Comparative proteomic analysis of Mycobacterium tuberculosis strain H37Rv versus H37Ra

Background: Mycobacterium tuberculosis (MTB) H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain MTB H37Rv. In spite of extensive study, variation in virulence between the MTB H37Rv and MTB H37Ra strains is still to be understood. The difference in protein expression or structure due to mutation may probably be an important factor for the virulence property of MTB H37Rv strain. Methods: In this study, a whole proteome comparison between these two strains was carried out using bioinformatics approaches to elucidate differences in their protein sequences. Results: On comparison of whole proteome using NCBI standalone BLAST program between these two strains, 3759 identical proteins in both the strains out of 4003 proteins were revealed in MTB H37Rv and 4034 proteins were revealed in MTB H37Ra; 244 proteins of MTB H37Rv and 260 proteins of MTB H37Ra were found to be non-identical. A total of 172 proteins were identified with mutations (Insertions/deletions/substitutions) in MTB H37Ra while 53 proteins of MTB H37Rv and 85 proteins of MTB H37Ra were found to be distinct. Among 244 non-identical proteins, 19 proteins were reported to have an important biological function; In this study, mutation was shown in these proteins of MTB H37Ra. Conclusion: This study reports the protein differences with mutations between MTB H37Rv and H37Ra, which may help in better understanding the pathogenesis and virulence properties of MTB H37Rv

N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (DapE) a Potential Biomarker of Mycobacterium Tuberculosis: Tag-Free Purification by Metal Affinity Chromatography: Tag-free purification of DapE by metal affinity chromatography

DapE is an enzyme which is highly essential in lysine biosynthetic pathway for the growth and survival of Mycobacterium tuberculosis (Mtb) and other bacterial species although absent in human host. However, the sequence identity of Mtb-DapE with other mycobacterium and bacterial species estimated between 93 to 75% and approximately 50%, respectively. DapE is a metallo-enzyme requires few of transition metals for activity and bacterial proliferation. The homology model based structural studies including published structures revealed an availability of zinc interacting conserved amino residues in Mtb-DapE. In this study, we purified full length recombinant Mtb-DapE as tag-free (Mtb-DapETagFree) protein from inclusion bodies using zinc-NTA column. The single step purified protein observed with 96-98% purity and high yield. The indirect ELISA had 40% sensitivity using Mtb-DapE as an antigen against bovine tuberculosis (bTB) serum samples. IFA analysis with clear fluorescent spots of Mtb native DapE antigen in (3+) human TB positive sputum samples and recombinant Mtb-DapE positive control against Mtb-DapE polyclonal antibody are highly encouraging. The ELISA and IFA results incite for the consideration of Mtb-DapE in future development of quick and ideal TB detection assay along with other mycobacterium antigens. The future advanced detailed structural and functional studies using this highly purified and tag-free Mtb-DapE may provide discovery of antitubercular drug(s) and promising inhibitory molecules. HIGHLIGHTS Expression and purification of recombinant Mtb-DapETagFree. On-column refolded protein with 98% purity from inclusion bodies purified by zinc-NTA column affinity chromatography. The purified protein observed no aggregation and degradation even after 8 months storage. ELISA and IFA results supports for development of detection assay for TB from tubercular samples

Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug

AbstractTuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (MTB), which remains a major cause for morbidity and mortality in several developing countries. Most drug-resistant MTB clinical strains are resistant to isoniazid (INH), a first-line anti-TB drug. Mutation in KatG, a catalase-peroxidase, of MTB is reported to be a major cause of INH resistance. Normally upon activation by KatG, INH is converted to an active intermediate which has antimycobacterial action in MTB. This INH intermediate in the presence of NADH forms INH-NAD adduct which inhibits inhA (2-trans-enoyl-acyl carrier protein reductase) of MTB, thus blocking the synthesis of mycolic acid, a major lipid of the mycobacterial cell wall. In this docking study, the high binding affinity of INH-NAD adduct towards InhA was observed in comparison with INH alone. In this study, two resistant mutants of KatG (S315T and S315N) were modeled using Modeller9v10 and docking analysis with INH was performed using AutoDock4.2 and the docking results of these mutants were compared with the wild type KatG. Docking results revealed the formation of a single hydrogen (H) bond between the secondary amine nitrogen (–NH) of INH with Thr or Asn residues in place of Serine at 315 position of KatG mutant strains respectively, whereas in the case of the wild type, there was no H-bond formation observed between INH and Ser315. The H-bond formation may prevent free radical formation by KatG in mutant strains thus the development of resistance to the drug. This in silico evidence may implicate the basis of INH resistance in KatG mutant strains

