Rifabutin loaded inhalable ß-glucan microparticle based drug delivery system for pulmonary TB.

Inhalable microparticle-based anti TB drug delivery systems are being investigated extensively for Tuberculosis [TB] treatment as they offer efficient and deep lung deposition with several advantages over conventional routes. It can reduce the drug dose, treatment duration and toxic effects and optimize the drug bioavailability. Yeast derived ß-glucan is a ß-[1-3/1-6] linked biocompatible polymer and used as carrier for various biomolecules. Due to presence of glucan chains, particulate glucans act as PAMP and thereby gets internalized via receptor mediated phagocytosis by the macrophages. In this study, ß-glucan microparticles were prepared by adding l-leucine as excipient, and exhibited 70% drug [Rifabutin] loading efficiency. Further, the sizing and SEM data of particles revealed a size of 2-4 µm with spherical dimensions. The FTIR and HPLC data confirmed the ß-glucan composition and drug encapsulations efficiency of the particles. The mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD] data indicated that these particles are inhalable in nature and have better thermal stability as per DSC thermogram. These particles were found to be non-toxic upto a concentration of 80 µg/ml and were found to be readily phagocytosed by human macrophage cells in-vitro as well as in-vivo by lung alveolar macrophage. This study provides a framework for future design of inhalable ß-glucan particle based host-directed drug delivery system against pulmonary TB.

Role of inflammatory cytokine burst in neuro-invasion of Japanese Encephalitis virus infection: an immunotherapeutic approaches.

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.

Toxin-Antitoxin system of Mycobacterium tuberculosis: Roles beyond stress sensor and growth regulator.

The advent of effective drug regimen and BCG vaccine has significantly decreased the rate of morbidity and mortality of TB. However, lengthy treatment and slower recovery rate, as well as reactivation of the disease with the emergence of multi-drug, extensively-drug, and totally-drug resistance strains, pose a serious concern. The complexities associated are due to the highly evolved and complex nature of the bacterium itself. One of the unique features of Mycobacterium tuberculosis [M.tb] is that it has undergone reductive evolution while maintaining and amplified a few gene families. One of the critical gene family involved in the virulence and pathogenesis is the Toxin-Antitoxin system. These families are believed to harbor virulence signature and are strongly associated with various stress adaptations and pathogenesis. The M.tb TA systems are linked with growth regulation machinery during various environmental stresses. The genes of TA systems are differentially expressed in the host during an active infection, oxidative stress, low pH stress, and starvation, which essentially indicate their role beyond growth regulators. Here in this review, we have discussed different roles of TA gene families in various stresses and their prospective role at the host-pathogen interface, which could be exploited to understand the M.tb associated pathomechanisms better and further designing the new strategies against the pathogen.

ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator.

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

Mycobacterium tuberculosis RipA Dampens TLR4-Mediated Host Protective Response Using a Multi-Pronged Approach Involving Autophagy, Apoptosis, Metabolic Repurposing, and Immune Modulation.

Reductive evolution has endowed Mycobacterium tuberculosis (M. tb) with moonlighting in protein functions. We demonstrate that RipA (Rv1477), a peptidoglycan hydrolase, activates the NFκB signaling pathway and elicits the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12, through the activation of an innate immune-receptor, toll-like receptor (TLR)4. RipA also induces an enhanced expression of macrophage activation markers MHC-II, CD80, and CD86, suggestive of M1 polarization. RipA harbors LC3 (Microtubule-associated protein 1A/1B-light chain 3) motifs known to be involved in autophagy regulation and indeed alters the levels of autophagy markers LC3BII and P62/SQSTM1 (Sequestosome-1), along with an increase in the ratio of P62/Beclin1, a hallmark of autophagy inhibition. The use of pharmacological agents, rapamycin and bafilomycin A1, reveals that RipA activates PI3K-AKT-mTORC1 signaling cascade that ultimately culminates in the inhibition of autophagy initiating kinase ULK1 (Unc-51 like autophagy activating kinase). This inhibition of autophagy translates into efficient intracellular survival, within macrophages, of recombinant Mycobacterium smegmatis expressing M. tb RipA. RipA, which also localizes into mitochondria, inhibits the production of oxidative phosphorylation enzymes to promote a Warburg-like phenotype in macrophages that favors bacterial replication. Furthermore, RipA also inhibited caspase-dependent programed cell death in macrophages, thus hindering an efficient innate antibacterial response. Collectively, our results highlight the role of an endopeptidase to create a permissive replication niche in host cells by inducing the repression of autophagy and apoptosis, along with metabolic reprogramming, and pointing to the role of RipA in disease pathogenesis.

