Identification of novel inhibitors for trigger factor (TF) of M. tb: an in silico investigation.

Trigger factor, as a chaperone protein, is required for survival of Mycobacterium tuberculosis (M.tb) in a stressed environment. This protein interacts with various partners in both the pre- and the post-translation processes, yet the crystal structures of the M.tb trigger factor remain unresolved. In this study, we developed a homology model of M.tb trigger factor to facilitate the discovery and design of inhibitors. To validate the model, we employed several methodologies, including Ramachandran plot and molecular dynamics simulations. The simulations showed a stable trajectory, indicating the accuracy of the model. The active site of M.tb Trigger Factor was identified based on site scores, and virtual screening of over 70,000 compounds led to the identification of two potential hits: HTS02984 (ethyl 2-(3-(4-fluorophenyl)ureido)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate) and S06856 ((E)-N-(4-((2-(4-(tert-butyl)benzoyl)hydrazono)methyl)phenyl) acetamide). These compounds showed strong binding affinity and energy scores, and their chemical descriptors were evaluated. Our study provides a reliable computational model for M.tb Trigger Factor and identifies two potential inhibitors for this crucial protein, which could aid in the development of novel therapies against tuberculosis.Communicated by Ramaswamy H. Sarma.

Macrophages in Microbial Pathogenesis: Commonalities of Defense Evasion Mechanisms.

Macrophages are key arsenals of the immune system against invaders. After compartmental isolation of a pathogen in phagosomes, the host immune response attempts to neutralize the pathogen. However, pathogens possess the ability to subvert these assaults and can also convert macrophages into their replicative niche. The multiple host defense evasion mechanisms employed by these pathogens include phagosome maturation arrest, molecular mimicry through secretory antigens, interference with host signaling, active radical neutralization, inhibition of phagosome acidification, alteration of programmed cell death, and other mechanisms. Macrophage biology as a part of the host-pathogen interaction has expanded rapidly in the past decade. The present review aims to shed some light upon the macrophage defense evasion strategies employed by pathogens. We have also incorporated recent knowledge in the field of macrophage dynamics during infection and evolutionary perspectives of macrophage dynamics.

M.tb-Rv2462c of Mycobacterium tuberculosis Shows Chaperone-like Activity and Plays a Role in Stress Adaptation and Immunomodulation.

Mycobacterium tuberculosis (M.tb)-encoded factors protect it against host-generated stresses and support its survival in the hostile host environment. M.tb possesses two peptidyl-prolyl cis-trans isomerases and a probable trigger factor encoded by Rv2462c which has an FKBP-like PPIase domain. PPIases are known to assist the folding of peptidyl-prolyl bonds and are involved in various cellular processes important for bacterial survival in host-generated stresses. In this study, we aim to functionally characterize Rv2462c of M.tb. Our data suggest that the trigger factor of M.tb exhibits chaperone activity both in vitro and in vivo. Heterologous expression of M.tb-Rv2462c locus into Mycobacterium smegmatis enhanced its survival within macrophages, adaptation to oxidative stress and biofilm formation. M.tb-trigger factor has strong immunomodulatory potential and modifies the cytokine profile of the host towards the proinflammatory axis.

Lessons from SARS-CoV-2 Pandemic: Evolution, Disease Dynamics and Future.

The COVID-19 pandemic is rising at an unprecedented rate. The surging number of deaths every day, global lockdown and travel restrictions have resulted in huge losses to society. The impact is massive and will leave a historical footprint. The Spanish Flu of 1918, which was the last pandemic that had a similar impact, was shadowed under the consequences of World War I. All the brilliance, strength and economies of countries worldwide are aimed at fighting the COVID-19 pandemic. The knowledge about coronavirus dynamics, its nature and epidemiology are expanding every day. The present review aims to summarize the structure, epidemiology, symptoms, statistical status of the disease status, intervention strategies and deliberates the lessons learnt during the pandemic. The intervention approaches, antiviral drug repurposing and vaccine trials are intensified now. Statistical interpretations of disease dynamics and their projections may help the decision-makers.

Resistin in metabolism, inflammation, and disease.

Resistin is a small secretory protein that has a pleiotropic role in rodents and humans. Both rodent resistin and human resistin have an extremely stable and high-order multimeric structure. Moreover, there is significant variation in the source of secretion and the diversity of functions of resistin. Mouse resistin resists insulin action and contributes to type 2 diabetes mellitus, while human resistin plays a role in inflammation and also functions as a small accessory chaperone. Currently, active research in the area identified a significant role for resistin in stress biology and as a biomarker in diagnostics to evaluate disease status and treatment outcome. This review summarizes recent developments within resistin biology including their association with obesity, inflammation, stress response mechanisms, and its role in clinical diagnostics.

