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Mycobacterium tuberculosis ( M. tb ) utilizes the multifunctionality of its protein factors to deceive the host. The unabated global incidence and prevalence of tuberculosis (TB) and the emergence of multidrug-resistant strains warrant the discovery of novel drug targets that can be exploited to manage TB. This study reports the role of M. tb AAA+ family protein MoxR1 in regulating host-pathogen interaction and immune system functions. We report that MoxR1 binds to TLR4 in macrophage cells and further reveal how this signal the release of proinflammatory cytokines. We show that MoxR1 activates the PI3K-AKT-MTOR signalling cascade by inhibiting the autophagy-regulating kinase ULK1 by potentiating its phosphorylation at serine 757, leading to its suppression. Using autophagy-activating and repressing agents such as rapamycin and bafilomycin A1 suggested that MoxR1 inhibits autophagy flux by inhibiting autophagy initiation. MoxR1 also inhibits apoptosis by suppressing the expression of MAPK JNK1/2 and cFOS, which play critical roles in apoptosis induction. Intriguingly, MoxR1 also induced robust disruption of cellular bioenergetics by metabolic reprogramming to rewire the citric acid cycle intermediates, as evidenced by the lower levels of citric acid and electron transport chain enzymes (ETC) to dampen host defence. These results point to a multifunctional role of M. tb MoxR1 in dampening host defences by inhibiting autophagy, apoptosis, and inducing metabolic reprogramming. These mechanistic insights can be utilized to devise strategies to combat TB and better understand survival tactics by intracellular pathogens.

Mycobacterium tuberculosis ( Mtb ) is the causative agent of human tuberculosis (TB) which primarily infects the macrophages. Nearly a quarter of the world's population is infected latently by Mtb . Only around 5%-10% of those infected develop active TB disease, particularly during suppressed host immune conditions or comorbidity such as HIV, hinting toward the heterogeneity of Mtb infection. The aerosolized Mtb first reaches the lungs, and the resident alveolar macrophages (AMs) are among the first cells to encounter the Mtb infection. Evidence suggests that early clearance of Mtb infection is associated with robust innate immune responses in resident macrophages. In addition to lung-resident macrophage subsets, the recruited monocytes and monocyte-derived macrophages (MDMs) have been suggested to have a protective role during Mtb infection. Mtb , by virtue of its unique cell surface lipids and secreted protein effectors, can evade killing by the innate immune cells and preferentially establish a niche within the AMs. Continuous efforts to delineate the determinants of host defense mechanisms have brought to the center stage the crucial role of macrophage phenotypical variations for functional adaptations in TB. The morphological and functional heterogeneity and plasticity of the macrophages aid in confining the dissemination of Mtb. However, during a suppressed or hyperactivated immune state, the Mtb virulence factors can affect macrophage homeostasis which may skew to favor pathogen growth, causing active TB. This mini-review is aimed at summarizing the interplay of Mtb pathomechanisms in the macrophages and the implications of macrophage heterogeneity and plasticity during Mtb infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ahmad, Rani, Alam, Zarin, Pandey, Singh, Hasnain and Ehtesham.)

Mycobacterium tuberculosis ( M. tb ) persists as latent infection in nearly a quarter of the global population and remains the leading cause of death among infectious diseases. While BCG is the only vaccine for TB, its inability to provide complete protection makes it imperative to engineer BCG such that it expresses immunodominant antigens that can enhance its protective potential. In-silico comparative genomic analysis of Mycobacterium species identified M. tb Rv1507A as a "signature protein" found exclusively in M. tb . In-vitro (cell lines) and in-vivo experiments carried out in mice, using purified recombinant Rv1507A revealed it to be a pro-inflammatory molecule, eliciting significantly high levels of IL-6, TNF-α, and IL-12. There was increased expression of activation markers CD69, CD80, CD86, antigen presentation molecules (MHC I/MHCII), and associated Th1 type of immune response. Rv1507A knocked-in M. smegmatis also induced significantly higher pro-inflammatory Th1 response and higher survivability under stress conditions, both in-vitro (macrophage RAW264.7 cells) and in-vivo (mice). Sera derived from human TB patients showed significantly enhanced B-cell response against M. tb Rv1507A. The ability of M. tb Rv1507A to induce immuno-modulatory effect, B cell response, and significant memory response, renders it a putative vaccine candidate that demands further exploration., (Copyright © 2020 Arora, Alam, Naqvi, Ahmad, Sheikh, Rahman, Hasnain and Ehtesham.)

Microscopy-based tuberculosis (TB) diagnosis i.e. Ziehl-Neelsen screening still remains the primary diagnostic method in resource poor and high TB burden countries, however this method has poor sensitivity (~60%). Bringing three million TB patients who are left undiagnosed under the treatment has been a major focus as part of END-TB strategy across the world. We have developed a portable set-up called 'SeeTB' that converts a bright-field microscope into fluorescence microscope (FM) with minimal interventions. SeeTB, a total internal reflection-based fluorescence excitation system allows visualization of auramine-O stained bacilli efficiently with high signal-to-noise ratio. Along with the device, we have developed a sputum-processing reagent called 'CLR' that homogenizes and digests the viscous polymer matrix of sputum. We have compared the performance of SeeTB system in 237 clinical sputum samples along with FM, GeneXpert and liquid culture. In comparison with culture as gold standard, FM has sensitivity of 63.77% and SeeTB has improved sensitivity to 76.06%. In comparison with GeneXpert, FM has sensitivity of 73.91% while SeeTB has improved sensitivity to 85.51%. However, there is no significant change in the specificity between FM and SeeTB system. In short, SeeTB system offers the most realistic option for improved TB case identification in resource-limited settings.

Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium -specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion-an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells., (Copyright © 2018 Ahmad et al.)

Through comparative analyses using BLASTp and BLASTn of the 25 target sequences, our research identified two unique post-transcriptional modifiers, Rv1509 and Rv2231A, which serve as distinctive and characteristic proteins of M.tb - the Signature Proteins. Here, we have characterized these two signature proteins associated with pathophysiology of M.tb which may prove to be therapeutically important targets. Dynamic Light Scattering and Analytical Gel Filtration Chromatography exhibited that Rv1509 exists as a monomer while Rv2231A as a dimer in solution. Secondary structures were determined using Circular Dichroism and further validated through Fourier Transform Infrared spectroscopy. Both the proteins are capable of withstanding a wide range of temperature and pH variations. Fluorescence spectroscopy based binding affinity experiments showed that Rv1509 binds to iron and may promote organism growth by chelating iron. In the case of Rv2231A, a high affinity for its substrate RNA was observed, which is facilitated in presence of Mg2+ suggesting it might have RNAse activity, supporting the prediction through in-silico studies. This is the first study on biophysical characterization of these two therapeutically important proteins, Rv1509 and Rv2231A, providing important insights into their structure -function correlations which are crucial for development of new drugs/ early diagnostics tools targeting these proteins. [ABSTRACT FROM AUTHOR]