Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense.

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and ß-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Are all VapC toxins of Mycobacterium tuberculosis endowed with enigmatic RNase activity?

Mycobacterium tuberculosis (M. tb) employs an extensive network of more than 90 toxin-antitoxin systems, and among them, VapC toxins are the most abundant. While most VapCs function as classical RNases with toxic effects, a significant number of them do not exhibit toxicity. However, these non-toxic VapCs may retain specific RNA binding abilities as seen in case of VapC16, leading to ribosome stalling at specific codons and reprofiling M. tb's proteome to aid in the bacterium's survival under different stressful conditions within the host. Here, we challenge the conventional classification of all VapC toxins as RNases and highlight the complexity of M. tb's strategies for survival and adaptation during infection.

Structural and Biophysical properties of therapeutically important proteins Rv1509 and Rv2231A of Mycobacterium tuberculosis.

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.

Mycobacterium tuberculosis protein MoxR1 enhances virulence by inhibiting host cell death pathways and disrupting cellular bioenergetics.

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.

Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: potential broad-spectrum therapeutic targets against infectious diseases.

The intracellular viral, bacterial, or parasitic pathogens evade the host immune challenges to propagate and cause fatal diseases. The microbes overpower host immunity at various levels including during entry into host cells, phagosome formation, phagosome maturation, phagosome-lysosome fusion forming phagolysosomes, acidification of phagolysosomes, and at times after escape into the cytosol. Phagolysosome is the final organelle in the phagocyte with sophisticated mechanisms to degrade the pathogens. The immune evasion strategies by the pathogens include the arrest of host cell apoptosis, decrease in reactive oxygen species, the elevation of Th2 anti-inflammatory response, avoidance of autophagy and antigen cross-presentation pathways, and escape from phagolysosomal killing. Since the phagolysosome organelle in relation to infection/cure is seldom discussed in the literature, we summarize here the common host as well as pathogen targets manipulated or utilized by the pathogens established in phagosomes and phagolysosomes, to hijack the host immune system for their benefit. These common molecules or pathways can be broad-spectrum therapeutic targets for drug development for intervention against infectious diseases caused by different intracellular pathogens.

Evolution of SARS-CoV-2: BA.4/BA.5 Variants Continues to Pose New Challenges.

The acquisition of a high number of mutations, notably, the gain of two mutations L452R and F486V in RBD, and the ability to evade vaccine/natural infection-induced immunity suggests that Omicron is continuing to use "immune-escape potential" as an evolutionary space to maintain a selection advantage within the population. Despite the low hospitalizations and lower death rate, the surges by these variants may offset public health measures and disrupt health care facilities as seen recently in Portugal and the USA. Interestingly these BA.4/BA.5 variants have been found to be more severe than the earlier-emerged Omicron variants. We believe that aggressive COVID-19 surveillance using affordable testing strategies might actually help understand the evolution and transmission pattern of new variants. The sudden dip in reporting of new cases in some of the low- and middle-income countries is an alarming situation and needs to be addressed as this could lead to undetected transmission of future variants of interest/concern of SARS-CoV-2 in large population settings, including advent of a 'super' virus. It would be interesting to examine the possible role/influence, if any, of the two different kinds of vaccines, the spike protein-based versus the inactivated whole virus, in the evolution of BA.4/BA.5.

Energetics of Spike Protein Opening of SARS-CoV-1 and SARS-CoV-2 and Its Variants of Concern: Implications in Host Receptor Scanning and Transmission.

The receptor binding domain(s) (RBD) of spike (S) proteins of SARS-CoV-1 and SARS-CoV-2 (severe acute respiratory syndrome coronavirus) undergoes closed to open transition to engage with host ACE2 receptors. In this study, using multi atomistic (equilibrium) and targeted (non-equilibrium) molecular dynamics simulations, we have compared energetics of RBD opening pathways in full-length (modeled from cryo-EM structures) S proteins of SARS-CoV-1 and SARS-CoV-2. Our data indicate that amino acid variations at the RBD interaction interface can culminate into distinct free energy landscapes of RBD opening in these S proteins. We further report that mutations in the S protein of SARS-CoV-2 variants of concern can reduce the protein-protein interaction affinity of RBD(s) with its neighboring domains and could favor its opening to access ACE2 receptors. The findings can also aid in predicting the impact of future mutations on the rate of S protein opening for rapid host receptor scanning.

Macrophage: A Cell With Many Faces and Functions in Tuberculosis.

