Multiple transcription factors co-regulate the Mycobacterium tuberculosis adaptation response to vitamin C.

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.

Genome analysis identifies a spontaneous nonsense mutation in ppsD leading to attenuation of virulence in laboratory-manipulated Mycobacterium tuberculosis.

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.

Genome-wide expression profiling establishes novel modulatory roles of vitamin C in THP-1 human monocytic cell line.

BACKGROUND: Vitamin C (vit C) is an essential dietary nutrient, which is a potent antioxidant, a free radical scavenger and functions as a cofactor in many enzymatic reactions. Vit C is also considered to enhance the immune effector function of macrophages, which are regarded to be the first line of defence in response to any pathogen. The THP-1 cell line is widely used for studying macrophage functions and for analyzing host cell-pathogen interactions. RESULTS: We performed a genome-wide temporal gene expression and functional enrichment analysis of THP-1 cells treated with 100 µM of vit C, a physiologically relevant concentration of the vitamin. Modulatory effects of vitamin C on THP-1 cells were revealed by differential expression of genes starting from 8 h onwards. The number of differentially expressed genes peaked at the earliest time-point i.e. 8 h followed by temporal decline till 96 h. Further, functional enrichment analysis based on statistically stringent criteria revealed a gamut of functional responses, namely, 'Regulation of gene expression', 'Signal transduction', 'Cell cycle', 'Immune system process', 'cAMP metabolic process', 'Cholesterol transport' and 'Ion homeostasis'. A comparative analysis of vit C-mediated modulation of gene expression data in THP-1cells and human skin fibroblasts disclosed an overlap in certain functional processes such as 'Regulation of transcription', 'Cell cycle' and 'Extracellular matrix organization', and THP-1 specific responses, namely, 'Regulation of gene expression' and 'Ion homeostasis'. It was noteworthy that vit C modulated the 'Immune system' process throughout the time-course. CONCLUSIONS: This study reveals the genome-wide effects of physiological levels of vit C on THP-1 gene expression. The multitude of effects impacted by vit C in macrophages highlights its role in maintaining homeostasis of several cellular functions. This study provides a rational basis for the use of the Vitamin C- THP-1 cell model, to study biochemical and cellular responses to stresses, including infection with M. tuberculosis and other intracellular pathogens.

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses.

Mycobacterium tuberculosis (Mtb) owes its success as a pathogen in large measure to its ability to exist in a persistent state of 'dormancy' resulting in a lifelong latent tuberculosis (TB) infection. An understanding of bacterial adaptation during dormancy will help in devising approaches to counter latent TB infection. In vitro models have provided valuable insights into bacterial adaptation; however, they have limitations because they do not disclose the bacterial response to the intracellular environment wherein the bacteria are simultaneously exposed to multiple stresses. We describe the pleiotropic response of Mtb in the vitamin C (vit C) model of dormancy developed in our laboratory. Vit C mediates a rapid regulation of genes representing ~14 % of the genome in Mtb cultures. The upregulated genes were better represented in lipid, intermediary metabolism and regulatory protein categories. The downregulated genes mainly related to virulence, detoxification, information pathways and cell wall processes. A comparison of this response to that in other models indicates that vit C generates a multiple-stress environment for axenic Mtb cultures that resembles a macrophage-like environment. The bacterial response to vit C resembles responses to gaseous stresses such as hypoxia and nitric oxide, oxidative and nitrosative stresses, nutrient starvation and, notably, the activated macrophage environment itself. These responses demonstrate that the influence of vit C on Mtb gene expression extends well beyond the DevR dormancy regulon. A detailed characterization of the response to vit C is expected to disclose useful strategies to counter the adaptive mechanisms essential to Mtb dormancy.

Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis.

The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A+K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevR-SigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.

The residue threonine 82 of DevR (DosR) is essential for DevR activation and function in Mycobacterium tuberculosis despite its atypical location.

