Search

Non-coding RNAs have emerged as critical regulators of the immune response to infection. MicroRNAs (miRNAs) are small non-coding RNAs which regulate host defense mechanisms against viruses, bacteria and fungi. They are involved in the delicate interplay between Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and its host, which dictates the course of infection. Differential expression of miRNAs upon infection with M. tuberculosis, regulates host signaling pathways linked to inflammation, autophagy, apoptosis and polarization of macrophages. Experimental evidence suggests that virulent M. tuberculosis often utilize host miRNAs to promote pathogenicity by restricting host-mediated antibacterial signaling pathways. At the same time, host- induced miRNAs augment antibacterial processes such as autophagy, to limit bacterial proliferation. Targeting miRNAs is an emerging option for host-directed therapies. Recent studies have explored the role of long non-coding RNA (lncRNAs) in the regulation of the host response to mycobacterial infection. Among other functions, lncRNAs interact with chromatin remodelers to regulate gene expression and also function as miRNA sponges. In this review we attempt to summarize recent literature on how miRNAs and lncRNAs are differentially expressed during the course of M. tuberculosis infection, and how they influence the outcome of infection. We also discuss the potential use of non-coding RNAs as biomarkers of active and latent tuberculosis. Comprehensive understanding of the role of these non-coding RNAs is the first step towards developing RNA-based therapeutics and diagnostic tools for the treatment of TB.

Mycobacterium tuberculosis can survive within its host for extended periods of time without any clinical symptoms of disease and reactivate when the immune system is weakened. A detailed understanding of how M. tuberculosis enters into and exits out of dormancy, is necessary in order to develop new strategies for tackling tuberculosis. Omics methodologies are unsupervised and unbiased to any hypothesis, making them useful tools for the discovery of new drug targets. This review summarizes the findings of transcriptomic and proteomic approaches toward understanding dormancy and reactivation of M. tuberculosis. Within the granuloma of latently infected individuals, the bacteria are dormant, with a marked slowdown of growth, division and metabolism. In vitro models have attempted to simulate these features by subjecting the bacterium to hypoxia, nutrient starvation, potassium depletion, growth in the presence of vitamin C, or growth in the presence of long-chain fatty acids. The striking feature of all the models is the upregulation of the DosR regulon, which includes the transcriptional regulator Rv0081, one of the central hubs of dormancy. Also upregulated are chaperone proteins, fatty acid and cholesterol degrading enzymes, the sigma factors SigE and SigB, enzymes of the glyoxylate and the methylcitrate cycle, the Clp proteases and the transcriptional regulator ClgR. Further, there is increased expression of genes involved in mycobactin synthesis, fatty acid degradation, the glyoxylate shunt and gluconeogenesis, in granulomas formed in vitro from peripheral blood mononuclear cells from latently infected individuals compared to naïve individuals. Genes linked to aerobic respiration, replication, transcription, translation and cell division, are downregulated during dormancy in vitro, but upregulated during reactivation. Resuscitation in vitro is associated with upregulation of genes linked to the synthesis of mycolic acids, phthiocerol mycocerosate (PDIM) and sulfolipids; ribosome biosynthesis, replication, transcription and translation, cell division, and genes encoding the five resuscitation promoting factors (Rpfs). The expression of proteases, transposases and insertion sequences, suggests genome reorganization during reactivation.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus induced disease-2019 (COVID-19), is a type of common cold virus responsible for a global pandemic which requires immediate measures for its containment. India has the world’s largest population aged between 10 and 40 years. At the same time, India has a large number of individuals with diabetes, hypertension and kidney diseases, who are at a high risk of developing COVID-19. A vaccine against the SARS-CoV-2, may offer immediate protection from the causative agent of COVID-19, however, the protective memory may be short-lived. Even if vaccination is broadly successful in the world, India has a large and diverse population with over one-third being below the poverty line. Therefore, the success of a vaccine, even when one becomes available, is uncertain, making it necessary to focus on alternate approaches of tackling the disease. In this review, we discuss the differences in COVID-19 death/infection ratio between urban and rural India; and the probable role of the immune system, co-morbidities and associated nutritional status in dictating the death rate of COVID-19 patients in rural and urban India. Also, we focus on strategies for developing masks, vaccines, diagnostics and the role of drugs targeting host-virus protein-protein interactions in enhancing host immunity. We also discuss India’s strengths including the resources of medicinal plants, good food habits and the role of information technology in combating COVID-19. We focus on the Government of India’s measures and strategies for creating awareness in the containment of COVID-19 infection across the country.

Two-component systems (TCSs) are central to the ability of Mycobacterium tuberculosis to respond to stress. One such paired TCS is SenX3-RegX3, which responds to phosphate starvation. Here we show that RegX3 is required for M. tuberculosis to withstand low pH, one of the challenges encountered by the bacterium in the host environment, and that RegX3 activates the cytosolic redox sensor WhiB3 to launch an appropriate response to acid stress. We show that the whiB3 promoter of M. tuberculosis harbors a RegX3 binding motif. Electrophoretic mobility shift assays (EMSAs) show that phosphorylated RegX3 (RegX3-P) (but not its unphosphorylated counterpart) binds to this motif, whereas a DNA binding mutant, RegX3 (K204A) fails to do so. Mutation of the putative RegX3 binding motif on the whiB3 promoter, abrogates the binding of RegX3-P. The significance of this binding is established by demonstrating that the expression of whiB3 is significantly attenuated under phosphate starvation or under acid stress in the regX3-inactivated mutant, ΔregX3. Green fluorescent protein (GFP)-based reporter assays further confirm the requirement of RegX3 for the activation of the whiB3 promoter. The compromised survival of ΔregX3 under acid stress and its increased trafficking to the lysosomal compartment are reversed upon complementation with either regX3 or whiB3, suggesting that RegX3 exerts its effects in a WhiB3-dependent manner. Finally, using an in vitro granuloma model, we show that granuloma formation is compromised in the absence of regX3, but restored upon complementation with either regX3 or whiB3. Our findings provide insight into an important role of RegX3 in the network that regulates the survival of M. tuberculosis under acid stress similar to that encountered in its intracellular niche. Our results argue strongly in favor of a role of the RegX3-WhiB3 axis in establishment of M. tuberculosis infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

BACKGROUND: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). METHODOLOGY/PRINCIPAL FINDINGS: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. CONCLUSIONS: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.