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Abstract Background Tuberculosis currently stands as the second leading cause of deaths worldwide due to single infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. Results We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at − 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. Conclusions The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Abstract Background Latent tuberculosis infection is attributed in part to the existence of Mycobacterium tuberculosis in a persistent non-replicating dormant state that is associated with tolerance to host defence mechanisms and antibiotics. We have recently reported that vitamin C treatment of M. tuberculosis triggers the rapid development of bacterial dormancy. Temporal genome-wide transcriptome analysis has revealed that vitamin C-induced dormancy is associated with a large-scale modulation of gene expression in M. tuberculosis. Results An updated transcriptional regulatory network of M.tuberculosis (Mtb-TRN) consisting of 178 regulators and 3432 target genes was constructed. The temporal transcriptome data generated in response to vitamin C was overlaid on the Mtb-TRN (vitamin C Mtb-TRN) to derive insights into the transcriptional regulatory features in vitamin C-adapted bacteria. Statistical analysis using Fisher’s exact test predicted that 56 regulators play a central role in modulating genes which are involved in growth, respiration, metabolism and repair functions. Rv0348, DevR, MprA and RegX3 participate in a core temporal regulatory response during 0.25 h to 8 h of vitamin C treatment. Temporal network analysis further revealed Rv0348 to be the most prominent hub regulator with maximum interactions in the vitamin C Mtb-TRN. Experimental analysis revealed that Rv0348 and DevR proteins interact with each other, and this interaction results in an enhanced binding of DevR to its target promoter. These findings, together with the enhanced expression of devR and Rv0348 transcriptional regulators, indicate a second-level regulation of target genes through transcription factor- transcription factor interactions. Conclusions Temporal regulatory analysis of the vitamin C Mtb-TRN revealed that there is involvement of multiple regulators during bacterial adaptation to dormancy. Our findings suggest that Rv0348 is a prominent hub regulator in the vitamin C model and large-scale modulation of gene expression is achieved through interactions of Rv0348 with other transcriptional regulators.

Background Near-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance. Methods We recently developed the “TB Concentration & Transport” kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the “TB DNA Extraction” kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing. In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain's line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207). Results Trans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52–98%), 77.7% for isoniazid (95% CI 52–94%), 85.7% for fluoroquinolones (95% CI 42–100%) and 66.6% for aminoglycosides (95% CI 9–99%), with a specificity range of 93.7% (95% CI 87–97) to 99.1% (95% CI 95–100) using phenotypic drug susceptibility testing (DST) as a reference standard. A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83–1.0)). Conclusions This study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective “Universal DST” services to TB subjects residing in remote areas.

Abstract Background A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. Results Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. Conclusions It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely 'TB Detect' (containing BioFM-Filter device), 'TB Concentration and Transport' (containing Trans-Filter device) and 'TB DNA Extraction' kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing.

DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be 'pre-adapted' to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under 'non-inducing' conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the mechanism of DevR overexpression-mediated induction of the regulon in the absence of the normal environmental cue and establishes the α10 helix as an universal and pivotal targeting interface for DevR inhibitor development.

Objective/Background: Bacterial persistence is the hallmark of tuberculosis (TB) and poses the biggest threat to the success of any antitubercular drug regimen. The DevR/DosR dormancy regulator of Mycobacterium tuberculosis belongs to the NarL subfamily of response regulators and is essential for M. tuberculosis persistence in macaque models of TB. The DevR/DosR crystal structure revealed a unique (αβ)4 topology instead of the classical (αβ)5 structure found in the receiver domain of other regulators in this subfamily. It was proposed that phosphorylation may culminate in the formation of a DNA-binding-competent dimeric species via α10–α10 helix interactions. Here, we deciphered the role of the α10 helix in activation of the DevR/DosR response regulator in M. tuberculosis. Methods: Wild-type (WT) and mutant DevR [α10-helix-deleted DevR (DevRΔα10)] proteins were cloned in suitable plasmids and expressed in Escherichia coli and M. tuberculosis strains. An in vitro phosphorylation assay was performed using acetyl phosphate, and the dimeric/oligomeric status of WT DevR and mutant proteins in the presence or absence of phosphorylation was assessed by glutaraldehyde-based in vitro cross-linking, followed by western blot analysis. Additionally, recombinant M. tuberculosis strains expressing WT and mutant DevR proteins were assessed for dormancy regulon gene expression under aerobic and hypoxic conditions by western blot analysis. An electrophoretic mobility shift assay was performed to assess the in vitro DNA-binding activity of DevR proteins to the target DNA, and biophysical characterization was performed using circular dichroism spectroscopy, fluorescence spectroscopy, and thermal shift assays. Results: Our results revealed that DevR structure and activity are modulated by phosphorylation-dependent α10 helix dimerization. In its hyperphosphorylated state, DevRΔα10 is defective in DNA binding and exhibits an open and less stable conformation. The combined results of in vitro cross-linking and genetic analysis established an essential role for the α10 helix in postphosphorylation dimerization of DevR and gene activation. The importance of the α10 helix for dormancy regulon induction in M. tuberculosis established the α10–α10 helix interaction as a novel target in the DevR-signaling pathway for developing inhibitors against DevR, a key regulator of hypoxia-triggered dormancy. Conclusion: This study established the importance of the α10 helix for DevR activation in M. tuberculosis and proposed a novel molecular tool to screen small-molecule inhibitors targeting dimerization of DevR in the absence (inactive state) or presence of phosphorylation (active state) to combat latent TB infection. This concept can be extended to screen inhibitors against response regulators where dimerization is crucial for their activation.

DevR activates the transcription of ~48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in Mycobacterium tuberculosis, tgs1 and Rv3131 encode triacyl-glycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent tgs1-Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at -42.5 and -63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate tgs1-Rv3131 transcription by ~210- and ~ll0-fold, respectively. The presence of a single P site significantly reduced activation of tgs1 expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished tgs1 but not Rv3131 transcription despite DevR occupancy at both sites. The lack of tgs1 expression is most likely due to disruption of its -35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and -35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of tgs1-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes. doi: 10.1128/JB.00310-09

Under hypoxic conditions or upon exposure to low concentrations of nitric oxide, DevR transcriptional regulator mediates the activation of ~50 genes that are believed to assist in dormancy development in Mycobacterium tuberculosis. Most of the strongly induced genes are characterized by the presence of one to four copies of a Dev box-like sequence at an upstream location. Among these are several gene pairs that are transcribed in opposite directions. This arrangement could provide for coordinated control of the adjacent genes under inducing conditions. In this work, we report the first detailed analysis of DevR-mediated hypoxic regulation of narK2-Rv1738 genes that are oppositely oriented in M. tuberculosis. Phosphorylated DevR interacts with intergenic sequences and protects ~80 bp of DNA that contains three binding sites, designated Dev boxes D1, D2, and D3. The hypoxia-specific transcription start points of narK2 and Rv1738 were mapped, and it was noted that the-35 elements of both promoters overlapped with the proximally placed Dev box. DevR bound cooperatively to adjacently placed D2 and D3 boxes while binding to D1 was independent of DevR interaction with the D2 and D3 boxes. Mutational analysis and green fluorescent protein reporter assays established that the three Dev boxes function synergistically to mediate maximal transcriptional induction of both narK2 and Rv1738 in hypoxic cultures of M. tuberculosis. Analysis of narK2 promoter activity indicates that it is under negative regulation in addition to DevR-mediated positive regulation and also reveals differences between M. tuberculosis and Mycobacterium bovis BCG.

The DevR-DevS two-component system of Mycobacterium tuberculosis mediates bacterial adaptation to hypoxia, a condition believed to be associated with the initiation and maintenance of dormant bacilli during latent tuberculosis. The activity of the Rv3134c-devRS operon was studied in M. tuberculosis using several transcriptional fusions comprised of promoter regions and the gfp reporter gene under inducing and aerobic conditions. Aerobic transcription was DevR independent, while hypoxic induction was completely DevR dependent. The hypoxia transcriptional start point, [T.sub.H, was mapped at -40 bp upstream of Rv3134c. In contrast, the divergently transcribed Rv3135 gene was not induced under hypoxic conditions. DNase I footprinting and mutational analyses demonstrated that induction required the interaction of DevR~P with binding sites centered at bp -42.5 and -63.5 relative to [T.sub.H]. Binding to the distal site (D) was necessary to recruit another molecule of DevR~P to the proximal site (P), and interaction with both sequences was essential for promoter activation. These sites did not bind to either unphosphorylated or phosphorylation-defective DevR protein, which was consistent with an essential role for DevR~P in activation. Phosphorylated DevR also bound to three copies of the motif at the hspX promoter. The Rv3134c and hspX promoters have a similar architecture, wherein the proximal DevR~P binding site overlaps with the promoter -35 element. A model for the likely mode of action of DevR at these promoters is discussed.