16 research outputs found
pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity
No full textPrephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity
The N-terminal domain of Mycobacterium tuberculosis PPE17 (Rv1168c) protein plays a dominant role in inducing antibody responses in active TB patients
No full textThe baculovirus antiapoptotic p35 gene also functions via an oxidant-dependent pathway
No full textCellular imbalance in the levels of antioxidants and reactive oxygen species resulting in apoptosis is directly associated with a number of parasitic infections, aging, and several genetic and multifactorial diseases. The baculovirus AcNPV-encoded antiapoptotic p35 gene prevents apoptosis induced by a variety of apoptotic agents in different systems. We demonstrate the ability of the p35 gene to inhibit oxidative stress-induced apoptosis. In vitro cultured Spodoptera frugiperda (Sf9) insect cells infected with wild-type AcNPV carrying the antiapoptotic p35 gene did not undergo apoptosis when subjected to oxidative stress generated by the exogenous application of oxidants or in vivo generation of reactive oxygen species or on direct exposure of cells to UV radiations. An AcNPV mutant carrying a deletion of the p35 gene failed to arrest cell death. Transfection of cells with a recombinant plasmid containing the p35 gene under the transcriptional control of a stress promoter (Drosophila hsp70) was also able to rescue cells from oxidative stress-induced cell death, demonstrating the direct involvement of P35. ESR spin-trapping studies conducted in vitro and in vivo demonstrated that P35 functions directly as an antioxidant by mopping out free radicals and consequently prevents cell death by acting at an upstream step in the reactive oxygen species-mediated cell death pathway
The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response.
No full textBACKGROUND: Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. METHODOLOGY/PRINCIPAL FINDING: We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15-22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8-10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-gamma, TNF-alpha and IL-2 cytokines. CONCLUSION: While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis
Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker
No full textIn multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence
The PE25 protein induces low B cell (IgG) response whereas the PPE41 and PE25/PPE41 protein complex induce high B cell response in mice.
No full text<p>ELISA was carried out with sera from mice immunized with the PE25, PPE41 and PE25/PPE41 recombinant proteins without any adjuvant, and screened for antibody against the respective recombinant proteins. The X-axis represents the sera from mice immunized with PBS, PE25, PPE41 and PE25/PPE41 proteins and the different bars (as indicated) represent the B cell response against each of the proteins. The p value is calculated between the groups of mice immunized with the PE25 and PPE41 proteins. The data shown represent three independent experiments carried out in triplicates.</p
The PPE41 and PE25/PPE41 complex proteins enhance CD4<sup>+</sup> and CD8<sup>+</sup> T cell activity.
No full text<p>Splenocytes from naïve mice or mice immunized with each of the recombinant proteins (PE25, PPE41 and PE25/PPE41) were stimulated with different concentration of the corresponding proteins <i>in-vitro</i>. The intracellular IFN-γ secreted by CD4<sup>+</sup> (a) and CD8<sup>+</sup> T cells (b) specific to the PE25, PPE41 and the PE25/PPE41 complex proteins was measured by flow cytometry and summarized as % increase in different experimental groups (c). The population of CD4<sup>+</sup> and CD8<sup>+</sup> T cells specific to the recombinant proteins was measured by stimulating the splenocytes with the respective proteins for 24–36 hrs. The data shown represent one of at least three similar independent experiments.</p
Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein
No full textAlthough the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H2O2), a major component of ROSs, inhibits interleukin-12 (IL-12) p40 and IL-12 p70 induction in murine macrophages and catalase pretreatment prevents H2O2-mediated down-regulation of IL-12. Endogenous accumulation of H2O2/ROSs in macrophages treated with alloxan resulted in IL-12 p40 inhibition. Although nuclear expression of both p50 and p65 NF-κB increased on H2O2 exposure, nuclear c-rel level was inhibited. Overexpression of c-rel restored IL-12 p40 on stimulation with lipopolysaccharide plus IFN-γ during H2O2 treatment. H2O2 did not inhibit c-rel induction in cytosol; however, it prevented the transport of c-rel from cytosol to the nucleus. H2O2 activated calmodulin (CaM) protein in the cytosol, which subsequently sequestered c-rel in the cytosol preventing its transport to the nucleus. The CaM inhibitor trifIuoperazine increased both nuclear c-rel and IL-12 p40 levels in H2O2-treated macrophages, emphasizing a role of CaM in these processes. H2O2/ROSs thus down-regulate IL-12 induction in macrophages by a novel pathway inhibiting c-rel translocation to the nucleus through activation of CaM protein
Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein
No full textThe PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage
No full textThe pathophysiological functions of proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins of Mycobacterium tuberculosis are not well understood. In this study, we demonstrate that one of the PPE proteins, PPE18 can stimulate macrophages to secrete IL-10, known to favor a Th2 type response. The recombinant PPE18 was found to specifically interact with the TLR2 leading to an early and sustained activation of p38 MAPK, which is critical for IL-10 induction. In silico docking analyses and mutation experiments indicate that PPE18 specifically interacts with the leucine rich repeat 11˜15 domain of TLR2 and the site of interaction is different from that of a synthetic lipopeptide Pam3CSK4 known to activate predominantly ERK 1/2. When PMA-differentiated THP-1 macrophages were infected with a mutant Mycobacterium tuberculosis strain lacking the PPE18, produced poorer levels of IL-10 as compared with those infected with the wild-type strain. In contrast, an M. smegmatis strain overexpressing the PPE18 induced higher levels of IL-10 in infected macrophages. Our data indicate that the PPE18 protein may trigger an anti-inflammatory response by inducing IL-10 production