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.

MycoVarP: Mycobacterium Variant and Drug Resistance Prediction Pipeline for Whole-Genome Sequence Data Analysis.

Whole-genome sequencing (WGS) provides a comprehensive tool to analyze the bacterial genomes for genotype-phenotype correlations, diversity of single-nucleotide variant (SNV), and their evolution and transmission. Several online pipelines and standalone tools are available for WGS analysis of Mycobacterium tuberculosis (Mtb) complex (MTBC). While they facilitate the processing of WGS data with minimal user expertise, they are either too general, providing little insights into bacterium-specific issues such as gene variations, INDEL/synonymous/PE-PPE (IDP family), and drug resistance from sample data, or are limited to specific objectives, such as drug resistance. It is understood that drug resistance and lineage-specific issues require an elaborate prioritization of identified variants to choose the best target for subsequent therapeutic intervention. Mycobacterium variant pipeline (MycoVarP) addresses these specific issues with a flexible battery of user-defined and default filters. It provides an end-to-end solution for WGS analysis of Mtb variants from the raw reads and performs two quality checks, viz, before trimming and after alignments of reads to the reference genome. MycoVarP maps the annotated variants to the drug-susceptible (DS) database and removes the false-positive variants, provides lineage identification, and predicts potential drug resistance. We have re-analyzed the WGS data reported by Advani et al. (2019) using MycoVarP and identified some additional variants not reported so far. We conclude that MycoVarP will help in identifying nonsynonymous, true-positive, drug resistance-associated variants more effectively and comprehensively, including those within the IDP of the PE-PPE/PGRS family, than possible from the currently available pipelines.

Mycobacterium smegmatis Bacteria Expressing Mycobacterium tuberculosis-Specific Rv1954A Induce Macrophage Activation and Modulate the Immune Response.

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Is Involved in Modulation of Macrophage Functions to Favor Bacterial Persistence.

Mycobacterium tuberculosis (M. tb) Rv0297-encoded PE_PGRS5 has been known to be expressed at the later stages of infection and in acidified phagosomes during transcriptome and proteomic studies. The possible role of Rv0297 in the modulation of phagosomal maturation and in providing protection against a microbicidal environment has been hypothesized. We show that Rv0297PGRS is involved in modulating the calcium homeostasis of macrophages followed by impedance of the phagolysosomal acidification process. This is evident from the downregulation of the late endosomal markers (Rab7 and cathepsin D) in the macrophages infected with recombinant Mycobacterium smegmatis (rM.smeg)-M.smeg_Rv0297 and M.smeg_Rv0297PGRS-or treated with recombinant Rv0297PGRS protein. Macrophages infected with rM.smeg expressing Rv0297 produce nitric oxide and undergo apoptosis, which may aid in the dissemination of pathogen in the later stages of infection. Rv0297 was also found to be involved in rescuing the bacterium from oxidative and hypoxic stress employed by macrophages and augmented the survivability of the recombinant bacterium. These results attribute to the functional significance of this protein in M.tb virulence mechanism. The fact that this protein gets expressed at the later stages of lung granulomas during M.tb infection suggests that the bacterium possibly employs Rv0297 as its dissemination and survival strategy.

Immunodominant Mycobacterium tuberculosis Protein Rv1507A Elicits Th1 Response and Modulates Host Macrophage Effector Functions.

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.

Emerging genetic diversity among clinical isolates of SARS-CoV-2: Lessons for today.

Considering the current pandemic of COVID-19, it is imperative to gauge the role of molecular divergence in SARS-CoV-2 with time, due to clinical and epidemiological concerns. Our analyses involving molecular phylogenetics is a step toward understanding the transmission clusters that can be correlated to pathophysiology of the disease to gain insight into virulence mechanism. As the infections are increasing rapidly, more divergence is expected followed possibly by viral adaptation. We could identify mutational hotspots which appear to be major drivers of diversity among strains, with RBD of spike protein emerging as the key region involved in interaction with ACE2 and consequently a major determinant of infection outcome. We believe that such molecular analyses correlated with clinical characteristics and host predisposition need to be evaluated at the earliest to understand viral adaptability, disease prognosis, and transmission dynamics.

SeeTB: A novel alternative to sputum smear microscopy to diagnose tuberculosis in high burden countries.

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.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein.

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.

Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity In-Vitro and In-Vivo.

Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H2O2 and CoCl2 thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence.