Since 2019, there has been a growing focus on mRNA vaccines for infectious disease prevention, particularly following the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). mRNA vaccines offer advantages such as rapid production and the ability to induce robust cellular and antibody responses, which are essential for combating infections that require cell-mediated immunity, including Tuberculosis (TB). This review explores recent progress in TB mRNA vaccines and addresses several key areas: (1) the urgent need for new TB vaccines; (2) current advancements in TB vaccine development, and the advantages and challenges of mRNA technology; (3) the design and characteristics of TB mRNA vaccines; (4) the immunological mechanisms of TB mRNA vaccines; (5) manufacturing processes for TB mRNA vaccines; and (6) safety and regulatory considerations. This interdisciplinary review aims to provide insights for researchers working to address critical questions in TB mRNA vaccine development.

Despite being a preventable and treatable disease, tuberculosis (TB) remained the second leading infectious cause of death globally in 2022, surpassed only by COVID-19. The death rate from TB is influenced by numerous factors that include antibiotic drug resistance, noncompliance with chemotherapy by patients, concurrent infection with the human immunodeficiency virus, delayed diagnosis, varying effectiveness of the Bacille-Calmette-Guerin vaccine, and other factors. Even with the recent advances in our knowledge of Mycobacterium tuberculosis and the accessibility of advanced genomic tools such as proteomics and microarrays, alongside modern methodologies, the pursuit of next-generation inhibitors targeting distinct or multiple molecular pathways remains essential to combat the increasing antimicrobial resistance. Hence, there is an urgent need to identify and develop new drug targets against TB that have unique mechanisms. Novel therapeutic targets might encompass gene products associated with various aspects of mycobacterial biology, such as transcription, metabolism, cell wall formation, persistence, and pathogenesis. This review focuses on the present state of our knowledge and comprehension regarding various inhibitors targeting key metabolic pathways of M. tuberculosis. The discussion encompasses small molecule, synthetic, peptide, natural product and microbial inhibitors and navigates through promising candidates in different phases of clinical development. Additionally, we explore the crucial enzymes and targets involved in metabolic pathways, highlighting their inhibitors. The metabolic pathways explored include nucleotide synthesis, mycolic acid synthesis, peptidoglycan biosynthesis, and energy metabolism. Furthermore, advancements in genetic approaches like CRISPRi and conditional expression systems are discussed, focusing on their role in elucidating gene essentiality and vulnerability in Mycobacteria.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the most ancient diseases recorded. In cases of bone TB, it significantly disrupts bone homeostasis, though the precise mechanisms are poorly understood and effective treatment targets are scarce. Our study investigated the role of Rv1509 in the pathogenesis of bone TB. We found that Rv1509 enhances the differentiation of bone marrow macrophages (BMMs) into osteoclasts by activating the TLR2 pathway, which stimulates the production of IL-6 and TNF-α. This, in turn, indirectly inhibits osteoblast differentiation and mineralization. Additionally, Rv1509 directly impairs osteoblast function and enhances the secretion of RANKL via TLR2 signaling, creating a detrimental RANKL/OPG imbalance that promotes osteoclast differentiation and bone degradation. Notably, the injection of Rv1509 into mouse skulls led to extensive bone damage, highlighting its significant role as a virulence factor in the pathogenesis of bone TB.

Among the critical priority pathogens listed by the World Health Organization, Mycobacterium tuberculosis strains resistant to rifampicin present a significant global threat. Consequently, the study of the mechanisms of resistance to new antitubercular drugs and the discovery of new effective molecules are two crucial points in tuberculosis drug discovery. In this study, we discovered a compound named RCB18350, which is active against M. tuberculosis growth and exhibits a minimum inhibitory concentration (MIC) of 1.25 μg/mL. It was also effective against multidrug-resistant isolates. We deeply studied the mechanism of resistance/action of RCB18350 by using several approaches. We found that Rv3406, an iron- and α-ketoglutarate-dependent sulfate ester dioxygenase, is capable of metabolizing the compound into its inactive metabolite. This finding highlights the role of this enzyme in the mechanism of resistance to RCB18350.

One of the primary healthcare problems in the world today is tuberculosis (TB), a chronic infectious illness brought on by Mycobacterium tuberculosis (M. tuberculosis). A distinct family of PE_PGRS proteins, encoded by the M. tuberculosis genome, has attracted more attention because of their involvement in immune evasion and bacterial pathogenicity. Nevertheless, the specific functions and mechanisms of action for the majority of PE_PGRS proteins remain largely unexplored. This study focuses on the Rv2741 (PE_PGRS47) gene, which is exclusively present in pathogenic mycobacteria. To examine the function of Rv2741 in host-pathogen interactions, we created recombinant strains of Mycobacterium smegmatis (M. smegmatis) that expressed the M. tuberculosis Rv2741 gene. IL-1α was found to be a key mediator of host response modulation by Rv2741. Rv2741 downregulates the secretion of IL-1α and inhibits the MAPK signaling pathway, particularly the p38 and ERK1/2 pathways, thereby cooperatively inhibiting macrophage autophagy and apoptosis. Meanwhile, the decrease in IL-1α secretion directly leads to changes in the cytokine secretion pattern and a reduction in nitric oxide (NO) production. This multifaceted regulatory mechanism ultimately favors the survival of M. smegmatis in macrophages. This research significantly expands our understanding of Rv2741 function, revealing its crucial role as a multifunctional virulence factor in the immune evasion of M. tuberculosis.

