pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate 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 Co-Operonic PE25/PPE41 Protein Complex of Mycobacterium tuberculosis Elicits Increased Humoral and Cell Mediated Immune Response

BACKGROUND: 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

The baculovirus antiapoptotic p35 gene also functions via an oxidant-dependent pathway

Cellular 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 N-terminal domain of <i>Mycobacterium tuberculosis</i> PPE17 (Rv1168c) protein plays a dominant role in inducing antibody responses in active TB patients

<div><p>The PPE (proline-proline-glutamic acid) proteins of <i>Mycobacterium tuberculosis</i> are characterized by a conserved N-terminal domain of approximately 180 amino acids and variable C-terminal domain. Since last decade, these proteins have gained much importance in the serodiagnosis of tuberculosis (TB) as they act as a source of antigenic variation. We have demonstrated earlier that one of the PPE proteins PPE17 (Rv1168c) induces strong B-cell and T-cell responses in active TB disease and also displays a higher antibody titer compared to immunodominant antigens such as ESAT-6, Hsp60 and PPD. However, the immunodominant domain of PPE17 (N-terminal or C-terminal) was not examined in detail. In the present study, we observed that antibody responses elicited in TB patients were directed mostly towards the N-terminal domain of PPE17 (N-PPE17). The antibody generated against N-PPE17 in TB patients did not significantly cross-react with N-terminal domains of other PPE proteins used in this study. Our data suggest that the N-terminal domain of PPE17 protein is immunodominant and could be used as a better serodiagnostic marker than the full-length PPE17 protein.</p></div

The N-terminal fragment of PPE17 (N-PPE17) shows better seroreactivity than full-length PPE17 in active TB patients (n = 143).

<p>(A) The full-length PPE17 or N-PPE17 protein was coated on EIA plate at various concentrations starting from 25 ng/well/50 μl to 1 μg/well/50 μl and antibody responses of TB patient sera were compared. The negative control (no antigen) displayed a background absorbance value of 0.056. Data shown in Fig 4A were re-plotted to compare the antibody levels of TB patients between PPE17 and N-PPE17 at 50 ng/well/50 μl (B1), 100 ng/well/50 μl (C1) and 250 ng/well/50 μl (D1) antigen coating concentrations. Data from Fig 4A were re-plotted to compare the PPE17 and N-PPE17-specific antibody levels in sera samples of smear positive (n = 58), smear negative (n = 38) and extrapulmonary TB (n = 47) patients at antigen concentration of 50 ng/well/50 μl (B2), 100 ng/well/50 μl (C2) and 250 ng/well/50 μl (D2).</p

N-PPE17-specific antibodies in sera obtained from TB patients did not significantly cross-react with N-terminal domains of other PPE proteins.

<p>Sera samples (n = 50) pre-incubated with recombinantly purified full-length PPE17 or PPE18 or PPE44 or PPE65 protein were added to EIA plates coated with recombinantly purified N-PPE17. The plates were further incubated with anti-human IgG-HRP and absorbance was read at 492 nm using the chromogenic substrate OPD.</p

The N-terminal fragment of PPE17 (N-PPE17) is more sensitive to detect TB cases than full-length PPE17 protein.

<p>(A) The percentages of high-level responders (patients showing antibody levels greater than or equal to the cut-off values) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179965#pone.0179965.g001" target="_blank">Fig 1A</a> were calculated using a cut-off value (mean OD₄₉₂ of healthy control sera plus 3 SD) for PPE17 and N-PPE17. (B) The percentage of high level responders was compared for pulmonary (smear-positive and smear-negative) and extrapulmonary TB cases.</p

The full-length PPE17 protein (PPE17) as well as the N-terminal fragment of PPE17 (N-PPE17) can discriminate active TB patients (n = 180) from BCG-vaccinated healthy controls (n = 20).

<p>(A) The mean absorbance values (OD₄₉₂) of TB patients <i>versus</i> healthy controls were compared for PPE17 and N-PPE17 proteins following EIA. (B) The EIA absorbance values shown in Fig 1A were re-plotted to compare the antibody levels of the smear positive pulmonary TB (n = 78), smear negative pulmonary TB (n = 47) and extrapulmonary TB (n = 55) patients between the PPE17 and N-PPE17 proteins. Horizontal line indicates the mean absorbance values.</p

Characteristics of study participants.

<p>Characteristics of study participants.</p