Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.

Targeting Mycobacterium tuberculosis pH-driven adaptation.

Mycobacterium tuberculosis (Mtb) senses and adapts to host environmental cues as part of its pathogenesis. One important cue sensed by Mtb is the acidic pH of its host niche - the macrophage. Acidic pH induces widespread transcriptional and metabolic remodelling in Mtb. These adaptations to acidic pH can lead Mtb to slow its growth and promote pathogenesis and antibiotic tolerance. Mutants defective in pH-dependent adaptations exhibit reduced virulence in macrophages and animal infection models, suggesting that chemically targeting these pH-dependent pathways may have therapeutic potential. In this review, we discuss mechanisms by which Mtb regulates its growth and metabolism at acidic pH. Additionally, we consider the therapeutic potential of disrupting pH-driven adaptations in Mtb and review the growing class of compounds that exhibit pH-dependent activity or target pathways important for adaptation to acidic pH.

Analysis of the components of Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) and its regulation of γδ T-cell function.

BACKGROUND: Mycobacterium tuberculosis heat-resistant antigen (Mtb-HAg) is a peptide antigen released from the mycobacterial cytoplasm into the supernatant of Mycobacterium tuberculosis (Mtb) attenuated H37Ra strain after autoclaving at 121 °C for 20 min. Mtb-HAg can specifically induce γδ T-cell proliferation in vitro. However, the exact composition of Mtb-HAg and the protein antigens that are responsible for its function are currently unknown. METHODS: Mtb-HAg extracted from the Mtb H37Ra strain was subjected to LC‒MS mass spectrometry. Twelve of the identified protein fractions were recombinantly expressed in Escherichia coli by genetic engineering technology using pET-28a as a plasmid and purified by Ni-NTA agarose resin to stimulate peripheral blood mononuclear cells (PBMCs) from different healthy individuals. The proliferation of γδ T cells and major γδ T-cell subset types as well as the production of TNF-α and IFN-γ were determined by flow cytometry. Their proliferating γδ T cells were isolated and purified using MACS separation columns, and Mtb H37Ra-infected THP-1 was co-cultured with isolated and purified γδ T cells to quantify Mycobacterium viability by counting CFUs. RESULTS: In this study, Mtb-HAg from the attenuated Mtb H37Ra strain was analysed by LC‒MS mass spectrometry, and a total of 564 proteins were identified. Analysis of the identified protein fractions revealed that the major protein components included heat shock proteins and Mtb-specific antigenic proteins. Recombinant expression of 10 of these proteins in by Escherichia coli genetic engineering technology was used to successfully stimulate PBMCs from different healthy individuals, but 2 of the proteins, EsxJ and EsxA, were not expressed. Flow cytometry results showed that, compared with the IL-2 control, HspX, GroEL1, and GroES specifically induced γδ T-cell expansion, with Vγ2δ2 T cells as the main subset, and the secretion of the antimicrobial cytokines TNF-α and IFN-γ. In contrast, HtpG, DnaK, GroEL2, HbhA, Mpt63, EsxB, and EsxN were unable to promote γδ T-cell proliferation and the secretion of TNF-α and IFN-γ. None of the above recombinant proteins were able to induce the secretion of TNF-α and IFN-γ by αß T cells. In addition, TNF-α, IFN-γ-producing γδ T cells inhibit the growth of intracellular Mtb. CONCLUSION: Activated γδ T cells induced by Mtb-HAg components HspX, GroES, GroEL1 to produce TNF-α, IFN-γ modulate macrophages to inhibit intracellular Mtb growth. These data lay the foundation for subsequent studies on the mechanism by which Mtb-HAg induces γδ T-cell proliferation in vitro, as well as the development of preventive and therapeutic vaccines and rapid diagnostic reagents.

MicroRNA-144-3p Inhibits Host Lipid Catabolism and Autophagy by Targeting PPARα and ABCA1 During Mycobacterium Tuberculosis Infection.