Author Correction: Artificial Intelligence and Machine learning based prediction of resistant and susceptible mutations in Mycobacterium tuberculosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Artificial Intelligence and Machine learning based prediction of resistant and susceptible mutations in Mycobacterium tuberculosis.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), causes highest number of deaths globally for any bacterial disease necessitating novel diagnosis and treatment strategies. High-throughput sequencing methods generate a large amount of data which could be exploited in determining multi-drug resistant (MDR-TB) associated mutations. The present work is a computational framework that uses artificial intelligence (AI) based machine learning (ML) approaches for predicting resistance in the genes rpoB, inhA, katG, pncA, gyrA and gyrB for the drugs rifampicin, isoniazid, pyrazinamide and fluoroquinolones. The single nucleotide variations were represented by several sequence and structural features that indicate the influence of mutations on the target protein coded by each gene. We used ML algorithms - naïve bayes, k nearest neighbor, support vector machine, and artificial neural network, to build the prediction models. The classification models had an average accuracy of 85% across all examined genes and were evaluated on an external unseen dataset to demonstrate their application. Further, molecular docking and molecular dynamics simulations were performed for wild type and predicted resistance causing mutant protein and anti-TB drug complexes to study their impact on the conformation of proteins to confirm the observed phenotype.

Disorder-to-order transition in PE-PPE proteins of Mycobacterium tuberculosis augments the pro-pathogen immune response.

A growing body of evidence supports the hypothesis that intrinsically disordered proteins often mediate host-pathogen interactions and modulate host functions for pathogen survival and virulence. Mycobacterium tuberculosis (M.tb) has evolved largely through reductive evolution, with a few exceptions such as the glycine-alanine-rich PE-PPE/PGRS protein family, which has been expanding in pathogenic mycobacteria. Here, our analyses of the M.tb proteome and secretome revealed that the PE-PGRS subfamily is enriched for disordered regions and disordered binding sites, pointing to their importance in host-pathogen interactions. As a case study, the secondary structure of PE35-PPE68 and PE32-PPE65 of the pathogenesis-related RD1 and RD8 regions was analyzed through Fourier-transform infrared spectroscopy. These disordered proteins displayed a considerable structural shift from disordered to ordered while engaged in the formation of complexes. While these proteins are immunogenic individually and enhance the pro-pathogen response, their corresponding complexes enhanced the responses manifold as displayed here by PE35 and PPE68. It is likely that M.tb exploits such disorder-order structural dynamics as a strategy to mount a pro-pathogen response and subvert host defense for productive infection. This functional gain also serves as a means to compensate genomic content loss due to reductive evolution.

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 Co-operonic PE32/PPE65 Proteins Alter Host Immune Responses by Hampering Th1 Response.

PE/PPE genes, present in cluster with ESAT-6 like genes, are suspected to have a role in antigenic variation and virulence of Mycobacterium tuberculosis. Their roles in immune evasion and immune modulation of host are also well documented. We present evidence that PE32/PPE65 present within the RD8 region are co-operonic, co-transcribed, and co-translated, and play role in modulating host immune responses. Experiments with macrophage cell lines revealed that this protein complex suppresses pro-inflammatory cytokines such as TNF-α and IL-6 whereas also inducing high expression of anti-inflammatory IL-10. Immunization of mice with these recombinant proteins dampens an effective Th1 response as evident from reduced frequency of IFN-γ and IL-2 producing CD4(+) and CD8(+) T cells. IgG sub-typing from serum of immunized mice revealed high levels of IgG1 when compared with IgG2a and IgG2b. Further IgG1/IgG2a ratio clearly demonstrated that the protein complex manipulates the host immune response favorable to the pathogen. Our results demonstrate that the co-transcribed and co-translated PE32 and PPE65 antigens are involved specifically in modulating anti-mycobacterial host immune response by hampering Th1 response.

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.

Interaction of Mycobacterium tuberculosis Virulence Factor RipA with Chaperone MoxR1 Is Required for Transport through the TAT Secretion System.

UNLABELLED: Mycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ß-lactam antibiotics, opening up new candidates for drug repurposing. IMPORTANCE: The virulence mechanism of mycobacteria is very complex. Broadly, the virulence factors can be classified as secretion factors, cell surface components, enzymes involved in cellular metabolism, and transcriptional regulators. The mycobacteria have evolved several mechanisms to secrete its proteins. Here, we have identified one of the virulence proteins of Mycobacterium tuberculosis, RipA, possessing peptidoglycan hydrolase activities secreted by the TAT secretion pathway. We also identified MoxR1 as a protein-protein interaction partner of RipA and demonstrated chaperone activity of this protein. We show that MoxR1-mediated folding is critical for the secretion of RipA within the TAT system. Inhibition of this export system in M. tuberculosis will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ß-lactam antibiotics, opening up new candidates for drug repurposing.