Mycobacterium tuberculosis Rv2324 is a multifunctional feast/famine regulatory protein involved in growth, DNA replication and damage control.

Feast/famine regulatory proteins (FFRPs) are multifunctional regulators. We show that Mtb Rv2324 is important for growth, survival, and countering DNA damage in Mycobacterium tuberculosis (Mtb). DNA-relaxation activity against linear and supercoiled substrates suggest its involvement in transcription activation, while its high affinity for recombination, replication and repair substrates suggest a role there too. Small-Angle-X-ray scattering supports the adoption of an 'open' quaternary association in response to amino-acid binding. Size-exclusion-chromatography and glutaraldehyde cross-linking identify the adoption of diverse oligomers modulated by amino-acid binding, and DNA interactions. We tested G52A, G101T and D104A mutants which correspond to highly conserved residues, distal to the DNA-binding site, and are important for amino acids binding. G101T exhibits increased DNA affinity, while G52A and D104A exhibit weak DNA-binding thereby suggesting that they mediate effector-binding, and DNA binding activities. Gain and loss-of-function studies show that Rv2324 overexpression promotes growth-rate, while its knock-down leads to retarded growth. Rv2324 down-regulation lowers Mtb survival inside resting and IFN-ϒ-activated macrophages. Rv2324 protects the pathogen from DNA damage, as evidenced by the reduction in the knockdown strain's survival following treatment with H2O2 and UV light. Overall, we show that Rv2324 plays a crucial role in regulating survival and growth of Mtb.

Rv1717 Is a Cell Wall - Associated ß-Galactosidase of Mycobacterium tuberculosis That Is Involved in Biofilm Dispersion.

Understanding the function of conserved hypothetical protein (CHP)s expressed by a pathogen in the infected host can lead to better understanding of its pathogenesis. The present work describes the functional characterization of a CHP, Rv1717 of Mycobacterium tuberculosis (Mtb). Rv1717 has been previously reported to be upregulated in TB patient lungs. Rv1717 belongs to the cupin superfamily of functionally diverse proteins, several of them being carbohydrate handling proteins. Bioinformatic analysis of the amino acid sequence revealed similarity to glycosyl hydrolases. Enzymatic studies with recombinant Rv1717 purified from Escherichia coli showed that the protein is a ß-D-galactosidase specific for pyranose form rather than the furanose form. We expressed the protein in Mycobacterium smegmatis (Msm), which lacks its ortholog. In Msm Rv1717 , the protein was found to localize to the cell wall (CW) with a preference to the poles. Msm Rv1717 showed significant changes in colony morphology and cell surface properties. Most striking observation was its unusual Congo red colony morphotype, reduced ability to form biofilms, pellicles and autoagglutinate. Exogenous Rv1717 not only prevented biofilm formation in Msm, but also degraded preformed biofilms, suggesting that its substrate likely exists in the exopolysaccharides of the biofilm matrix. Presence of galactose in the extracellular polymeric substance (EPS) has not been reported before and hence we used the galactose-specific Wisteria floribunda lectin (WFL) to test the same. The lectin extensively bound to Msm and Mtb EPS, but not the bacterium per se. Purified Rv1717 also hydrolyzed exopolysaccharides extracted from Msm biofilm. Eventually, to decipher its role in Mtb, we downregulated its expression and demonstrate that the strain is unable to disperse from in vitro biofilms, unlike the wild type. Biofilms exposed to carbon starvation showed a sudden upregulation of Rv1717 transcripts supporting the potential role of Rv1717 in Mtb dispersing from a deteriorating biofilm.

Effect of various drugs on differentially detectable persisters of Mycobacterium tuberculosis generated by long-term lipid diet.

Persisters of Mycobacterium tuberculosis (Mtb) that fail to form colonies on agar media when de-stressed are termed as differentially detectable (DD) persisters. Since in the host, Mtb primarily survives by utilizing lipids, we used a long-term lipid diet model to induce DD persisters of M. tuberculosis. Persisters were induced by replacing the dextrose-containing medium with one containing fatty acids instead of dextrose (FAM). After 2, 4 or 6 weeks, CFU and most probable number assays were performed; the difference between the two gave an estimate of DD persisters. Since rifampicin has been shown to induce formation of DD persisters in vitro, one set of FAM cultures were also given short-term rifampicin stress after 2, 4 or 6 weeks. Fraction of DD persisters increased with time and rifampicin treatment enhanced the effect of fatty acids, at 2 and 4 weeks. At six weeks, even in the absence of rifampicin, ∼95% population were DD persisters. The DD persisters were vulnerable to drugs interfering with bacterial respiration such as thioridazine, bedaquiline and clofazimine. The study indicates potential formation of DD persisters of Mtb in a lipid-rich microenvironment in the host even before antibiotic therapy.

Alternate pathway to ascorbate induced inhibition of Mycobacterium tuberculosis.

Ascorbate has been demonstrated to interfere with the growth of Mycobacterium tuberculosis. It scavenges oxygen in the culture medium to induce dormancy of M. tuberculosis. It kills the mycobacteria by generating reactive oxygen intermediates via iron mediated Fenton reactions. In this study, we observed that ascorbate can inhibit M. tuberculosis isocitrate lyase (MtbICL) with an IC50 of 2.15 mΜ. MtbICL is an essential enzyme for the survival of M. tuberculosis under dormancy. We studied the effect of ascorbate on the growth of M. tuberculosis H37Rv metabolizing through citric acid cycle or glyoxylate cycle with glucose or acetate respectively as the sole carbon source. It was observed that 4 mM ascorbate inhibited ∼89% of the growth in glucose medium, which was confirmed to be mediated by Fenton reaction, as the inhibition was significantly lesser (61%) under low iron condition. On the other hand, in acetate medium, ∼97% of the growth was inhibited and the inhibition was uninfluenced by the iron levels. 3-nitropropionate, a known inhibitor of MtbICL, was seen to cause significantly higher inhibition in the acetate medium than in the glucose medium; however it was indifferent to iron levels in either medium. Molecular docking and dynamic simulation studies confirmed stable binding of ascorbate to MtbICL leading to its inhibition. These observations suggest an additional pathway for ascorbate induced inhibition of M. tuberculosis through inhibition of glyoxylate cycle. Since human immune cells can accumulate ascorbate in millimolar concentrations, the in vitro activity range (1-4 mM) of ascorbate against M. tuberculosis could be extrapolated in vivo. Our result supports the possible benefits of adding high vitamin C diet in TB-treated patients.

Isocitrate lyase of Mycobacterium tuberculosis is inhibited by quercetin through binding at N-terminus.

Combating tuberculosis requires new therapeutic strategies that not only target the actively dividing bacilli but also the dormant bacilli during persistent infection. Isocitrate lyase (ICL) is a key enzyme of the glyoxylate shunt, crucial for the survival of bacteria in macrophages and mice. MtbICL is considered as one of the potential and attractive drug targets against persistent infection. We report the inhibition of MtbICL by quercetin with IC50 of 3.57 µM. In addition, quercetin strongly inhibited the growth of Mtb H37Rv utilizing acetate, rather than glucose as the sole carbon source, suggesting the inhibition of glyoxylate shunt. Quercetin binds at the N-terminus of MtbICL (Kd - 6.68 µM).

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45µM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300µg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51µM, mammalian ATPase IC50>100µM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12µg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100µg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5µg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173µmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Coupling reporter expression to respiration detects active as well as dormant mycobacteria in vitro and in mouse tissues.

BACKGROUND: Mycobacterium tuberculosis is known to slow down its transcriptional activity during dormancy. Hence, while using reporter strains, it is important to couple the reporter gene to a promoter that is strong and sensitive both in active and dormant M. tuberculosis. Since respiration is an indispensable process even in dormant bacteria, validation of the promoters of respiratory chain genes - type II NADH dehydrogenase (Pndh) and adenosine triphosphate (ATP) synthase operon (Patps) - of MTB was undertaken for this purpose. METHODS: Putative promoter containing sequences were cloned upstream of a red fluorescent protein (RFP) gene. Mycobacterium smegmatis or M. tuberculosis carrying episomal constructs were validated for growth, fitness and fluorescence in different models in vitro and in vivo. RESULTS: Either promoter can drive stable and strong expression of RFP in actively growing and dormant M. smegmatis in vitro without significantly affecting growth or viability. Fluorescence due to Pndh and Patps was significantly higher than Phsp60. The fitness of M. tuberculosis H37Rv counterparts was unaffected inside J774 macrophages. In immunocompetent mice, despite an initial attenuation in the lungs, both strains reached loads similar to wild type during chronic infection. In the spleen, the fluorescent strain counts were similar to wild type counts throughout. RFP fluorescence in tissue homogenates was more homogenous among mice due to Pndh compared with Patps. CONCLUSIONS: Coupling an appropriate reporter to the promoter of ndh-2 gene of M. tuberculosis can make the reporter expression respiration sensitive and thereby reliably detect both active and dormant populations of the reporter strain.

Biological evaluation of novel substituted chloroquinolines targeting mycobacterial ATP synthase.

The ATP synthase of Mycobacterium tuberculosis is a validated drug target against which a diarylquinoline drug is under clinical trials. The enzyme is crucial for the viability both of actively replicating and non-replicating/dormant M. tuberculosis. Enzyme levels drop drastically as the bacilli enter dormancy and hence an inhibitor would make the dormant bacilli even more vulnerable. In this study, a set of 18 novel substituted chloroquinolines were screened against Mycobacterium smegmatis ATP synthase; 6 compounds with the lowest 50% inhibitory concentration (IC(50)) values (0.36-1.83 µM) were selected for further in vitro studies. All six compounds inhibited the growth of M. tuberculosis H37Rv in vitro, with minimum inhibitory concentrations (MICs) of 3.12 µg/mL (two compounds) or 6.25 µg/mL (four compounds). All of them were bactericidal to non-replicating M. tuberculosis H37Rv in hypoxic culture; three compounds caused a >2 log(10) reduction in CFU counts in 4 days at concentrations of 16× or 32× their MICs, compared with a 0.2 log(10) reduction by isoniazid and a >4 log(10) reduction by rifampicin at 100× their MICs. The compounds also contributed to a greater reduction in total cellular ATP of the bacilli compared with isoniazid and rifampicin during an exposure time of 18 h. The compounds at 100 µM caused only 5-35% inhibition of mouse liver mitochondrial ATP synthase, leading to selectivity indices ranging from >55-fold to >278-fold. In vitro cytotoxicity to the Vero cell line measured as the 50% cytotoxic concentration (CC(50)) of the compounds ranged between 55 µg/mL and >300 µg/mL.

Insights into the mechanism of action of hyaluronate lyase: role of C-terminal domain and Ca2+ in the functional regulation of enzyme.

Hyaluronate lyases (HLs) cleave hyaluronan and certain other chondroitin/chondroitin sulfates. Although native HL from Streptococcus agalactiae is composed of four domains, it finally stabilizes after autocatalytic conversion as a 92-kDa enzyme composed of the N-terminal spacer, middle alpha-, and C-terminal domains. These three domains are independent folding/unfolding units of the enzyme. Comparative structural and functional studies using the enzyme and its various fragments/domains suggest a relatively insignificant role of the N-terminal spacer domain in the 92-kDa enzyme. Functional studies demonstrate that the alpha-domain is the catalytic domain. However, independently it has a maximum of only about 10% of the activity of the 92-kDa enzyme, whereas its complex with the C-terminal domain in vitro shows a significant enhancement (about 6-fold) in the activity. It has been previously proposed that the C-terminal domain modulates the enzymatic activity of HLs. In addition, one of the possible roles for calcium ions was suggested to induce conformational changes in the enzyme loops, making HL more suitable for catalysis. However, we observed that calcium ions do not interact with the enzyme, and its role actually is in modulating the hyaluronan conformation and not in the functional regulation of enzyme.