Mycobacterium tuberculosis (Mtb), a leading cause of death from infection, completes its life cycle entirely in humans except for transmission through the air. To begin to understand how Mtb survives aerosolization, we mimicked liquid and atmospheric conditions experienced by Mtb before and after exhalation using a model aerosol fluid (MAF) based on the water-soluble, lipidic, and cellular constituents of necrotic tuberculosis lesions. MAF induced drug tolerance in Mtb, remodeled its transcriptome, and protected Mtb from dying in microdroplets desiccating in air. Yet survival was not passive: Mtb appeared to rely on hundreds of genes to survive conditions associated with transmission. Essential genes subserving proteostasis offered most protection. A large number of conventionally nonessential genes appeared to contribute as well, including genes encoding proteins that resemble antidesiccants. The candidate transmission survival genome of Mtb may offer opportunities to reduce transmission of tuberculosis.

Bovine tuberculosis (bTB) is a respiratory disease caused by Mycobacterium bovis, posing a significant threat to animal health and the livestock industry. Current control strategies for bTB rely on diagnostic tests and slaughter policies. However, the limitations of existing diagnostic methods, which depend on PPD antigens, necessitate the exploration of alternative antigens to enhance the accuracy and reliability of bTB diagnosis. This study aimed to identify, produce, and evaluate novel antigens for use in the intradermal skin test for bTB diagnosis. A pangenome analysis of four Mycobacterium species identified 12 unique genes specific to M. bovis SP38. Further integrated bioinformatic analysis revealed 224 genomic islands associated with virulence and pathogenesis. Among these, a highly antigenic protein, termed HP28, was selected for in vivo testing. The recombinant HP28 protein (rHP28) was expressed in E. coli and assessed for its ability to induce intradermal skin reactions in guinea pigs. The rHP28 protein elicited a skin reaction of 6.6 mm at 72 h post-injection, whereas negative controls showed no reaction. This study presents a pipeline for the selection of antigens using integrated bioinformatic analysis to identify diagnostic targets that can effectively distinguish between sensitized and non-sensitized animals, offering a promising approach for improving bTB diagnostics.

This article reports on the complete genome of Pseudomonas sp. strain Stari2, which was shown to have the ability to degrade carbon tetrachloride (CCl4) in aerobic conditions. A single circular sequence of 6,310,573 bp, a GC content of 60.3%, and 5,680 coding sequences were obtained. In silico analysis revealed an average nucleotide identity of 94.06% to its closest species.

Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (105 to 102 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

In every bacterium, nucleoid-associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions. Their central role in controlling the chromatin dynamics and transcription has been well-appreciated in several well-studied organisms. Here, we review the diversity, distribution, structure, and function of NAPs from the genus Mycobacterium. We highlight the progress made in our understanding of the effects of these proteins on various processes and in responding to environmental stimuli and stress of mycobacteria in their free-living as well as during distinctive intracellular lifestyles. We project them as potential drug targets and discuss future studies to bridge the information gap with NAPs from well-studied systems.

Introduction. The adhesin, Mycobacterium tuberculosis curli pili (MTP), assists the pathogen in attachment, invasion and disease progression. Previously, this adhesin was demonstrated to contribute to the pathogen's cell wall functions and fatty acid metabolism and affects total metabolite abundance in central carbon metabolism and fatty acid metabolism of the host. The accumulation/depletion of metabolites is reliant on the gene expression of proteins involved in the import, transport and breakdown of substrates.Gap statement. MTP has not been investigated in relation to genes involved in import/transport/breakdown of substrates.Aim. This study aimed to investigate the possible regulatory role of MTP in modulating metabolic changes of the pathogen in different microenvironments.Methods. Ribonucleic acid was harvested from bacterial broth cultures of adhesin-proficient and adhesin-deficient M. tuberculosis. These strains were also used to infect differentiated THP-1 macrophages for 4 h prior to isolation of intracellular bacteria, RNA extraction and reverse transcription real-time quantitative PCR. The expression levels of selected genes involved in fatty acid transport (lucA, mce1D, mceG, Rv2799, Rv0966c and omamB), β-oxidation (fadA5 and fadB), lactate oxidation (lldD1 and lldD2) and gluconeogenic carbon flow (pckA) were analysed by absolute quantification.Results. The gene expression levels of lucA, mce1D and pckA were significantly lower, and those of Rv2799, Rv0966c, mceG, fadA5 and lldD2 were significantly higher in the adhesin-proficient cultured bacterial strains relative to the Δmtp strain. The intracellular adhesin-proficient bacteria displayed significantly higher gene expression levels of Rv2799 and significantly lower gene expression levels of Rv0966c, fadA5, lldD1 and pckA relative to the Δmtp strain. Interestingly, during early infection, the intracellular Δmtp displayed significantly increased expression of omamB, mceG, fadB, lldD1 and lldD2 relative to the broth culture. This trend was inverted in the WT models.Conclusion. MTP are significantly associated with the regulation of genes involved in lipid transport, β-oxidation and lactate oxidation.