MicroRNA-mediated metabolic reprogramming recently has been identified as an important strategy for Mycobacterium tuberculosis (Mtb) to evade host immune responses. However, it is unknown what role microRNA-144-3p (miR-144-3p) plays in cellular metabolism during Mtb infection. Here, we report the meaning of miR-144-3p-mediated lipid accumulation for Mtb-macrophage interplay. Mtb infection was shown to upregulate the expression of miR-144-3p in macrophages. By targeting peroxisome proliferator-activated receptor α (PPARα) and ATP-binding cassette transporter A1 (ABCA1), miR-144-3p overexpression promoted lipid accumulation and bacterial survival in Mtb-infected macrophages, while miR-144-3p inhibition had the opposite effect. Furthermore, reprogramming of host lipid metabolism by miR-144-3p suppressed autophagy in response to Mtb infection. Our findings uncover that miR-144-3p regulates host metabolism and immune responses to Mtb by targeting PPARα and ABCA1, suggesting a potential host-directed tuberculosis therapy by targeting the interface of miRNA and lipid metabolism.

[Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis]. / ç»“æ ¸åˆ†æžæ†èŒRv3432cè›‹ç™½çš„åŽŸæ ¸è¡¨è¾¾ä¸Žç”Ÿç‰©ä¿¡æ¯å­¦åˆ†æž.

Objective: To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb. Methods: The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL. Results: pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured. Conclusion: We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Synchronization of Mycobacterium life cycle: A possible novel mechanism of antimycobacterial drug resistance evolution and its manipulation.

Mycobacterium Tuberculosis (Mtb) causing Tuberculosis (TB) is a widespread disease infecting millions of people worldwide. Additionally, emergence of drug resistant tuberculosis is a major challenge and concern in high TB burden countries. Most of the drug resistance in mycobacteria is attributed to developing acquired resistance due to spontaneous mutations or intrinsic resistance mechanisms. In this review, we emphasize on the role of bacterial cell cycle synchronization as one of the intrinsic mechanisms used by the bacteria to cope with stress response and perhaps involved in evolution of its drug resistance. The importance of cell cycle synchronization and its function in drug resistance in cancer cells, malarial and viral pathogens is well understood, but its role in bacterial pathogens has yet to be established. From the extensive literature survey, we could collect information regarding how mycobacteria use synchronization to overcome the stress response. Additionally, it has been observed that most of the microbial pathogens including mycobacteria are responsive to drugs predominantly in their logarithmic phase, while they show resistance to antibiotics when they are in the lag or stationary phase. Therefore, we speculate that Mtb might use this novel strategy wherein they regulate their cell cycle upon antibiotic pressure such that they either enter in their low metabolic phase i.e., either the lag or stationary phase to overcome the antibiotic pressure and function as persister cells. Thus, we propose that manipulating the mycobacterial drug resistance could be possible by fine-tuning its cell cycle.

Tuberculosis and its clinical consequences on Women's health.

Mycobacterium tuberculosis causes tuberculosis, a fatal infection resulting in widespread illness and death. In 2020, approximately 10 million people were diagnosed with tuberculosis. The top 30 tuberculosis-endemic countries accounted for 86% of all estimated occurrence cases worldwide. In this context, eight of these accounted for two-thirds of the global total, with India having a prevalence of 26%. Aside from lung inflammation, the risk factors for tuberculosis in women include extra-pulmonary infection, particularly genital tuberculosis, tuberculous mastitis, and tuberculous in the peritoneum, intestine, and spine. Depending on the epidemiologic context and screening methods, different tuberculosis symptoms and disease diagnoses are more or less common among expectant mothers. The disease is almost certainly going to have a global impact. The social stigma and anxiety associated with tuberculosis may have a much more significant negative impact on women's health behaviors than men. Notably, the abdominal sites of miliary tuberculosis could mimic tumor likely, carcinoma and lymphoma. Also, the results of the diagnostic accuracy tests for the condition demonstrate that extra-pulmonary tuberculosis can be quickly and accurately diagnosed in various sites using both the T-SPOT assay and the GeneXpert/PCR test. Therefore, this review exemplified the prevalence of extra-pulmonary tuberculosis at various points in women's lives. On the contrary, it also illustrated the symptoms and dangers of TB as they relate to women's health.

Lipoarabinomannan mediates localized cell wall integrity during division in mycobacteria.

The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. The phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM) are cell envelope components critical for host-pathogen interactions, but their physiological functions in mycobacteria remained elusive. In this work, using biosynthetic mutants of these lipoglycans, we examine their roles in maintaining cell envelope integrity in Mycobacterium smegmatis and Mycobacterium tuberculosis. We find that mutants defective in producing mature LAM fail to maintain rod cell shape specifically at the new pole and para-septal regions whereas a mutant that produces a larger LAM becomes multi-septated. Therefore, LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell wall integrity and septal placement.

Indole Propionic Acid Disturbs the Normal Function of Tryptophanyl-tRNA Synthetase in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.

Iron-related gene mutations driving global Mycobacterium tuberculosis transmission revealed by whole-genome sequencing.

BACKGROUND: Iron plays a crucial role in the growth of Mycobacterium tuberculosis (M. tuberculosis). However, the precise regulatory mechanism governing this system requires further elucidation. Additionally, limited studies have examined the impact of gene mutations related to iron on the transmission of M. tuberculosis globally. This research aims to investigate the correlation between mutations in iron-related genes and the worldwide transmission of M. tuberculosis. RESULTS: A total of 13,532 isolates of M. tuberculosis were included in this study. Among them, 6,104 (45.11%) were identified as genomic clustered isolates, while 8,395 (62.04%) were classified as genomic clade isolates. Our results showed that a total of 12 single nucleotide polymorphisms (SNPs) showed a positive correlation with clustering, such as Rv1469 (ctpD, C758T), Rv3703c (etgB, G1122T), and Rv3743c (ctpJ, G676C). Additionally, seven SNPs, including Rv0104 (T167G, T478G), Rv0211 (pckA, A302C), Rv0283 (eccB3, C423T), Rv1436 (gap, G654T), ctpD C758T, and etgB C578A, demonstrated a positive correlation with transmission clades across different countries. Notably, our findings highlighted the positive association of Rv0104 T167G, pckA A302C, eccB3 C423T, ctpD C758T, and etgB C578A with transmission clades across diverse regions. Furthermore, our analysis identified 78 SNPs that exhibited significant associations with clade size. CONCLUSIONS: Our study reveals the link between iron-related gene SNPs and M. tuberculosis transmission, offering insights into crucial factors influencing the pathogenicity of the disease. This research holds promise for targeted strategies in prevention and treatment, advancing research and interventions in this field.

P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum.

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-ß secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-ß levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-ß to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-ß from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-ß response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble Mycobacterium tuberculosis. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with M. tuberculosis in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, morbidity, and mortality. Although a high-resolution LC-MS/MS-based multiprotein panel assay can be developed due to its specificity and sensitivity, it needs a library of species-specific peptides as a platform. Toward this, we performed an analysis of proteomes of 9 NTM species with more than 20 million peptide spectrum matches gathered from 26 proteome data sets. Our metaproteomic analyses determined 48,172 species-specific proteotypic peptides across 9 NTMs. Notably, M. smegmatis (26,008), M. abscessus (12,442), M. vaccae (6487), M. fortuitum (1623), M. avium subsp. paratuberculosis (844), M. avium subsp. hominissuis (580), and M. marinum (112) displayed >100 species-specific proteotypic peptides. Finally, these peptides and corresponding spectra have been compiled into a spectral library, FASTA, and JSON formats for future reference and validation in clinical cohorts by the biomedical community for further translation.

Mycobacterium tuberculosis-THP-1 like macrophages protein-protein interaction map revealed through dual RNA-seq analysis and a computational approach.

Introduction. Infection caused by Mycobacterium tuberculosis (M. tb) is still a leading cause of mortality worldwide with estimated 1.4 million deaths annually.Hypothesis/Gap statement. Despite macrophages' ability to kill bacterium, M. tb can grow inside these innate immune cells and the exploration of the infection has traditionally been characterized by a one-sided relationship, concentrating solely on the host or examining the pathogen in isolation.Aim. Because of only a handful of M. tb-host interactions have been experimentally characterized, our main goal is to predict protein-protein interactions during the early phases of the infection.Methodology. In this work, we performed an integrative computational approach that exploits differentially expressed genes obtained from Dual RNA-seq analysis combined with known domain-domain interactions.Results. A total of 2381 and 7214 genes were identified as differentially expressed in M. tb and in THP-1-like macrophages, respectively, revealing different transcriptional profiles in response to infection. Over 48 h of infection, the host-pathogen network revealed 25 016 PPIs. Analysis of the resulting predicted network based on cellular localization information of M. tb proteins, indicated the implication of interacting nodes including the bacterial PE/PPE/PE_PGRS family. In addition, M. tb proteins interacted with host proteins involved in NF-kB signalling pathway as well as interfering with the host apoptosis ability via the potential interaction of M. tb TB16.3 with human TAB1 and M. tb GroEL2 with host protein kinase C delta, respectively.Conclusion. The prediction of the full range of interactions between M. tb and host will contribute to better understanding of the pathogenesis of this bacterium and may provide advanced approaches to explore new therapeutic targets against tuberculosis.

Deciphering the molecular mechanism and regulation of formaldehyde detoxification in Mycobacterium smegmatis.

The build-up of formaldehyde, a highly reactive molecule is cytotoxic and must be eliminated for the organism's survival. Formaldehyde detoxification system is found in nearly all organisms including both pathogenic and non-pathogenic mycobacteria. MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis (Msm), is an indispensable part of this system and forms a bicistronic operon with its downstream uncharacterized gene, fmh. We here show that Fmh, a putative metallo-beta-lactamase, is essential in tolerating higher amounts of formaldehyde when co-overexpressed with mscR in vivo. Our NMR studies indicate that MscR, along with Fmh, enhances formate production through a mycothiol (MSH)-dependent pathway, emphasizing the importance of Fmh in detoxifying formaldehyde. Although another aldehyde dehydrogenase, MSMEG_1543, induces upon formaldehyde addition, it is not involved in its detoxification. We also show that the expression of the mscR operon is constitutive and remains unchanged upon formaldehyde addition, as displayed by the promoter activity of PmscR and by the transcript and protein levels of MscR. Furthermore, we establish the role of a thiol-responsive sigma factor SigH in formaldehyde detoxification. We show that SigH, and not SigE, is crucial for formaldehyde detoxification, even though it does not directly regulate mscR operon expression. In addition, sensitivity to formaldehyde in sigH-knockout could be alleviated by overexpression of mscR. Taken together, our data demonstrate the importance of MSH-dependent pathways in detoxifying formaldehyde in a mycobacterial system. An absence of such MSH-dependent proteins in eukaryotes and its complete conservation in M. tuberculosis, the causative agent of tuberculosis, further unravel new drug targets for this pathogen.IMPORTANCEExtensive research has been done on formaldehyde detoxification in different bacteria. However, our current understanding of the mechanisms underlying this process in mycobacteria remains exceedingly little. We previously showed that MscR, a formaldehyde dehydrogenase from Mycobacterium smegmatis, plays a pivotal role in this detoxification pathway. Here, we present a potential S-formyl-mycothiol hydrolase named Fmh, thought to be a metallo-beta-lactamase, which functions along with mycothiol (MSH) and MscR to enhance formate production within this detoxification pathway. Co-expression of Fmh with MscR significantly enhances the efficiency of formaldehyde detoxification in M. smegmatis. Our experiments establish that Fmh catalyzes the final step of this detoxification pathway. Although an alternative sigma factor SigH was found to be involved in formaldehyde detoxification, it did not directly regulate the expression of mscR. Since formaldehyde detoxification is essential for bacterial survival, we envisage this process to be a potential drug target for M. tuberculosis eradication.

BACH1 promotes tissue necrosis and Mycobacterium tuberculosis susceptibility.

Oxidative stress triggers ferroptosis, a form of cellular necrosis characterized by iron-dependent lipid peroxidation, and has been implicated in Mycobacterium tuberculosis (Mtb) pathogenesis. We investigated whether Bach1, a transcription factor that represses multiple antioxidant genes, regulates host resistance to Mtb. We found that BACH1 expression is associated clinically with active pulmonary tuberculosis. Bach1 deletion in Mtb-infected mice increased glutathione levels and Gpx4 expression that inhibit lipid peroxidation. Bach1-/- macrophages exhibited increased resistance to Mtb-induced cell death, while Mtb-infected Bach1-deficient mice displayed reduced bacterial loads, pulmonary necrosis and lipid peroxidation concurrent with increased survival. Single-cell RNA-seq analysis of lungs from Mtb-infected Bach1-/- mice revealed an enrichment of genes associated with ferroptosis suppression. Bach1 depletion in Mtb-infected B6.Sst1S mice that display human-like necrotic lung pathology also markedly reduced necrosis and increased host resistance. These findings identify Bach1 as a key regulator of cellular and tissue necrosis and host resistance in Mtb infection.

Mycobacterial PE12 protein promotes bacterial survival through inhibiting cell apoptosis.

Mycobacterial PE_PGRS family proteins play key roles in pathogen-host interaction. However, the function of most PE_PGRS proteins remains unknown. In this study, we characterized the role of PE12 of Mycobacterium bovis (M. bovis) on bacterial growth, bacterial survival, and host cell apoptosis. Transcriptome sequencing of infected THP-1 cells was also performed. Compared to Ms_Vec, we found that M. bovis PE12 did not alter the colony morphology of M. smegmatis. The survival of Ms_PE12 was obviously higher than that of Ms_Vec. Furthermore, PE12 significantly suppressed the apoptosis of THP-1 induced by M. smegmatis infection. Transcriptome analysis results showed that there were 70 downregulated genes in the Ms_PE12 infection group in comparison with the Ms_Vec infection group, and these differentially expressed genes were enriched in 240 downregulated GO terms and 6 KEGG pathways. The downregulated expression genes are involved in cell adhesion, phagocytosis, apoptosis, inflammatory response, glycolysis and transmembrane transporter activity. Taken together, our study reveals that PE12 can suppress apoptosis and inhibit proinflammatory cytokine response. We propose that PE12 is related to macrophage phagocytosis and apoptosis, providing useful information to the pathogenic mechanisms of M. bovis.

Understanding the structural basis of the binding specificity of c-di-AMP to M. smegmatis RecA using computational biology approach.

Mycobacterium tuberculosis RecA (MtRecA), a protein involved in DNA repair, homologous recombination and SOS pathway, contributes to the development of multidrug resistance. ATP binding-site in RecA has been a drug target to disable RecA dependent DNA repair. For the first time, experiments have shown the existence and binding of c-di-AMP to a novel allosteric site in the C-terminal-Domain (CTD) of Mycobacterium smegmatis RecA (MsRecA), a close homolog of MtRecA. In addition, it was observed that the c-di-AMP was not binding to Escherichia coli RecA (EcRecA). This article analyses the possible interactions of the three RecA homologs with the various c-di-AMP conformations to gain insights into the structural basis of the natural preference of c-di-AMP to MsRecA and not to EcRecA, using the structural biology tools. The comparative analysis, based on amino acid composition, homology, motifs, residue types, docking, molecular dynamics simulations and binding free energy calculations, indeed, conclusively indicates strong binding of c-di-AMP to MsRecA. Having very similar results as MsRecA, it is highly plausible for c-di-AMP to strongly bind MtRecA as well. These insights from the in-silico studies adds a new therapeutic approach against TB through design and development of novel allosteric inhibitors for the first time against MtRecA.Communicated by Ramaswamy H. Sarma.