The DevR (DosR) response regulator initiates the bacterial adaptive response to a variety of signals, including hypoxia in in vitro models of dormancy. Its receiver domain works as a phosphorylation-mediated switch to activate the DNA binding property of its output domain. Receiver domains are characterized by the presence of several highly conserved residues, and these sequence features correlate with structure and hence function. In response regulators, interaction of phosphorylated aspartic acid at the active site with the conserved threonine is believed to be crucial for phosphorylation-mediated conformational change. DevR contains all the conserved residues, but the structure of its receiver domain in the unphosphorylated protein is strikingly different, and key threonine (T82), tyrosine (Y101), and lysine (K104) residues are placed uncharacteristically far from the D54 phosphorylation site. In view of the atypical location of T82 in DevR, the present study aimed to examine the importance of this residue in the activation mechanism. Mycobacterium tuberculosis expressing a DevR T82A mutant protein is defective in autoregulation and supports hypoxic induction of the DevR regulon only very weakly. These defects are ascribed to slow and partial phosphorylation and the failure of T82A mutant protein to bind cooperatively with DNA. Our results indicate that the T82 residue is crucial in implementing conformational changes in DevR that are essential for cooperative binding and for subsequent gene activation. We propose that the function of the T82 residue in the activation mechanism of DevR is conserved in spite of the unusual architecture of its receiver domain.

In vivo assessment of a single adenine mutation in 5'UTR of Endothelin-1 gene in paediatric cases with severe pulmonary hypertension: an observational study.

OBJECTIVE: Endothelin-1 plays an important role in the pathogenesis of severe pulmonary hypertension. The + 139 'A', adenine insertion variant in 5'UTR of edn1 gene has been reported to be associated with increased expression of Endothelin-1 in vitro. The aim of present study was to explore the association of this variant with the circulating levels of Endothelin-1 in vivo using archived DNA and plasma samples from 38 paediatric congenital heart disease (cyanotic and acyanotic) patients with severe pulmonary hypertension. RESULTS: The plasma Endothelin-1 levels were highly varied ranging from 1.63 to75.16 pg/ml. The + 139 'A' insertion variant in 5'UTR of edn1 was seen in 8 out of 38 cases with only one acyanotic sample demonstrating homozygosity of inserted 'A' allele at + 139 site (4A/4A genotype). The plasma Endothelin-1 levels in children with homozygous variant 3A/3A genotype were comparable in cyanotic and acyanotic groups. Lone 4A/4A acyanotic sample had ET-1 levels similar to the median value of ET-1 associated with 3A/3A genotype and was absent in cyanotic group presumably due to deleterious higher ET-1 levels. The discussed observations, limited by the small sample size, are suggestive of homozygous adenine insertion variant posing a risk in cyanotic babies with Severe Pulmonary Hypertension.

Development of a highly effective low-cost vaporized hydrogen peroxide-based method for disinfection of personal protective equipment for their selective reuse during pandemics.

BACKGROUND: Personal Protective Equipment (PPE) is required to safely work with biological agents of bacterial (i.e. Mycobacterium tuberculosis) or viral origin (Ebola and SARS). COVID-19 pandemic especially has created unforeseen public health challenges including a global shortage of PPE needed for the safety of health care workers (HCWs). Although sufficient stocks of PPE are currently available, their critical shortage may develop soon due to increase in demand and depletion of existing supply lines. To empower our HCWs and ensure their continued protection, proactive measures are urgently required to develop procedures to safely decontaminate the PPEs to allow their "selective reuse" during contingency situations. METHODS: Herein, we have successfully developed a decontamination method based on vaporized hydrogen peroxide (VHP). We have used a range of concentration of hydrogen peroxide to disinfect PPE (coveralls, face-shields, and N-95 masks). To ensure a proper disinfection, we have evaluated three biological indicators namely Escherichia coli, Mycobacterium smegmatis and spores of Bacillus stearothermophilus, considered as the gold standard for disinfection processes. We next evaluated the impact of repeated VHP treatment on physical features, permeability, and fabric integrity of coveralls and N-95 masks. Next, we performed Scanning Electron Microscopy (SEM) to evaluate microscopic changes in fiber thickness of N-95 masks, melt blown layer or coverall body suits. Considering the fact that any disinfection procedure should be able to meet local requirements, our study included various regionally procured N-95 masks and coveralls available at our institute All India Institute of Medical Sciences (AIIMS), New Delhi, India. Lastly, the practical utility of VHP method developed herein was ascertained by operationalizing a dedicated research facility disinfecting used PPE during COVID-19. RESULTS: Our prototype studies show that a single VHP cycle (7-8% Hydrogen peroxide) could disinfect PPE and PPE housing room of about 1200 cubic feet (length10 ft × breadth 10 ft × height 12 ft) in less than 10 min, as noted by a complete loss of B. stearothermophilus spore revival. The results are consistent and reproducible as tested in over 10 cycles in our settings. Further, repeated VHP treatment did not result in any physical tear, deformity or other appreciable change in the coverall and N-95 masks. Our permeation tests evaluating droplet penetration did not reveal any change in permeability post-VHP treatments. Also, SEM analysis indeed revealed no significant change in fiber thickness or damage to fibers of coveralls or melt blown layer of N-95 masks essential for filtration. There was no change in user comfort and experience following VHP treatment of PPE. Based on results of these studies, and parameters developed and optimized, an institutional research facility to disinfect COVID-19 PPE is successfully established and operationalized with more than 80% recovery rate for used PPE post-disinfection. CONCLUSIONS: Our study, therefore, successfully establishes the utility of VHP to effectively disinfect PPE for a possible reuse as per the requirements. VHP treatment did not damage coveralls, cause physical deformity and also did not alter fabric architecture of melt blown layer. We observed that disinfection process was successful consistently and therefore believe that the VHP-based decontamination model will have a universal applicability and utility. This process can be easily and economically scaled up and can be instrumental in easing global PPE shortages in any biosafety facility or in health care settings during pandemic situation such as COVID-19.

Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets.

G-quadruplex structure forming motifs are among the most studied evolutionarily conserved drug targets that are present throughout the genome of different organisms and susceptible to influencing various biological processes. Here we report highly conserved potential G-quadruplex motifs (PGQs) in three essential genes (espK, espB, and cyp51) among 160 strains of the Mycobacterium tuberculosis genome. Products of these genes are involved in pathways that are responsible for virulence determination of bacteria inside the host cell and its survival by maintaining membrane fluidity. The espK and espB genes are essential players that prevent the formation of mature phagolysosome and antigen presentation by host macrophages. The cyp51 is another PGQ-possessing gene involved in sterol biosynthesis pathway and membrane formation. In the present study, we revealed the formation of stable intramolecular parallel G-quadruplex structures by Mycobacterium PGQs using a combination of techniques (NMR, circular dichroism [CD], and gel electrophoresis). Next, isothermal titration calorimetry (ITC) and CD melting analysis demonstrated that a well-known G-quadruplex ligand, TMPyP4, binds to and stabilizes these PGQ motifs. Finally, polymerase inhibition and qRT-PCR assays highlight the biological relevance of PGQ-possessing genes in this pathogen and demonstrate that G-quadruplexes are potential drug targets for the development of effective anti-tuberculosis therapeutics.

Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis.

The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

Multifaceted remodeling by vitamin C boosts sensitivity of Mycobacterium tuberculosis subpopulations to combination treatment by anti-tubercular drugs.

Bacterial dormancy is a major impediment to the eradication of tuberculosis (TB), because currently used drugs primarily target actively replicating bacteria. Therefore, decoding of the critical survival pathways in dormant tubercle bacilli is a research priority to formulate new approaches for killing these bacteria. Employing a network-based gene expression analysis approach, we demonstrate that redox active vitamin C (vit C) triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving ~ 67% of the genome. Vit C-adapted bacteria display well-described features of dormancy, including growth stasis and progression to a viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, dissipation of reductive stress through triglyceride (TAG) accumulation, protective response to oxidative stress, and tolerance to first line TB drugs. VBNC bacteria are reactivatable upon removal of vit C and they recover drug susceptibility properties. Vit C synergizes with pyrazinamide, a unique TB drug with sterilizing activity, to kill dormant and replicating bacteria, negating any tolerance to rifampicin and isoniazid in combination treatment in both in-vitro and intracellular infection models. Finally, the vit C multi-stress redox models described here also offer a unique opportunity for concurrent screening of compounds/combinations active against heterogeneous subpopulations of Mtb. These findings suggest a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment.

Sustained expression of DevR/DosR during long-term hypoxic culture of Mycobacterium tuberculosis.

DevR/DosR is a key mediator of 'dormancy' adaptation in Mycobacterium tuberculosis in response to gaseous stresses such as hypoxia that inhibit aerobic mode of respiration. In the present study, a temporal analysis over a 1 year period has revealed robust expression of representative DevR regulon genes devR, hspX and tgs1, during long-term 'dormancy' adaptation to hypoxia. Notably, a predominant proportion of long-term hypoxia-adapted bacteria were characterized by their inability to grow on solid media, accumulation of triacylglycerols and recovery of growth in liquid media. Persistent expression of HspX and the accumulation of triacylglycerols reveal a previously underappreciated role of DevR during adaptation to extended hypoxia, and endorse DevR as an effective target for thwarting the sustained survival of 'dormant' subpopulation of M. tuberculosis.