Single-nucleotide variants (SNVs) and polymorphisms are characteristic biomarkers in various biological contexts, including pathogen drug resistances and human diseases. Tools that lower the implementation barrier of molecular SNV detection methods would provide greater leverage of the expanding single-nucleotide polymorphism/SNV database. The oligonucleotide ligation assay (OLA) is a highly specific means for detection of known SNVs and is especially powerful when coupled with PCR. Yet, the OLA design process remains intensive, and criteria for success are uncertain. To assist in the design process, this study describes OLAgen, an open-source tool to automate development of OLAs and their coupled PCR assays. The software facilitates alignment of sequences surrounding SNVs and generates ligation probes while screening for dimerization potential. OLAgen successfully produced ligation probes that closely matched previously validated designs for HIV-1, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and KRAS, confirming its reliability and potential for clinical applications. The tool was used to generate new assays targeting Mycobacterium tuberculosis drug resistance and variants in the human JAK2, BRAF, and factor V genes, all of which demonstrated 100% sensitivity and specificity in controlled laboratory experiments. The OLAgen predicted assay designs detected mutant frequencies as low as 1% to 5% in wild-type backgrounds in proof-of-concept laboratory studies. OLAgen represents a significant advancement in accessible assay design, promoting the broader application of OLA technology in clinical and research settings.

PE_PGRS domain proteins represent a family of proteins found in pathogenic and non-pathogenic mycobacteria such as M. smegmatis. This conserved family is characterized by two distinct regions denoted as the variable PGRS domain defined by glycine-rich repeats, and a PE domain consisting of two antiparallel alpha-helices. There are many indications that PE_PGRS proteins are involved in immunopathogenesis and virulence by evading or triggering the host immune response. However, there is not yet any information on their degree of specialization or redundancy. Computational analysis and structural annotation using AlphaFold3 combined with other tools reveals an exceptionally powerful and unprecedented ability to undergo phase separation by the PGRS domain. This suggests that PGRS's glycine-rich, multivalent, low-complexity composition supports phase separation while adopting a structured conformation, contrary to the disordered nature typical of such domains. While previously never reported, the hypothesized role of PGRS in virulence indicates a novel window into the seemingly ubiquitous role of phase separation in cellular compartmentalization and molecular dynamics. This review aims to summarize the current understanding of the PE_PGRS family and its various biological roles in the context of bioinformatic analyses of some interesting representatives of M. marinum that are under control by host sterols. Based on the structural bioinformatics analysis, we discuss future approaches to uncover the mechanistic role of this intriguing family of mycobacterial proteins in both pathogenic and non-pathogenic mycobacteria.

The Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in mycobacteria plays a crucial role in pathogenesis and immune evasion. These proteins characterized by unique structures with conserved sequences. This study elucidated the specific immunological functions of MMAR_1296 from marine mycobacterium. Expressing MMAR_1296 in Mycobacterium smegmatis (M. smegmatis) led to significant alterations in bacterial morphology, as well as reduced survival of M. smegmatis under adverse in vitro conditions and within macrophages. Furthermore, transcriptome analysis of mouse macrophages indicated that natural immunity-related pathways were upregulated in the group infected with M. smegmatis recombinantly expressing MMAR_1296. Moreover, the mycobacterium Growth Inhibition Assays(MGIA)in mice demonstrated that M. smegmatis expressing MMAR_1296 exerted a significant inhibitory effect against Mycobacterium abscessus (M. abscessus) and Mycobacterium marinum (M. marinum) infections. Immunization challenge experiments in mice further confirmed its protective effects, showing a reduction in organ bacterial loads by 1 log10 value compared to the positive control group. These findings indicate that MMAR_1296 is a promising vaccine candidate for M. abscessus and M. marinum. Given that PE/PPE protein family is also a crucial component of Mycobacterium tuberculosis (M. tuberculosis) antigens, further exploration of sequence functions based on MMAR_1296 could reveal broader applications of PE/PPE proteins family for M. tuberculosis treatment. This study supported vaccine development targeting PE/PPE proteins in mycobacteria and paves the way for broader applications.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host's immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor, a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3Mt (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3Mt, as well as native GlnA3Mt, prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3Mt expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria.

Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3Mt can indeed covalently modify spermine via glutamylation. However, GlnA3Mt is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3Mt and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis.

Changes in cell shape have been shown to be an integral part of the mycobacterial life cycle; however, systematic investigations into its patterns of pleomorphic behaviour in connection with stages or conditions of growth are scarce. We have studied the complete growth cycle of Mycobacterium monacense cultures, a Non-Tuberculous Mycobacterium (NTM), in solid as well as in liquid media. We provide data showing changes in cell shape from rod to coccoid and occurrence of refractive cells ranging from Phase Grey to phase Bright (PGB) in appearance upon ageing. Changes in cell shape could be correlated to the bi-phasic nature of the growth curves for M. monacense (and the NTM Mycobacterium boenickei) as measured by the absorbance of liquid cultures while growth measured by colony-forming units (CFU) on solid media showed a uniform exponential growth. Based on the complete M. monacense genome we identified genes involved in cell morphology, and analyses of their mRNA levels revealed changes at different stages of growth. One gene, dnaK_3 (encoding a chaperone), showed significantly increased transcript levels in stationary phase cells relative to exponentially growing cells. Based on protein domain architecture, we identified that the DnaK_3 N-terminus domain is an MreB-like homolog. Endogenous overexpression of M. monacense dnaK_3 in M. monacense was unsuccessful (appears to be lethal) while exogenous overexpression in Mycobacterium marinum resulted in morphological changes with an impact on the frequency of appearance of PGB cells. However, the introduction of an anti-sense "gene" targeting the M. marinum dnaK_3 did not show significant effects. Using dnaK_3-lacZ reporter constructs we also provide data suggesting that the morphological differences could be due to differences in the regulation of dnaK_3 in the two species. Together these data suggest that, although its regulation may vary between mycobacterial species, the dnaK_3 might have a direct or indirect role in the processes influencing mycobacterial cell shape.

Understanding the mechanisms of allosteric regulation in response to second messengers is crucial for advancing basic and applied research. This study focuses on the differential allosteric regulation by the ubiquitous signaling molecule, cAMP, in the cAMP receptor protein from Escherichia coli (CRPEcoli) and from Mycobacterium tuberculosis (CRPMTB). By introducing structurally homologous mutations from allosteric hotspots previously identified in CRPEcoli into CRPMTB and examining their effects on protein solution structure, stability and function, we aimed to determine the factors contributing to their differential allosteric regulation. Our results demonstrate that the mutations did not significantly alter the overall fold, assembly and thermodynamic stability of CRPMTB, but had varying effects on cAMP binding affinity and cooperativity. Interestingly, the mutations had minimal impact on the specific binding of CRPMTB to DNA promoter sites. However, we found that cAMP primarily reduces nonspecific CRPMTB-DNA complexes and that the mutants largely lose this ability. Furthermore, our experiments revealed that CRPMTB-DNA complexes serve as a nucleation point for additional binding of CRPMTB proteins to form high-order oligomers with the DNA. Overall, our findings highlight the importance of both cAMP and DNA interactions in modulating the allosteric regulation of CRPMTB and provide insights into the differential responses of CRPEcoli and CRPMTB to cAMP.

Glutamate decarboxylase (Gad), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA), consuming a proton in the process and thereby contributing to intracellular pH homeostasis in bacteria. However, the presence and function of the Gad-dependent mechanism in mycobacteria remain largely unexplored. This study aimed to characterize Gad activity in Mycobacterium tuberculosis (Mtb). We detected Gad activity in live cells of both Mtb and Mycobacterium smegmatis (MS). Gad activity and GABA was also detected in cell lysates of Mtb and MS. The gadB gene from Mtb was cloned, expressed, and GadB protein was purified under native conditions using MS as an expression host. Initial attempts to express GadB in Escherichia coli (E. coli) resulted in the overexpressed protein being present in the insoluble fraction and was enzymatically inactive when purified under denaturing conditions. Subsequently, an acetamide-inducible expression system was employed in MS for successful overexpression and purification of recombinant GadB. 6 × His-GadB was purified using immobilized metal affinity chromatography, and its molecular weight was determined to be ~ 51.2 kDa by SDS-PAGE. The purified 6 × His-GadB enzyme was active at both neutral and acidic pH. Its activity was found to be PLP-dependent, with optimal activity at pH 7.2 and 50°C. These findings suggest that Gad is expressed in Mtb both in normal and in acidic medium, supporting the possible existence of a Gad-dependent acid resistance mechanism in Mtb.

Developing new classes of drugs that are active against infections caused by Mycobacterium tuberculosis is a priority for treating and managing this deadly disease. Here, we describe screening a small library of 20 DNA gyrase inhibitors and identifying new lead compounds. Three structurally diverse analogues were identified with minimal inhibitory concentrations of 0.125 μg/mL against both drug-susceptible and drug-resistant strains of M. tuberculosis. These lead compounds also demonstrated antitubercular activity in ex vivo studies using infected THP-1 macrophages with minimal cytotoxicity in THP-1, HeLa, and HepG2 cells (IC50 ≥ 128 μg/mL). The molecular target of the lead compounds was validated through biochemical studies of select analogues with purified M. tuberculosis gyrase and the generation of resistant mutants. The lead compounds were assessed in combination with bedaquiline and pretomanid to determine the clinical potential, and the select lead (158) demonstrated in vivo efficacy in an acute model of TB infection in mice, reducing the lung bacterial burden by approximately 3 log10 versus untreated control mice. The advancement of DNA gyrase inhibitors expands the field of innovative therapies for tuberculosis and may offer an alternative to fluoroquinolones in future therapeutic regimens.

We conducted an exploration of the 3-aminothieno[2,3-b]pyridine-2-carboxamide (TPA) series for its potential as a drug scaffold against Mycobacterium tuberculosis. Existing analogs were active against a recombinant strain of M. tuberculosis with reduced expression of the sole signal peptidase LepB, but with poor activity against the wild-type strain. Our aim was to improve potency and explore the structure-activity relationship of the series. We identified two subsets of TPA. The first subset of compounds had equipotent activity against wild-type and LepB hypomorph strains and may represent a series with a different target. The second subset of compounds had increased activity against the LepB hypomorph strain and thus appears to be pathway-specific. Among this latter set we identified 17af as a potent inhibitor (IC90 = 1.2 μM) with some cytotoxicity (IC50 = 19 μM) and which retained increased activity against the LepB hypomorph (IC90 = 0.41 μM).

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiology of M. tuberculosis (Mtb), which may enable the pathogen to withstand treatment. While antibiotic-treated Mtb has been evaluated in in vitro experiments, it is unclear if and how long-term in vivo treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affects Mtb physiologic processes differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the Mtb transcriptome in the BALB/c high-dose aerosol infection mouse model following 4 weeks of treatment with three sterilizing and three non-sterilizing antibiotics. Certain transcriptional changes were shared among most antibiotics, including decreased expression of genes associated with protein synthesis and metabolism and the induction of certain genes associated with stress responses. However, the magnitude of this shared response differed between antibiotics. Sterilizing antibiotics rifampin, pyrazinamide, and bedaquiline generated a more quiescent Mtb state than did non-sterilizing antibiotics isoniazid, ethambutol, and streptomycin, as indicated by the decreased expression of genes associated with translation, transcription, secretion of immunogenic proteins, metabolism, and cell wall synthesis. Additionally, we identified distinguishing transcriptional effects specific to each antibiotic, indicating that different mechanisms of action induce distinct patterns in response to cellular injury. In addition to elucidating the Mtb physiologic changes associated with antibiotic stress, this study demonstrates the value of SEARCH-TB as a highly granular pharmacodynamic assay that reveals antibiotic effects that are not apparent based on culture alone.

