Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR-γ dependant pexophagy mechanism to control redox levels in macrophages.

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis-1 and DLP-1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down-regulating the expression of pexophagy associated proteins (p-AMPKα, p-ULK-1, Atg5, Atg7, Beclin-1, LC3-II, TFEB and Keap-1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor-γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages.

Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette-Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.

Epigenetic orchestration of host immune defences by Mycobacterium tuberculosis.

Being among the top 10 causes of adult deaths, tuberculosis (TB) disease is considered a major global public health concern to address. The human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb), is an extremely competent and well-versed pathogen that promotes pathogenesis by evading the host immune systems through numerous tactics. Investigations revealed that Mtb could evade the host defense mechanisms by reconfiguring the host gene transcription and causing epigenetic changes. Although results indicate the link between epigenetics and disease manifestation in other bacterial infections, little is known regarding the kinetics of the epigenetic alterations in mycobacterial infection. This literature review discusses the studies in Mtb-induced epigenetic alterations inside the host and its contribution in the host immune evasion strategies. It also discusses how the Mtb-induced alterations could be used as 'epibiomarkers' to diagnose TB. Additionally, this review also discusses therapeutic interventions to be enhanced through remodification by 'epidrugs'.

An Immunoinformatics-Based Study of Mycobacterium tuberculosis Region of Difference-2 Uncharacterized Protein (Rv1987) as a Potential Subunit Vaccine Candidate for Preliminary Ex Vivo Analysis.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein's capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1ß, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.

Host-mycobacteria conflict: Immune responses of the host vs. the mycobacteria TLR2 and TLR4 ligands and concomitant host-directed therapy.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the most grievous infectious diseases with long-term morbidity and unpredicted mortality rates globally. Thus, understanding the host-pathogen interactions to develop potential drugs is the most focused area of research. Mtb has many antigens communicating with host cells via various pattern recognition receptors (PRRs). From which, toll-like receptors-2 and 4 (TLR2 and 4) are two major PRRs that provide the primary immune response to Mtb infection of the respiratory tract. As a result, the TLR-mycobacterium antigen interaction triggers a variety of crucial innate immune signalling mechanisms such as phagosome maturation, oxidative stress, elicitation of cell deaths, production of proinflammatory cytokines, and eventually associates with the adaptive immune response to establish infection. Despite the extensive investigations on TLR2 and 4 Mtb ligands that have a significant role in the immune defence system, there are still many unsolved concerns driving researchers to explore the obscures. This review focuses on the host immune modulation due to Mtb-TLR2 and 4 ligand interaction. Subsequently, the host TLR2 and 4 immune signals in cooperation with other PRRs and successive cytokine expressions are discussed. Also highlighted are some recent findings on host-directed therapy related to TLRs that aid in developing novel immunotherapeutic prospects for the better control of Mtb infection.

Mycobacterium tuberculosis EsxL induces TNF-α secretion through activation of TLR2 dependent MAPK and NF-κB pathways.

Mycobacterium tuberculosis (Mtb) employs distinct strategies to circumvent host immune responses during the infection process. Various Mtb cell-wall associated and secretory proteins are known to play a critical role in the orchestration of host innate immune responses through modulation of signaling pathways. Mtb genome encodes for 23 (EsxA-EsxW) proteins belonging to the ESAT-6 like family; however, most of them are functionally unknown. Here, we show that Mtb EsxL induces tumor necrosis factor-alpha (TNF-α) production by activating nuclear translocation of nuclear factor-κB (NF-κB) via interaction with Toll-like Receptor 2 (TLR2). Blocking or silencing of TLR2 abrogated nuclear translocation of NF-kB and TNF-α production. Treatment with recombinant purified EsxL (rEsxL) activated mitogen-activated protein kinase (MAPK) pathway by inducing the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase (p38) pathways. At the same time, inhibition of ERK and p38 down-regulated the expression of TNF-α in rEsxL exposed murine macrophages. Besides TNF-α, EsxL also induced the production of IL-6 proinflammatory cytokine. Taken together, these results suggest that EsxL is able to induce TNF-α secretion via TLR2 through activation of NF-κB and MAPK signaling. This study will help in deducing therapeutic strategies for better control of the disease.

Identification and evaluation of immunogenic MHC-I and MHC-II binding peptides from Mycobacterium tuberculosis.

Due to several limitations of the only available BCG vaccine, to generate adequate protective immune responses, it is important to develop potent and cost-effective vaccines against tuberculosis (TB). In this study, we have used an immune-informatics approach to identify potential peptide based vaccine targets against TB. The proteome of Mycobacterium tuberculosis (Mtb), the causative agent of TB, was analyzed for secretory or surface localized antigenic proteins as potential vaccine candidates. The T- and B-cell epitopes as well as MHC molecule binding efficiency were identified and mapped in the modelled structures of the selected proteins. Based on antigenicity score and molecular dynamic simulation (MD) studies two peptides namely Pep-9 and Pep-15 were analyzed, modelled and docked with MHC-I and MHC-II structures. Both peptides exhibited no cytotoxicity and were able to induce proinflammatory cytokine secretion in stimulated macrophages. The molecular docking, MD and in-vitro studies of the predicted B and T-cell epitopes of Pep-9 and Pep-15 peptides with the modelled MHC structures exhibited strong binding affinity and antigenic properties, suggesting that the complex is stable, and that these peptides can be considered as a potential candidates for the development of vaccine against TB.

Computational discovery and ex-vivo validation study of novel antigenic vaccine candidates against tuberculosis.

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.