Induced TGF-beta and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-beta signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-beta in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-beta in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-beta followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-beta treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-beta together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-beta induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut + TGF-beta treated hGF cells. In concordance, areca nut along with TGF-beta enhanced fibroblast activation as demonstrated by potentiation of alpha SMA, gamma SMA and collagen gel contraction by hGF cells. Furthermore, TGF-beta secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

Dual role of arginine metabolism in establishing pathogenesis

Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.

Transglutaminase-2 Regulation by Arecoline in Gingival Fibroblasts

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P = 0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors

Background and Objective: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Material and Methods: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription polymerase chain reaction analysis. Results: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Conclusion: Taken together, our data highlight the importance of arecoline-induced epithelial changes in the pathogenesis of oral submucous fibrosis.