Drivers of tuberculosis (TB) transmission in India, the country estimated to carry a quarter of the world's burden, are not well studied. We conducted a genomic epidemiology study to compare epidemiological success, host factors, and drug resistance among the 4 major Mycobacterium tuberculosis (Mtb) lineages (L1-L4) circulating in Pune, India.

We performed whole-genome sequencing (WGS) of Mtb sputum culture-positive isolates from participants in two prospective cohort studies and predicted genotypic susceptibility using a validated random forest model. We compared lineage-specific phylogenetic and time-scaled metrics to assess epidemiological success.

Of the 612 isolates that met sequence quality criteria, Most were L3 (44.6%). The majority (61.1%) of multidrug-resistant isolates were L2 (P < .001) and L2 demonstrated a higher rate and more recent resistance acquisition. L4 and/or L2 demonstrated higher clustering and time-scaled haplotypic density (THD) compared to L3 and/or L1, suggesting higher epidemiological success. L4 demonstrated higher THD and clustering (odds ratio, 5.1 [95% confidence interval, 2.3-12.3]) in multivariate models controlling for host factors and resistance.

L2 shows a higher frequency of resistance, and both L2 and L4 demonstrate evidence of higher epidemiological success than L3 or L1 in Pune. Contact tracing around TB cases and heightened surveillance of TB DR in India is a public health priority.

Arginine, glutamic acid and selenocysteine based codon bias has been shown to regulate the translation of specific mRNAs for proteins that participate in stress responses, cell cycle and transcriptional regulation. Defining codon-bias in gene networks has the potential to identify other pathways under translational control. Here we have used computational methods to analyze the ORFeome of all unique human (19,711) and mouse (22,138) open-reading frames (ORFs) to characterize codon-usage and codon-bias in genes and biological processes. We show that ORFeome-wide clustering of gene-specific codon frequency data can be used to identify ontology-enriched biological processes and gene networks, with developmental and immunological programs well represented for both humans and mice. We developed codon over-use ontology mapping and hierarchical clustering to identify multi-codon bias signatures in human and mouse genes linked to signaling, development, mitochondria and metabolism, among others. The most distinct multi-codon bias signatures were identified in human genes linked to skin development and RNA metabolism, and in mouse genes linked to olfactory transduction and ribosome, highlighting species-specific pathways potentially regulated by translation. Extreme codon bias was identified in genes that included transcription factors and histone variants. We show that re-engineering extreme usage of C- or U-ending codons for aspartic acid, asparagine, histidine and tyrosine in the transcription factors CEBPB and MIER1, respectively, significantly regulates protein levels. Our study highlights that multi-codon bias signatures can be linked to specific biological pathways and that extreme codon bias with regulatory potential exists in transcription factors for immune response and development.

Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram-negative sepsis and COVID-19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin-antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome-wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery.

Macrophages are the primary targets of mycobacterial infection, which plays crucial roles both in nonspecific defence (innate immunity) as well as specific defence mechanisms (adaptive immunity) by secreting various cytokines, antimicrobial mediators and presenting antigens to T-cells. Sequencing of the mycobacterial genome revealed that 10% of its coding ability is devoted to the Pro-Glu motif-containing (PE) and Pro-Pro-Glu motif-containing (PPE) family proteins. While the function of most of the genes belonging to the PE-PPE family initially remained unannotated, recent studies have shown that many proteins of this family play critical roles in bacterial growth and cell functions, and manipulation of host immune responses, indicating their potential roles in mycobacterial virulence. In this review, we have focussed on describing the immunological importance of particularly the PE group of proteins in the context of 'virulence' determinants and outcome of tuberculosis disease. Additionally, we have discussed about the roles of these proteins on host-pathogen-interaction and how some of these genes can be targeted which may help us in designing effective anti-TB therapeutics.

While many novel candidates for tuberculosis vaccines are presently undergoing pre-clinical or clinical trials, none of them have been able to eliminate infection entirely. In this study, we engineered a potent chimeric protein vaccine candidate, Rv2299cD2D3-ESAT-6-Ag85B (REA), which induced Th1 and Th17 responses via dendritic cell maturation. REA-activated macrophages operated the killing mechanisms of Mycobacterium tuberculosis (MTB), such as phagosomal maturation and phagolysosome fusion, through the (PI3K)-p38 MAPK-Ca2+-NADPH oxidase pathway. Dendritic cells and macrophages activated by REA elicited synergistic anti-mycobacterial responses. Notably, REA-immunized mice suppressed MTB growth to undetectable levels at 16 weeks post-infection, which was supported by gross and pathologic findings and acid-fast staining of the lung tissues, and maintained antigen-specific multifunctional IFN-γ+IL-2+TNF-α CD4+ T and long-lasting T cells producing cytokines in the tissues. Our findings suggest that REA is an outstanding prime prophylactic vaccine candidate against tuberculosis.

MraY, a bacterial enzyme crucial for the synthesis of peptidoglycans, represents a promising yet underexplored target for the development of effective antibacterial agents. Nature has provided several classes of nucleoside inhibitors of MraY and scientists have modified these structures further to obtain natural product-like inhibitors of MraY. The natural products and their synthetic analogs suffer from non-optimal in vivo efficacy, and the synthetic complexity of the structures renders the synthesis and structure-activity relationship (SAR) studies of these molecules particularly challenging. In this study, we present our findings on the discovery of first-in-class 1,2,4-triazole-based MraY inhibitors that are not nucleoside-derived. A series of 1,2,4-triazole analogous were identified by a structure-activity-relationship (SAR) study using a structure-based drug design strategy. Compound 1 , with an IC 50 of 171 µM against MraY from Staphylococcus aureus (MraY SA ), was optimized to compound 12a , exhibiting an IC 50 of 25 µM. Molecular docking studies against MraY SA provided insights into these compounds' binding interactions and activity. Furthermore, screening against the ESKAPE bacterial panel was also conducted, through which we discovered compounds demonstrating broad-spectrum antibacterial activity against E. faecium , methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococci (VRE) strains and Mycobacterium tuberculosis . The novel, first-in-class non-nucleoside inhibitors of MraY highlighted in this work provide a strong proof-of-concept of how to leverage structural information of the protein to develop future antibacterial agents targeting MraY.

The global escalation in tuberculosis (TB) cases accompanied by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (M.tb) emphasizes the critical requirement for novel potent drugs. The M.tb demonstrates extraordinary adaptability, thriving in diverse conditions, and always finds itself in win-win situations regardless of whether the environment is favorable or unfavorable; no matter the magnitude of the challenge, it can endure and survive. This review aims to uncover the role of multiple stress sensors of M.tb that assist bacteria in remaining viable within the host for years against various physiological stresses offered by the host. M.tb is an exceptionally triumphant pathogen, primarily due to its adeptness in developing defense mechanisms against stressful situations. The recent advances emphasize the significance of M.tb stress sensors, including chaperones, proteases, transcription factors, riboswitches, inteins, etc., employed in responding to a spectrum of physiological stresses imposed by the host, encompassing surface stress, host immune responses, osmotic stress, oxidative and nitrosative stresses, cell envelope stress, environmental stress, reductive stress, and drug pressure. These sensors act as silent defenders orchestrating adaptive strategies, with limited comprehensive information in current literature, necessitating a focused review. The M.tb strategies utilizing these stress sensors to mitigate the impact of traumatic conditions demand attention to neutralize this pathogen effectively. Moreover, the intricacies of these stress sensors provide potential targets to design an effective TB drug using structure-based drug design against this formidable global health threat.

Background: Tuberculosis is the second largest public health threat caused by pathogens. Understanding Mycobacterium tuberculosis's transmission, virulence, and resistance profile is crucial for outbreak control. This study aimed to investigate the pangenome composition of Mycobacterium tuberculosis clinical isolates classified as L4 derived from Ecuador. Methods: We analyzed 88 clinical isolates of Mycobacterium tuberculosis by whole-genome sequencing (WGS) and bioinformatic tools for Lineage, Drug-resistance and Pangenome analysis. Results: In our analysis, we identified the dominance of the LAM lineage (44.3%). The pangenomic analysis revealed a core genome of approximately 3200 genes and a pangenome that differed in accessory and unique genes. According to the COG database, metabolism-related genes were the most representative of all partitions. However, differences were found within all lineages analyzed in the metabolic pathways described by KEGG. Isolates from Ecuador showed variations in genomic regions associated with beta-lactamase susceptibility, potentially leading to epistatic resistance to other drugs commonly used in TB treatment, warranting further investigation. Conclusions: Our findings provide valuable insights into the genetic diversity of Mycobacterium tuberculosis populations in Ecuador. These insights may be associated with increasing adaptation within host heterogeneity, variable latency periods, and reduced host damage, collectively contributing to disease spread. The application of WGS is essential to elucidating the epidemiology of TB in the country.

The inadequate efficacy of the Bacillus Calmette-Guérin (BCG) vaccine against adult pulmonary tuberculosis (TB) necessitates the development of new and effective vaccines. Human adenovirus serotype 5 (Ad5), which induces T-cell response, is a widely used viral vector. In this study, we aimed to evaluate the efficacy of a multi-antigenic recombinant Ad5 vectored vaccine and determine the optimal immunization route for enhanced immune response against Mycobacterium tuberculosis.

We constructed a multi-antigenic recombinant Ad5 vectored vaccine expressing four antigens (Ag85B-ESAT6-MPT64-Rv2660c) of M. tuberculosis (rAd-TB4), immunized with rAd-TB4 (5 × 107 infectious virus units/mouse) twice at an interval of 4 weeks starting at 10 weeks after BCG priming, and evaluated its boosting efficacy in a BCG-primed mouse model, and determined the optimal immunization route.

Compared with the BCG-only (2 × 105 colony forming units/mouse), subcutaneous injection of rAd-TB4 (1 × 107 infectious virus units/mL; two doses) elicited a T-cell response and cytokine production in lung lymphocytes and splenocytes. rAd-TB4 immunization significantly reduced bacterial loads and inflamed lung areas compared to BCG immunization (p < 0.01) and protected against the H37Rv challenge performed at 17 weeks of BCG priming. RNA sequencing of the whole blood of rAd-TB4-vaccinated mice collected pre- and, 1 and 4 weeks post-infection, identified differentially expressed genes associated with immune and inflammatory responses, especially those in the Wnt signaling pathway.