Unveiling the Significance of LysE in Survival and Virulence of <i>Mycobacterium tuberculosis</i>: A Review Reveals It as a Potential Drug Target, Diagnostic Marker, and a Vaccine Candidate

Tuberculosis (TB) remains a global health threat, necessitating innovative strategies for control and prevention. This comprehensive review explores the Mycobacterium tuberculosis Lysine Exporter (LysE) gene, unveiling its multifaceted roles and potential uses in controlling and preventing tuberculosis (TB). As a pivotal player in eliminating excess L-lysine and L-arginine, LysE contributes to the survival and virulence of M. tuberculosis. This review synthesizes findings from different electronic databases and includes 13 studies focused on the LysE of M. tuberculosis. The research unveils that LysE can be a potential drug target, a diagnostic marker for TB, and a promising candidate for vaccine development. The absence of LysE in the widely used BCG vaccine underscores its uniqueness and positions it as a novel area for TB prevention. In conclusion, this review underscores the significance of LysE in TB pathogenesis and its potential as a drug target, diagnostic marker, and vaccine candidate. The multifaceted nature of LysE positions it at the forefront of innovative approaches to combat TB, calling for sustained research efforts to harness its full potential in the global fight against this infectious disease

Combined Src/EGFR Inhibition Targets STAT3 Signaling and Induces Stromal Remodeling to Improve Survival in Pancreatic Cancer

Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics which hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src and EGFR pathways. Although it is well-established that concurrent EGFR and Src inhibition exert these antineoplastic properties through direct inhibition of mitogenic pathways in tumor cells, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In this study, we demonstrate in both orthotopic tumor xenograft and(PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, increases microvessel density, and prevents tissue fibrosis. Furthermore, the stromal changes induced by parallel EGFR and Src pathway inhibition resulted in improved overall survival in PKT mice when combined with gemcitabine. As a phase I clinical trial utilizing concurrent EGFR and Src inhibition with gemcitabine has recently concluded, these data provide timely translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. IMPLICATIONS: These findings demonstrate that Src/EGFR inhibition targets STAT3, remodels the tumor stroma, and results in enhanced delivery of gemcitabine to improve overall survival in a mouse model of PDAC

Tobacco Carcinogen-Induced Production of GM-CSF Activates CREB to Promote Pancreatic Cancer

Although smoking is a significant risk factor for pancreatic cancer (PDAC), the molecular mechanisms underlying PDAC development and progression in smokers are still unclear. Here we show the role of cAMP response element-binding protein (CREB) in the pathogenesis of smoking-induced PDAC. Smokers had significantly higher levels of activated CREB when compared to non-smokers. Cell lines derived from normal pancreas and pancreatic intraepithelial neoplasm (PanIN) exhibited low baseline pCREB levels compared to PDAC cell lines. Furthermore, elevated CREB expression correlated with reduced survival in PDAC patients. Depletion of CREB significantly reduced tumor burden after tobacco-specific nitrosamine 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment, suggesting a CREB-dependent contribution to PDAC growth and progression in smokers. Conversely, NNK accelerated PanIN lesion and PDAC formation via GM-CSF mediated activation of CREB in a PDAC mouse model. CREB inhibition (CREBi) in mice more effectively reduced primary tumor burden compared to control or GM-CSF blockade alone following NNK exposure. GM-CSF played a role in the recruitment of tumor associated macrophages (TAM) and Treg expansion and promotion, whereas CREBi significantly reduced TAM and Treg populations in NNK-exposed mice. Overall, these results suggest that NNK exposure leads to activation of CREB through GM-CSF, promoting inflammatory and Akt pathways. Direct inhibition of CREB, but not GM-CSF, effectively abrogates these effects and inhibits tumor progression, offering a viable therapeutic strategy for PDAC patients

Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6Ksuccessfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1a;LSL-Kras;Tgfbr2(PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway