Pathogenic Tuberculosis Research Articles

Diversification of gene content in the Mycobacterium tuberculosis complex is determined by phylogenetic and ecological signatures.

We analyzed the pan-genome and gene content modulation of the most diverse genome data set of the Mycobacterium tuberculosis complex (MTBC) gathered to date. The closed pan-genome of the MTBC was characterized by reduced accessory and strain-specific genomes, compatible with its clonal nature. However, significantly fewer gene families were shared between MTBC genomes as their phylogenetic distance increased. This effect was only observed in inter-species comparisons, not within-species, which suggests that species-specific ecological characteristics are associated with changes in gene content. Gene loss, resulting from genomic deletions and pseudogenization, was found to drive the variation in gene content. This gene erosion differed among MTBC species and lineages, even within M. tuberculosis, where L2 showed more gene loss than L4. We also show that phylogenetic proximity is not always a good proxy for gene content relatedness in the MTBC, as the gene repertoire of Mycobacterium africanum L6 deviated from its expected phylogenetic niche conservatism. Gene disruptions of virulence factors, represented by pseudogene annotations, are mostly not conserved, being poor predictors of MTBC ecotypes. Each MTBC ecotype carries its own accessory genome, likely influenced by distinct selective pressures such as host and geography. It is important to investigate how gene loss confer new adaptive traits to MTBC strains; the detected heterogeneous gene loss poses a significant challenge in elucidating genetic factors responsible for the diverse phenotypes observed in the MTBC. By detailing specific gene losses, our study serves as a resource for researchers studying the MTBC phenotypes and their immune evasion strategies.IMPORTANCEIn this study, we analyzed the gene content of different ecotypes of the Mycobacterium tuberculosis complex (MTBC), the pathogens of tuberculosis. We found that changes in their gene content are associated with their ecological features, such as host preference. Gene loss was identified as the primary driver of these changes, which can vary even among different strains of the same ecotype. Our study also revealed that the gene content relatedness of these bacteria does not always mirror their evolutionary relationships. In addition, some genes of virulence can be variably lost among strains of the same MTBC ecotype, likely helping them to evade the immune system. Overall, our study highlights the importance of understanding how gene loss can lead to new adaptations in these bacteria and how different selective pressures may influence their genetic makeup.

Impact of helminth infections on clinical and epidemiological features of microbial infections, and vaccine responses in ruminants: a systematic review.

Abstract Helminth infections are ubiquitous in animal populations, and their interactions with concurrent or subsequent microbial infections can alter clinical and epidemiological features of infectious diseases and outcomes of vaccines. We used the PRISMA 2009 guidelines to conduct a systematic review of helminth-microbial-pathogen interactions and their impact on epidemiology and clinical outcomes of microbial infections in ruminants. Of the 1,375 probable studies identified through Google Scholar and PubMed databases, 20 studies met our inclusion criteria and were qualitatively analysed. Fourteen studies were on domestic ruminants, while six were on African buffaloes. Individual helminths investigated were Fasciola hepatica (six studies) Haemonchus contortus (three studies), Ostertagia ostertagi (one study) and Dicrocoelium dendriticum (one study), while the rest looked at gastrointestinal nematodes in general. Thirteen studies evaluated interactions between helminths and bovine tuberculosis (BTB), trypanosomes and theileria pathogens while four focused on responses to vaccination against rabies, bovine respiratory pathogens and Bacillus of Calmette-Guérin. Helminths interacted with Trypanosoma spp , Mycobacterium bovis , Theileria parva synergistically to aggravate clinical anaemia as measured by PCV and mortality. Furthermore, concurrent helminth infections were reported to lower the sensitivity of BTB diagnostic tests, but they did not appear to affect antibody responses to vaccines. We envision that helminths may interact with microbial pathogens in a number of ways, but primarily through immune pathways to modify clinical and epidemiological features of infections in ruminants. The implications of these interactions include missed diagnosis, underestimation of disease burden and increased severity of disease outcomes.

The advances in adjuvant therapy for tuberculosis with immunoregulatory compounds.

Tuberculosis (TB) is a chronic bacterial disease, as well as a complex immune disease. The occurrence, development, and prognosis of TB are not only related to the pathogenicity of Mycobacterium tuberculosis (Mtb), but also related to the patient's own immune state. The research and development of immunotherapy drugs can effectively regulate the body's anti-TB immune responses, inhibit or eliminate Mtb, alleviate pathological damage, and facilitate rehabilitation. This paper reviews the research progress of immunotherapeutic compounds for TB, including immunoregulatory compounds and repurposing drugs, and points out the existing problems and future research directions, which lays the foundation for studying new agents for host-directed therapies of TB.

Evolution of nucleic acid extraction methods and its role in development of complex human diagnostic solutions with special reference to tuberculosis: A review

Nucleic acid extraction from biological sources has evolved with time and is currently one of the most important contributors to the development of modern human molecular diagnostics. Pure and sufficient quantity of DNA and RNA from limited quantity of cells, which may be fresh or archived, holds key to successful detection of source and cause of many diseases. The challenges of cell disruption to release nucleic acids and the wide range of strategies applied to purify them by removing various enzyme inhibitors are discussed. The demographic spread of the disease of tuberculosis, hurdles faced during transportation of the clinical samples for nucleic acid extraction of Mycobacterium tuberculosis pathogen and different detection technologies supported by the extracted pathogen nucleic acid are reviewed.

Baktericidna svojstva nekih organskih kiselina u odnosu na mikobakterije

Despite current successes in fighting animal tuberculosis, sporadic cases of infection with both the disease’s causative agents and atypical mycobacteria still occur in animals. Since animal mycobacteriosis cannot be treated with medication, priority must be given to their prevention and elimination as non-specific measures, including disinfection. The unique structure of the mycobacteria cell de- termines their high resistance to antimicrobial agents, therefore the constant search for effective disinfectants is an urgent task of veterinary science. This study aimed to determine the bactericidal properties of several inorganic acids against mycobacteria. Experiments were carried out following modern methodological aspects using the atypical mycobacteria M. fortuitum, pathogens of tuberculosis M. bovis and M. avium. Orthophosphoric (phosphoric) acid (H3PO4) and nitric acid (HNO3) were used in the experiments. Using the suspension method, orthophosphoric acid was found to have a bactericidal effect against atypical mycobacteria- ria M. fortuitum and the pathogen of tuberculoussis M. avium when tested at a concentration of 1.5% (24 hours) and 2.0% (1–24 hours), against the causative agent of tuberculosis M. bovis at a concentration of 1.5% (5–24 hours) and 2.0% (1–24 hours). Nitric acid showed a bactericidal effect when tested in solution against mycobacteriaria M. fortuitum and M. avium at a concentration of 2.0% (5–24 hours), and against M. bovis at a concentration of 2.0% after 1 hour exposure. A different level of mycobacterial growth intensity in test tubes was observed after the action of inorganic acids in sub-bactericidal and bacteriostatic concentrations, which was directly dependent on the concentration and exposure of the acid, and on the type of test culture of microorganisms. It has been shown that inorganic acids can disinfect surfaces contaminated with the tuberculosis-causing agent. This refers to various surfaces like wood, tile, fabric, glass, and metal. It has been found that orthophosphoric acid (1.5% for 24 hours) and nitric acid (2.0% for 5 hours) can be used to disinfect in cases of tuberculosis infection. These findings have been confirmed through bioassays on laboratory animals. Future research will focus on discovering and developing new medications with strong bactericidal properties against mycobacteria.

Association between fatty acid metabolism gene mutations and Mycobacterium tuberculosis transmission revealed by whole genome sequencing

BackgroundFatty acid metabolism greatly promotes the virulence and pathogenicity of Mycobacterium tuberculosis (M.tb). However, the regulatory mechanism of fatty acid metabolism in M.tb remains to be elucidated, and limited evidence about the effects of gene mutations in fatty acid metabolism on the transmission of M.tb was reported.ResultsOverall, a total of 3193 M.tb isolates were included in the study, of which 1596 (50%) were genomic clustered isolates. Most of the tuberculosis isolates belonged to lineage2(n = 2744,85.93%), followed by lineage4(n = 439,13.75%) and lineage3(n = 10,0.31%).Regression results showed that the mutations of gca (136,605, 317G > C, Arg106Pro; OR, 22.144; 95% CI, 2.591-189.272), ogt(1,477,346, 286G > C ,Gly96Arg; OR, 3.893; 95%CI, 1.432–10.583), and rpsA (1,834,776, 1235 C > T, Ala412Val; OR, 3.674; 95% CI, 1.217–11.091) were significantly associated with clustering; mutations in gca and rpsA were also significantly associated with clustering of lineage2. Mutation in arsA(3,001,498, 885 C > G, Thr295Thr; OR, 6.278; 95% CI, 2.508–15.711) was significantly associated with cross-regional clusters. We also found that 20 mutation sites were positively correlated with cluster size, while 11 fatty acid mutation sites were negatively correlated with cluster size.ConclusionOur research results suggested that mutations in genes related to fatty acid metabolism were related to the transmission of M.tb. This research could help in the future control of the transmission of M.tb.

Association between two-component systems gene mutation and Mycobacterium tuberculosis transmission revealed by whole genome sequencing

BackgroundTwo-component systems (TCSs) play a crucial role in the growth of Mycobacterium tuberculosis (M. tuberculosis). However, the precise regulatory mechanism of their contribution remain to be elucidated, and only a limited number of studies have investigated the impact of gene mutations within TCSs on the transmission of M. tuberculosis. Therefore, this study aims to explore the relationship between TCSs gene mutation and the global transmission of M. tuberculosis.ResultsA total of 13531 M.tuberculosis strains were enrolled in the study. Most of the M.tuberculosis strains belonged to lineage4 (n=6497,48.0%), followed by lineage2 (n=5136,38.0%). Our results showed that a total of 36 single nucleotide polymorphisms (SNPs) were positively correlated with clustering of lineage2, such as Rv0758 (phoR, C820G), Rv1747(T1102C), and Rv1057(C1168T). A total of 30 SNPs showed positive correlation with clustering of lineage4, such as phoR(C182A, C1184G, C662T, T758G), Rv3764c (tcrY, G1151T), and Rv1747 C20T. A total of 19 SNPs were positively correlated with cross-country transmission of lineage2, such as phoR A575C, Rv1028c (kdpD, G383T, G1246C), and Rv1057 G817T. A total of 41 SNPs were positively correlated with cross-country transmission of lineage4, such as phoR(T758G, T327G, C284G), kdpD(G1755A, G625C), Rv1057 C980T, and Rv1747 T373G.ConclusionsOur study identified that SNPs in genes of two-component systems were related to the transmission of M. tuberculosis. This finding adds another layer of complexity to M. tuberculosis virulence and provides insight into future research that will help to elucidate a novel mechanism of M. tuberculosis pathogenicity.

A simple assay for inhibitors of mycobacterial oxidative phosphorylation

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and adenosine triphosphate (ATP) synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and non-specific membrane uncoupling. In this assay, IMVs from M. smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Non-specific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III2IV2 inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III2IV2 while thioridazine uncouples the mycobacterial membrane non-specifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

Exploring the Potential of Cytomegalovirus-Based Vectors: A Review.

Viral vectors have emerged as powerful tools for delivering and expressing foreign genes, playing a pivotal role in gene therapy. Among these vectors, cytomegalovirus (CMV) stands out as a promising viral vector due to its distinctive attributes including large packaging capacity, ability to achieve superinfection, broad host range, capacity to induce CD8+ T cell responses, lack of integration into the host genome, and other qualities that make it an appealing vector candidate. Engineered attenuated CMV strains such as Towne and AD169 that have a ~15 kb genomic DNA deletion caused by virus passage guarantee human safety. CMV's large genome enables the efficient incorporation of substantial foreign genes as demonstrated by CMV vector-based therapies for SIV, tuberculosis, cancer, malaria, aging, COVID-19, and more. CMV is capable of reinfecting hosts regardless of prior infection or immunity, making it highly suitable for multiple vector administrations. In addition to its broad cellular tropism and sustained high-level gene expression, CMV triggers robust, virus-specific CD8+ T cell responses, offering a significant advantage as a vaccine vector. To date, successful development and testing of murine CMV (MCMV) and rhesus CMV (RhCMV) vectors in animal models have demonstrated the efficacy of CMV-based vectors. These investigations have explored the potential of CMV vectors for vaccines against HIV, cancer, tuberculosis, malaria, and other infectious pathogens, as well as for other gene therapy applications. Moreover, the generation of single-cycle replication CMV vectors, produced by deleting essential genes, ensures robust safety in an immunocompromised population. The results of these studies emphasize CMV's effectiveness as a gene delivery vehicle and shed light on the future applications of a CMV vector. While challenges such as production complexities and storage limitations need to be addressed, ongoing efforts to bridge the gap between animal models and human translation continue to fuel the optimism surrounding CMV-based vectors. This review will outline the properties of CMV vectors and discuss their future applications as well as possible limitations.

Understanding Elongasome Unit of Mycobacterium and its Comparative Analysis with Other Model Organisms

The reported incidences of 10.6 million tuberculosis cases worldwide with 1.6 million deaths in 2021 indicate that this disease, caused by Mycobacterium tuberculosis pathogen is difficult to treat and requires exploring newer possible therapeutic interventions. To identify novel drug targets, it is important to understand the basic physiological processes of each pathogen in detail. Cell division is the fundamental physiological process which maintains the replicative state of bacteria. This process requires remodelling of the cell wall, which is performed by two spatio-temporal organized complexes, elongasome and divisome. These two complexes, elongasome and divisome function in synthesis of peptidoglycan (PG) at poles or septum of the cell, respectively. This review article is focused on illustrating differential features and composition of mycobacterial elongasome complex. This sort of understanding would allow identification of new drug targets and design of Mycobacterium specific drugs.

Mycobacterium tuberculosis Rv1043c regulates the inflammatory response by inhibiting the phosphorylation of TAK1.

Mycobacterium tuberculosis can manipulate the host immunity through its effectors to ensure intracellular survival and colonization. Rv1043c has been identified as an effector potentially involved in M. tuberculosis pathogenicity. To explore the function of M. tuberculosis Rv1043c during infection, we overexpressed this protein in M. smegmatis, a non-pathogenic surrogate model in tuberculosis research. Here, we reported that Rv1043c enhanced mycobacterial survival and down-regulated the release of pro-inflammatory cytokines in macrophages and mice. In addition, Rv1043c inhibited the activation of MAPK and NF-κB signaling by preventing the phosphorylation of TAK1 indirectly. In conclusion, these data suggest that Rv1043c regulates the immune response and enhances the survival of recombinant M. smegmatis in vitro and in vivo.

Application of Nanopore Sequencing in the Diagnosis and Treatment of Pulmonary Infections.

This review provides an in-depth discussion of the development, principlesand utility of nanopore sequencing technology and its diverse applications in the identification of various pulmonary pathogens. We examined the emergence and advancements of nanopore sequencing as a significant player in this field. We illustrate the challenges faced in diagnosing mixed infections and further scrutinize the use of nanopore sequencing in the identification of single pathogens, including viruses (with a focus on its use in epidemiology, outbreak investigation, and viral resistance), bacteria (emphasizing 16S targeted sequencing, rare bacterial lung infections, and antimicrobial resistance studies), fungi (employing internal transcribed spacer sequencing), tuberculosis, and atypical pathogens. Furthermore, we discuss the role of nanopore sequencing in metagenomics and its potential for unbiased detection of all pathogens in a clinical setting, emphasizing its advantages in sequencing genome repeat areas and structural variant regions. We discuss the limitations in dealing with host DNA removal, the inherent high error rate of nanopore sequencing technology, along with the complexity of operation and processing,while acknowledging the possibilities provided by recent technological improvements. We compared nanopore sequencing with the BioFire system, a rapid molecular diagnostic system based on polymerase chain reaction. Although the BioFire system serves well for the rapid screening of known and common pathogens, it falls short in the identification of unknown or rare pathogens and in providing comprehensive genome analysis. As technological advancements continue, it is anticipated that the role of nanopore sequencing technology in diagnosing and treating lung infections will become increasingly significant.

Aminocoumarin derivatives grafted on graphene oxide - new antimicrobial agents to combat the resistance of Mycobacterium tuberculosis and ESKAPE pathogens

Two hybrid materials, based on 7-amino-4-(trifluoromethyl)coumarin and 7-amino-4-methyl-coumarin that attach covalently to graphene oxide (GO) have been synthesized in two steps: i) increasing the number of carboxyl groups required for functionalization with aminocoumarin derivatives by derivatization of the hydroxyls groups with chloroacetic acid, transforming GO into GO-COOH material; ii) activation of carboxylic groups using the carbodiimide-promoted reaction. The obtained composites were characterized by numerous methods that highlighted their successful obtaining. The antimicrobial activity was evaluated on resistant Mycobacterium tuberculosis strains, as well as on ESKAPE pathogens ((Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) in planktonic and biofilm growth states. The biocompatibility of the materials has been assayed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay as well as by measuring the level of nitric oxide/total reactive oxygen species and superoxide release in treated cells. The results have shown that materials exhibited improved inhibitory activity against resistant M. tuberculosis strains in comparison with GO, but also against planktonic and adherent strains. Also, the tested composites have been proved to be biocompatible on the MRC-5 fibroblast cells, demonstrating their promising potential to develop novel agents effective against multidrug resistant pathogens, including M. tuberculosis, as well as by the activation of a pro-inflammatory and of an oxidative response in the mammalian cells.