Our results indicate that rAd-TB4 immunization enhances the immune response to the vaccine boosting antigens in BCG-primed mice, making it a potential adult pulmonary TB vaccine candidate.

Tuberculosis (TB) remains a prominent global health challenge, with the World Health Organization documenting over 1 million annual fatalities. Despite the deployment of the Bacille Calmette-Guérin (BCG) vaccine and available therapeutic agents, the escalation of drug-resistant Mycobacterium tuberculosis strains underscores the pressing need for more efficacious vaccines and treatments. This review meticulously maps out the contemporary landscape of TB vaccine development, with a focus on antigen identification, clinical trial progress, and the obstacles and future trajectories in vaccine research. We spotlight innovative approaches, such as multi-antigen vaccines and mRNA technology platforms. Furthermore, the review delves into current TB therapeutics, particularly for multidrug-resistant tuberculosis (MDR-TB), exploring promising agents like bedaquiline (BDQ) and delamanid (DLM), as well as the potential of host-directed therapies. The hurdles in TB vaccine and therapeutic development encompass overcoming antigen diversity, enhancing vaccine effectiveness across diverse populations, and advancing novel vaccine platforms. Future initiatives emphasize combinatorial strategies, the development of anti-TB compounds targeting novel pathways, and personalized medicine for TB treatment and prevention. Despite notable advances, persistent challenges such as diagnostic failures and protracted treatment regimens continue to impede progress. This work aims to steer future research endeavors toward groundbreaking TB vaccines and therapeutic agents, providing crucial insights for enhancing TB prevention and treatment strategies.

It was a general belief that drug resistance in Mycobacterium tuberculosis (Mtb) was associated with lesser virulence, particularly rifampicin resistance, which is usually produced by mutations in the RNA polymerase Beta subunit (RpoB). Interestingly, this kind of bacterial mutations affect gene transcription with significant effects on bacterial physiology and metabolism, affecting also the bacterial antigenic constitution that in consequence can produce diverse immune responses and disease outcome. In the present study, we show the results of the Mtb clinical isolate A96, which is resistant to rifampicin and when used to infect BALB/c mice showed hypervirulence, apparently by rapidly polarization of the Th2 immune response through early and high production of IL-4. The 2D-PAGE analysis of the secretome of Mtb A96 showed 204 spots, and by immunoproteome, seven proteins that were differentially recognized with the sera of infected mice on day 28 were identified by LC-MS/MS. The proteins correspond to surface antigens, virulence factors, and energy metabolism enzymes. Some of them are immunodominant antigens, such as LpqH lipoprotein that induces IL-4 secretion in cell suspensions from the lung and spleen of mice infected with Mtb A96 at 28 days postinfection, suggesting that LpqH could be one of the main antigens involved in the Th2 polarization. The reduction of Mtb A96 hypervirulence in IL-4Rα-/- BALB/c mice highlights the importance of IL-4 induction and Th2 response polarization and the immunopathological response. Thus, high and rapid bias to Th2 response is a mechanism of Mtb virulence, which could be mediated by rifampicin-resistant Mtb isolates, probably by high production and secretion of specific antigens.

Tuberculosis (TB) continues to be a major cause of death worldwide despite having an effective combinatorial therapeutic regimen and vaccine. Being one of the most successful human pathogens, Mycobacterium tuberculosis retains the ability to adapt to diverse intracellular and extracellular environments encountered by it during infection, persistence, and transmission. Designing and developing new therapeutic strategies to counter the emergence of multidrug-resistant and extensively drug-resistant TB remains a major task. DNA topoisomerases make up a unique class of ubiquitous enzymes that ensure steady-state level supercoiling and solve topological problems occurring during DNA transactions in cells. They continue to be attractive targets for the discovery of novel classes of antibacterials and to develop better molecules from existing drugs by virtue of their reaction mechanism. The limited repertoire of topoisomerases in M. tuberculosis, key differences in their properties compared to topoisomerases from other bacteria, their essentiality for the pathogen's survival, and validation as candidates for drug discovery provide an opportunity to exploit them in drug discovery efforts. The present review provides insights into their organization, structure, function, and regulation to further efforts in targeting them for new inhibitor discovery. First, the structure and biochemical properties of DNA gyrase and Topoisomerase I (TopoI) of mycobacteria are described compared to the well-studied counterparts from other bacteria. Next, we provide an overview of known inhibitors of DNA gyrase and emerging novel bacterial topoisomerase inhibitors (NBTIs). We also provide an update on TopoI-specific compounds, highlighting mycobacteria-specific inhibitors.

FadD32, a fatty acyl-AMP ligase, plays an indispensable role in mycobacterial mycolic acid synthesis and is a validated target for tuberculosis (TB) drug development. The crystal structure of Mycobacterium tuberculosis (Mtb)FadD32 has laid the foundation of structure-based drug discovery against this crucial enzyme. Here, we screened the "isoxazole" scaffold containing molecules against MtbFadD32 and identified a compound 2,4-dibromo-6-[3-(trifluoromethyl)-1,2-oxazol-5-yl]phenol (M1) with specific inhibitory activity against Mtb. Kinetics experiments showed that M1 inhibits MtbFadD32 and MtbFadD28 activity. The transcriptomics response of Mtb disclosed M1-mediated regulation of mycobacterial decisive genes involved in cell wall synthesis, consequently creating unfavorable conditions for Mtb survival. Further, M1 curtails the Mtb survival in infected macrophages and reduces Mtb burden and tubercular granulomas in a chronic infection model of BALB/c mice. Our findings provide an effective chemical scaffold to inhibit MtbFadD32 with the potential to inhibit multiple MtbFadD family of enzymes for further development as a promising candidate for treating TB.

Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium abscessus (M. abscessus) are important pathogens that can cause lung diseases. Given the abundance of shared antigens between these two pathogens, evaluating the cross-immunization between Mycobacterium tuberculosis and Mycobacterium abscessus has implications for the assessment of tuberculosis vaccines based on nontuberculous mycobacteria (NTM). The whole-cell proteins of Mycobacterium abscessus were lysed via ultrasonication and then were subcutaneously injected into BALB/c mice either alone or mixed with adjuvant for three times at a 10-day interval. After the final immunization, cross-immune antigens were analysed via genomic comparison and Mycobacterium tuberculosis proteome microarrays. BALB/c mice splenic lymphocytes were stimulated with TB-PPD to assess the cross-immunity of the cellular immune response. The effect of cross-immunity on the growth of Mycobacterium tuberculosis was evaluated using a Mycobacterium tuberculosis growth inhibition assay. Despite the presence of 1,953 homologous gene clusters between Mycobacterium tuberculosis and Mycobacterium abscessus, only 302 Mycobacterium tuberculosis antigens exhibited cross-immunoreactivity after three immunizations. Compared with the PBS group, TB-PPD stimulation significantly increased the secretion of TNF-α, IL-4, and IL-6 by sensitized mouse splenic lymphocytes, and significantly affected the proliferation of IL-2+CD4 T and TNF-α+CD4 T cells in the immunized group (P < 0.05), but had no impact on IFN-γ and IFN-γ+ CD4 T cells. Furthermore, there was no significant difference in the proliferation of Mycobacterium tuberculosis between the immunized group and the PBS group in spleen cells. These data indicate that proteins from Mycobacterium abscessus are highly immunogenic in mice. However, the cross-immune response between Mycobacterium abscessus and Mycobacterium tuberculosis was inadequate to effectively inhibit the proliferation of Mycobacterium tuberculosis.

Macrophages are the primary host cell type for infection by Mycobacterium tuberculosis in vivo. Macrophages are also key immune effector cells that mediate the control of bacterial growth. However, the specific macrophage phenotypes that are required for optimal immune control of M. tuberculosis infection in vivo remain poorly defined. There are two distinct macrophage lineages in the lung, comprising embryonically derived, tissue-resident alveolar macrophages and recruited, blood monocyte-derived interstitial macrophages. Recent studies have shown that these lineages respond divergently to similar immune environments within the tuberculosis granuloma. Here, we discuss how the differing responses of macrophage lineages might affect the control or progression of tuberculosis disease. We suggest that the ability to reprogramme macrophage responses appropriately, through immunological or chemotherapeutic routes, could help to optimize vaccines and drug regimens for tuberculosis.

Tuberculosis (TB) is a chronic wasting infectious disease caused by Mycobacterium tuberculosis (MTB) or Mycobacterium bovis that can be transmitted among people and domestic animals. During the development of TB, macrophages of the innate immune system can act against MTB via autophagy and apoptosis to prevent the spread of the disease. Among the many autophagy regulatory pathways, the adenosine monophosphate (AMP)-activated protein kinase (AMPK)-mammalian rapamycin target protein (mTOR)-Unc-51-like kinase 1 (ULK1) pathway has received considerable attention. This study investigates the regulatory role of the AMPK-mTOR-ULK1 pathway in attenuating M. bovis Bacillus Calmette-Guérin (BCG)-induced autophagy and apoptosis in murine monocyte macrophages (RAW264.7). Changes in macrophage autophagy and apoptosis were analyzed using the AMPK activator AICAR and inhibitor Compound C to interfere with the AMPK-mTOR-ULK1 pathway and siRNA to silence the pathway. Consequently, BCG stimulation of macrophages significantly activated the AMPK-mTOR-ULK1 pathway while BCG-induced macrophage AMPK activation promoted macrophage autophagy and apoptosis. Activation of the AMPK-mTOR-ULK1 pathway by AICAR significantly improved autophagy occurrence in BCG-induced macrophages and increased apoptosis while Compound C with siRNA produced opposing effects by attenuating autophagy and apoptosis in BCG-induced macrophages. Thus, the AMPK-mTOR-ULK1 pathway has a dual regulatory role in BCG-induced macrophage autophagy and apoptosis and may have synergistic effects. This study analyzes the mechanism of resistance of host cells to MTB and provides a theoretical basis for new therapeutic strategies and related drug development.

Bacteria encounter various stressful conditions within a variety of dynamic environments, which they must overcome for survival. One way they achieve this is by developing phenotypic heterogeneity to introduce diversity within their population. Such distinct subpopulations can arise through endogenous fluctuations in regulatory components, wherein bacteria can express diverse phenotypes and switch between them, sometimes in a heritable and reversible manner. This switching may also lead to antigenic variation, enabling pathogenic bacteria to evade the host immune response. Therefore, phenotypic heterogeneity plays a significant role in microbial pathogenesis, immune evasion, antibiotic resistance, host niche tissue establishment, and environmental persistence. This heterogeneity can result from stochastic and responsive switches, as well as various genetic and epigenetic mechanisms. The development of phenotypic heterogeneity may create clonal populations that differ in their level of virulence, contribute to the formation of biofilms, and allow for antibiotic persistence within select morphological variants. This review delves into the current understanding of the molecular switching mechanisms underlying phenotypic heterogeneity, highlighting their roles in establishing infections caused by select bacterial pathogens.