Arnab China

Molecular dissection of the mycobacterial stringent response protein Rel.

Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram‐negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of RelMsm is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full‐length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Full‐length Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full‐length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA‐mediated synthesis of ppGpp.

Synthesis and antimycobacterial activities of novel 6-nitroquinolone-3-carboxylic acids.

Various 1-(substituted)-1,4-dihydro-6-nitro-4-oxo-7-(sub-secondary amino)-quinoline-3-carboxylic acids were synthesized from 2,4-dichlorobenzoic acid by six step synthesis. The compounds were evaluated for antimycobacterial in vitro and in vivo against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC(2)) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the 48 synthesized compounds, 7-(4-((benzo[d][1,3]dioxol-5-yl)methyl)piperazin-1-yl)-1-cyclopropyl-1,4-dihydro-6-nitro-4-oxoquinoline-3-carboxylic acid (8c) was found to be the most active compound in vitro with MIC of 0.08 and 0.16 microM against MTB and MDR-TB, respectively. In the in vivo animal model 8c decreased the bacterial load in lung and spleen tissues with 2.78 and 4.15-log10 protections, respectively, at the dose of 50 mg/kg body weight.

Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid.

Various 2-(sub)-3-fluoro/nitro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid derivatives were synthesized from 2-aminothiophenol by a five-step reaction, evaluated for in-vitro and in-vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2), and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the thirty-four synthesized compounds, 2-(3-(diethylcarbamoyl)piperidin-1-yl)-)-3-fluoro-5,12-dihydro-5-oxobenzothiazolo[3,2-a]quinoline-6-carboxylic acid (7l) was found to be the most active compound in vitro with MIC of 0.18 and 0.08 microM against MTB and MTR-TB, respectively. Compound 7l was found to be 2 and 570 times more potent than isoniazid against MTB and MDR-TB, respectively. In the in-vivo animal model 7l decreased the bacterial load in lung and spleen tissues with 2.78 and 3.12-log10 protections, respectively, at the dose of 50 mg/kg body weight.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA-protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter-RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P(rrnAPCL1), P(rrnB) and P(gyr) of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Synthesis, Antimycobacterial Activities and Phototoxic Evaluation of 5H-thiazolo[3,2-a]quinoline-4-carboxylic Acid Derivatives

Thirty four novel 7-fluoro/nitro-1,2-dihydro-5-oxo-8-(sub)-5H-thiazolo[3,2-a]quinoline-4-carboxylic acids were synthesized from 2,4-dichlorobenzoic acid and 2,4-dichloro-5-fluoroacetophenone by multi step reaction, evaluated for in vitro and in vivo antimycobacterial activities against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis (MC2) and also tested for the ability to inhibit the supercoiling activity of DNA gyrase from M. smegmatis. Among the synthesized compounds, 8-[6-[[(1,1-dimethylethoxy)carbonyl]amino]-3-azabicyclo[3.1.0]hex-3-yl]-1,2-dihydro-7-nitro-5-oxo-5H-thiazolo[3,2-a]quinoline-4-carboxylic acid (10q) was found to be the most active compound in vitro with MIC of 0.08 microM and <0.08 microM against MTB and MDR-TB respectively. Compound 10q was found to be 4.5 and >570 times more potent than isoniazid against MTB and MDR-TB respectively. In the in vivo animal model 10q decreased the bacterial load in lung and spleen tissues with 2.51 and 3.71-log10 protections respectively at the dose of 50 mg/kg body weight.

Distinct and contrasting transcription initiation patterns at Mycobacterium tuberculosis promoters.

Although sequencing of Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz . PgyrB1, PgyrR, PrrnPCL1 and PmetU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.

Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription.

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Inhibition of Mycobacterium tuberculosis RNA Polymerase by Binding of a Gre Factor Homolog to the Secondary Channel

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elaborate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and several inhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind the RNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor of transcription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channel of RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcription and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has a Gre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction with RNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from Mycobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of the former in influencing the lifestyle of M. tuberculosis.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.