A new approach to the epidemiological control of infectious diseases in the Russian population

The article describes a development related to the field of public health and molecular biology, in particular, medical diagnostics. We present the Isascreen-8 portable analyzer (Registration Certificate No. RZN 2022/17322, May 24, 2022) and its application for rapid diagnostics of a new coronavirus infection with the use of commercially available sets IsoAmp SARS-CoV-2 (Lytech Co. Ltd., Russia), and also our own development TB-IZATEST (Invention Application No. 2022133809, December 22, 2022) for diagnostics of tuberculosis pathogen. The approach proposed in this article can be used for rapid detection of genetic material (DNA/RNA) of Mycobacterium tuberculosis and SARS-CoV-2 in biological samples using the method of loop isothermal amplification in less than 20 minutes, and also as a universal platform for detection of genetic material of other pathogens in a biological sample. Using the proposed approach, various research tasks for epidemiological monitoring of tuberculosis and new coronavirus infection, qualitative and quantitative analysis of bacterial and viral load in samples, including evaluation of the effectiveness of the prescribed antibiotic therapy regimen, can be solved. The advantage is that this method can be used not only in diagnostic laboratories, but also in field laboratories in the most remote regions of Russia if the mandatory sanitary and epidemiological requirements are observed. With sufficient distribution, the proposed approach could help ensure epidemiological control of the prevalence of these socially significant infections.

Immunopathological response of the body in patients with chemosensitive and drug-resistant pulmonary tuberculosis

Immunological methods are important for diagnosing tuberculosis, evaluating the process activity, and forecasting the course of the disease and recovery.
 Materials and methods. 47 patients with first diagnosed destructive sputum smear-positive pulmonary tuberculosis underwent a complex immunoassay. The patients were divided into two groups based on the sensitivity/resistance of mycobacterium tuberculosis to antimycobacterial agents. The first group consisted of 22 patients with first-diagnosed chemosensitive tuberculosis with preserved sensitivity to antimycobacterial agents. The second group consisted of 25 patients with multi-drug resistant tuberculosis pulmonary tuberculosis (MDR-TBP). The research was conducted during the 2018-2021 years.
 Results Specific cell response disorders in patients with pulmonary tuberculosis are associated with the multi-structural T-cell protection misbalance caused by the quantitative changes of its components, the increase/decrease in the quantity of certain lymphocyte pools specifying the immune response vector.
 In cases of tuberculosis, phagocytosis plays an important role. Phagocytosis might release cells from the tuberculosis pathogen. To achieve this, the activation of cells should reach a certain level. However, the initial protective nature of cell activation might become aggressive.
 The T-cell immunity disorders were more evident in patients with MDR-TBP versus donors and patients with chemosensitive tuberculosis. The apparent decrease in СD3+СD56+, СD3+СD4+ pools and the increase in СD3+СD8+ were revealed in cases of MDR-TBP tuberculosis versus chemosensitive tuberculosis. The difference in СD3+СD4+, СD3+СD8+, СD3+СD4+/СD3+СD8+, CD3+СD8+HLA-DR+, СD16/56+8+ between the study and observational groups was statistically confirmed. The evident specific cell immunity disorders in patients with MDR-TBP aggravate the clinical course of the disease, causing destructive changes and acute and extensive processes.
 Conclusions Changes in different components of the immune system might occur during pulmonary tuberculosis (in T- and B-cells, phagocytic cells), specific and enzymatic processes are activated, and autoimmunization is evident. The intensity of the changes varies at different stages of the disease. Most immune disorders caused by the specific inflammation process require immune correction.

Wild mouse gut microbiota limits initial tuberculosis infection in BALB/c mice.

Mouse models are critical tools in tuberculosis (TB) research. Recent studies have demonstrated that the wild mouse gut microbiota promotes host fitness and improves disease resistance. Here we examine whether the wild mouse gut microbiota alters the immunopathology of TB in BALB/c mice. Conventional BALB/c mice (LabC) and mice born to germ-free BALB/c mothers reconstituted with the wild mouse gut microbiota (WildR) were used in our studies. WildR mice controlled initial TB infection better than LabC mice. The microbial gut communities of LabC mice and WildR mice had similar richness but significantly different composition prior to infection. TB reduced the gut community richness in both cohorts while differences in community composition remained indicating a general TB-induced dysbiosis. The wild mouse gut microbiota did not alter the typical lung histopathology of TB in the BALB/c model that includes unstructured immune cell infiltrates with infected foamy macrophages invading alveolar spaces. Animals of both cohorts mounted robust T cell responses in lungs and spleen with lower absolute counts of CD4 and CD8 T cells in lungs of WildR mice during acute infection, corresponding with observed differences in pathogen load. In summary, LabC mice and WildR mice showed largely overlapping TB immunopathology and pathogen kinetics, with WildR mice controlling early acute infection better than LabC mice.

Polypharmacological repurposing approach identifies approved drugs as potential inhibitors of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tb), the causative pathogen of tuberculosis (TB) remains the leading cause of death from single infectious agent. Furthermore, its evolution to multi-drug resistant (MDR) and extremely drug-resistant (XDR) strains necessitate de novo identification of drug-targets/candidates or to repurpose existing drugs against known targets through drug repurposing. Repurposing of drugs has gained traction recently where orphan drugs are exploited for new indications. In the current study, we have combined drug repurposing with polypharmacological targeting approach to modulate structure-function of multiple proteins in M. tb. Based on previously established essentiality of genes in M. tb, four proteins implicated in acceleration of protein folding (PpiB), chaperone assisted protein folding (MoxR1), microbial replication (RipA) and host immune modulation (S-adenosyl dependent methyltransferase, sMTase) were selected. Genetic diversity analyses in target proteins showed accumulation of mutations outside respective substrate/drug binding sites. Using a composite receptor-template based screening method followed by molecular dynamics simulations, we have identified potential candidates from FDA approved drugs database; Anidulafungin (anti-fungal), Azilsartan (anti-hypertensive) and Degarelix (anti-cancer). Isothermal titration calorimetric analyses showed that the drugs can bind with high affinity to target proteins and interfere with known protein-protein interaction of MoxR1 and RipA. Cell based inhibitory assays of these drugs against M. tb (H37Ra) culture indicates their potential to interfere with pathogen growth and replication. Topographic assessment of drug-treated bacteria showed induction of morphological aberrations in M. tb. The approved candidates may also serve as scaffolds for optimization to future anti-mycobacterial agents which can target MDR strains of M. tb.

ResR/McdR-regulated protein translation machinery contributes to drug resilience in Mycobacterium tuberculosis

Survival response of the human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb) to a diverse environmental cues is governed through its versatile transcription regulatory mechanisms with the help of a large pool of transcription regulators (TRs). Rv1830 is one such conserved TR, which remains uncharacterized in Mtb. It was named as McdR based on an effect on cell division upon its overexpression in Mycobacterium smegmatis. Recently, it has been implicated in antibiotic resilience in Mtb and reannotated as ResR. While Rv1830 affects cell division by modulating the expression of M. smegmatis whiB2, the underlying cause of its essentiality and regulation of drug resilience in Mtb is yet to be deciphered. Here we show that ResR/McdR, encoded by ERDMAN_2020 in virulent Mtb Erdman, is pivotal for bacterial proliferation and crucial metabolic activities. Importantly, ResR/McdR directly regulates ribosomal gene expression and protein synthesis, requiring distinct disordered N-terminal sequence. Compared to control, bacteria depleted with resR/mcdR exhibit delayed recovery post-antibiotic treatment. A similar effect upon knockdown of rplN operon genes further implicates ResR/McdR-regulated protein translation machinery in attributing drug resilience in Mtb. Overall, findings from this study suggest that chemical inhibitors of ResR/McdR may be proven effective as adjunctive therapy for shortening the duration of TB treatment.

SecretoMyc, a web-based database on mycobacteria secreted proteins and structure-based homology identification using bio-informatics tools

To better understand the interaction between the host and the Mycobacterium tuberculosis pathogen, it is critical to identify its potential secreted proteins. While various experimental methods have been successful in identifying proteins under specific culture conditions, they have not provided a comprehensive characterisation of the secreted proteome. We utilized a combination of bioinformatics servers and in-house software to identify all potentially secreted proteins from six mycobacterial genomes through the three secretion systems: SEC, TAT, and T7SS. The results are presented in a database that can be crossed referenced to selected proteomics and transcriptomics studies (https://secretomyc.cbs.cnrs.fr). In addition, thanks to the recent availability of Alphafold models, we developed a tool in order to identify the structural homologues among the mycobacterial genomes.