Peptidyl-prolyl isomerase-B is involved in Mycobacterium tuberculosis biofilm formation and a generic target for drug repurposing-based intervention.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (M.tb), takes one human life every 15 s globally. Disease relapse occurs due to incomplete clearance of the pathogen and reactivation of the antibiotic tolerant bacilli. M.tb, like other bacterial pathogens, creates an ecosystem of biofilm formed by several proteins including the cyclophilins. We show that the M.tb cyclophilin peptidyl-prolyl isomerase (PpiB), an essential gene, is involved in biofilm formation and tolerance to anti-mycobacterial drugs. We predicted interaction between PpiB and US FDA approved drugs (cyclosporine-A and acarbose) by in-silico docking studies and this was confirmed by surface plasmon resonance (SPR) spectroscopy. While all these drugs inhibited growth of Mycobacterium smegmatis (M.smegmatis) when cultured in vitro, acarbose and cyclosporine-A showed bacteriostatic effect while gallium nanoparticle (GaNP) exhibited bactericidal effect. Cyclosporine-A and GaNP additionally disrupted M.tb H37Rv biofilm formation. Co-culturing M.tb in their presence resulted in significant (2-4 fold) decrease in dosage of anti-tubercular drugs- isoniazid and ethambutol. Comparison of the cyclosporine-A and acarbose binding sites in PpiB homologues of other biofilm forming infectious pathogens revealed that these have largely remained unaltered across bacterial species. Targeting bacterial biofilms could be a generic strategy for intervention against bacterial pathogens.

Protein adaptations in extremophiles: An insight into extremophilic connection of mycobacterial proteome.

The biological paradox about how extremophiles persist at extreme ecological conditions throws a fascinating picture of the enormous potential of a single cell to adapt to homeostatic conditions in order to propagate. Unicellular organisms face challenges from both environmental factors and the ecological niche provided by the host tissue. Although the existence of extremophiles and their physiological properties were known for a long time, availability of whole genome sequence has catapulted the study on mechanisms of adaptation and the underlying principles that have enabled these unique organisms to withstand evolutionary and environmental pressures. Comparative genomics has shown that extremophiles possess the unique set of genes and proteins that empower them with biochemical machinery necessary to thrive in extreme environments. The presence of these proteins safeguards the cell against a wide array of extreme conditions such as temperature, pressure, radiations, chemicals, drugs etc. An insight into these adaptive mechanisms in extremophiles may help us to devise strategies to alter the genes and proteins that may have therapeutic potential and commercial value. Here we present an overview of the various adaptations in extremophiles. We also try to explain how mycobacterium channelizes its proteome to survive in stress conditions posed by host immune system.

Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Are Immunogenic, Alter Cytokine Profile and Aid in Intracellular Survival.

Mycobacterium tuberculosis (M. tb) has two peptidyl-prolyl isomerases (Ppiases) PpiA and PpiB, popularly known as cyclophilin A and cyclophilin B. The role of cyclophilins in processes such as signaling, cell surface recognition, chaperoning, and heat shock response has been well-documented. We present evidence that M. tb Ppiases modulate the host immune response. ELISA results revealed significant presence of antibodies to M. tb Ppiases in patient sera as compared to sera from healthy individuals. Treatment of THP-1 cells with increasing concentrations of rPpiA, induced secretion of pro-inflammatory cytokines TNF-α and IL-6. Alternatively, treatment with rPpiB inhibited secretion of TNF-α and induced secretion of IL-10. Furthermore, heterologous expression of M. tb PpiA and PpiB in Mycobacterium smegmatis increased bacterial survival in THP-1 cells as compared to those transformed with the vector control. Our results demonstrate that M. tb Ppiases are immunogenic proteins that can possibly modulate host immune response and enhance persistence of the pathogen within the host by subverting host cell generated stresses.

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.

Low expression level of glnA1 accounts for absence of cell wall associated poly-l-glutamate/glutamine in Mycobacterium smegmatis.

Cell wall associated poly-l-glutamine (PLG) layer synthesis is directly linked to glutamine synthetase (GS) encoded by glnA1 in tuberculosis causing mycobacteria. Avirulent Mycobacterium smegmatis (M. smegmatis) despite of having a glnA1 homolog lacks cell wall associated PLG layer. In the present study, we complemented a ΔglnA1 mutant of Mycobacterium bovis (lack PLG in cell wall) with M. smegmatis glnA1 cloned under M. bovis glnA1 promoter. PLG synthesis was restored in the cell wall of complemented strain. The complemented strain also showed increased resistance to physical stresses such as lysozyme, SDS and increased survival in THP-1 macrophages in comparison to the knockout. Further, in ß-galactosidase reporter assay M. smegmatis glnA1 promoter showed ten times less activity as compared to M. bovis glnA1 promoter. GACT-8-11 → TGAC mutations in the M. smegmatis glnA1 promoter restored its activity by 60% as compared to the activity of glnA1 promoter of M. bovis. This mutation also showed increased GS expression and produced cell wall associated PLG in M. smegmatis. The results of this study demonstrate that glnA1 promoter of M. smegmatis accounts for low expression level of GS and apparently responsible for absence of cell wall associated PLG layer.

The conserved hypothetical protein Rv0574c is required for cell wall integrity, stress tolerance, and virulence of Mycobacterium tuberculosis.

The virulence of Mycobacterium tuberculosis is intimately related to its distinctive cell wall. The biological significance of poly-α-L-glutamine (PLG), a component in the cell wall of virulent mycobacteria, has not been explored adequately. The focus of this study is to investigate the role of a locus, Rv0574c, coding for a polyglutamate synthase-like protein, in the synthesis of poly-α-L-glutamine in the context of mycobacterial virulence. Evaluation of Rv0574c gene expression in M. tuberculosis demonstrated its growth-phase-linked induction with concomitant accumulation of poly-α-L-glutamine in the cell wall. Rv0574c was activated under conditions prevalent in the tubercular granuloma, e.g., hypoxia, nitric oxide, and CO2. For functional characterization, we produced a deletion mutant of the Rv0574c gene by allelic exchange. The mutant produced smaller amounts of poly-α-L-glutamine in the cell wall than did the wild-type bacterium. Additionally, the increased sensitivity of the mutant to antitubercular drugs, SDS, lysozyme, and mechanical stress was accompanied by a drastic reduction in the ability to form biofilm. Growth of the ΔRv0574c strain was normal under in vitro conditions but was retarded in THP-1 macrophages and in the lungs and spleen of BALB/c mice. This was in agreement with histopathology of the lungs showing slow growth and less severe pathology than that of the wild-type strain. In summary, this study demonstrates that the protein encoded by the Rv0574c locus, by virtue of modulating PLG content in the cell wall, helps in maintaining cellular integrity in a hostile host environment. Also, its involvement in protecting the pathogen from host-generated lethal factors contributes to the infectious biology of M. tuberculosis.

Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability.

BACKGROUND: The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis. RESULTS: We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain. CONCLUSIONS: The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.

Enzymatic characterization of Catalase from Bacillus anthracis and prediction of critical residues using information theoretic measure of Relative Entropy.

In order to cope up with the reactive oxygen species (ROS) generated by host innate immune response, most of the intracellular organisms express Catalase for the enzymatic destruction/detoxification of hydrogen peroxide, to combat its deleterious effects. Catalase thus, scavenges ROS thereby playing a pivotal role in facilitating the survival of the pathogen within the host, and thus contributes to its pathogenesis. Bacillus anthracis harbors five isoforms of Catalase, but none of them has been studied so far. Thus, this study is the first attempt to delineate the biochemical and functional characteristics of one of the isoforms of Catalase (Cat1.4) of B. anthracis, followed by identification of residues critical for catalysis. The general strategy used, so far for mutational analysis in Catalases is structure based, i.e. the residues in the vicinity of heme were mutated to decipher the enzymatic mechanism. However, in the present study, protein sequence analysis was used for the prediction of catalytically important residues of Catalase. Essential measures were adopted to ensure the accuracy of predictions like after retrieval of well-annotated sequences from the database with EC 1.11.1.6, preprocessing was done to remove irrelevant sequences. The method used for multiple alignment of sequences, was guided by structural alignment and thereafter, an information theoretic measure, Relative Entropy was used for the critical residue prediction. By exploiting this strategy, we identified two previously known essential residues, H55 and Y338 in the active site which were demonstrated to be crucial for the activity. We also identified six novel crucial residues (Q332, Y117, H215, W257, N376 and H146) located distantly from the active site. Thus, the present study highlights the significance of this methodology to identify not only those crucial residues which lie in the active site of Catalase, but also the residues located distantly.