Synthesis and Antimycobacterial Activity of 2,1′-Dihydropyridomycins

Dihydropyridomycins <b>2</b> and <b>3</b>, which lack the characteristic enol ester moiety of the potent antimycobacterial natural product pyridomycin (<b>1</b>), have been prepared from l-Thr, <i>R</i>- and <i>S</i>-hydroxy isovaleric acid, and 3-pyridinecarboxaldehyde. The 2<i>R</i> isomer <b>2</b> shows only 4-fold lower anti-<i>Mtb</i> activity than <b>1</b>, indicating that the enol ester moiety in the natural product is not critical for its biological activity. This finding establishes <b>2</b> as a potent and more practical lead for anti-TB drug discovery

Susceptibilities of <i>M. smegmatis</i> strains overexpressing <i>cycA</i> alleles to DCS.

<p>(<b>A</b>) CFUs of recombinant <i>M. smegmatis</i> strains after 24 hours of treatment with indicated concentrations of DCS. (<b>C</b>) CFUs of recombinant <i>M. smegmatis</i> strains obtained over time upon exposure 200 µg/mL DCS. <i>M. smegmatis</i> harbouring pMD31 (black bars), pMDcycA_Mtb (white bars), pMDcycA_Mbov (light-gray bars) and pMDcycA_BCG (dark-gray bars). Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05). Representative of three independent experiments performed in duplicate.</p

Characterization of <i>cycA</i> merodiploid BCG and <i>M. smegmatis</i> strains.

<p>(<b>A</b>) Sequence of the SNP containing region in <i>cycA</i> from BCG-Pasteur::I425 and BCG-Pasteur::pYUB412 clones from the 5′ end. (<b>B</b>) CFUs of BCG-Pasteur::I425 (white bars) and BCG-Pasteur::pYUB412 (black bars) 4 days after treatment with indicated concentrations of DCS. (<b>C</b>) CFUs of BCG::I425 (solid squares) and BCG::pYUB412 (solid diamonds) obtained over time upon exposure to 32 µg/mL DCS. (<b>D</b>) CFUs of <i>M. smegmatis</i>::I425 (white bars) and <i>M. smegmatis</i>::pYUB412 (black bars) 24 hours after treatment with indicated concentrations of DCS. (<b>E</b>) CFUs of <i>M. smegmatis</i>::I425 (solid squares) and <i>M. smegmatis</i>::pYUB412 (solid diamonds) obtained over time upon exposure to 200 µg/mL DCS. Data points and error bars are means ± standard deviations. Statistically significant differences are denoted by asterisks (*, P<0.05; **, P<0.01). Representative of 3 independent experiments performed in duplicate.</p

Analyses of CycA amino acid sequences.

<p>(<b>A</b>) Partial CycA amino acid sequence alignment of various mycobacteria (the conserved G122 residue is indicated by the arrow). (<b>B</b>) 2-dimensional topological representation of <i>M. bovis</i> CycA with the G122S mutation found in all BCG (circled) in the extracellular loop (boxed) between the 3^rd and 4^th trans-membrane helices from the amino (N) terminus.</p

DNA and bacterial strains used in this work.

<p>DNA and bacterial strains used in this work.</p

Topoisomerases occupancy on <i>Mtb</i> genome.

<p><b>(A)</b> Twin supercoiled domain model. The movement of RNA polymerase machine on the genome/TUs generates wave of negative supercoils upstream and positive supercoils downstream to the transcription machine. The supercoils have to be removed by the action of topoisomerases allowing the unobstructed translocation of RNAP. The <i>in vivo</i> interaction of topoisomerases with the twin supercoiled domains is yet to be demonstrated. <b>(B)</b> ChIP-Seq analysis of RNA polymerase (RNAPCS), DNA gyrase (GyrCS) and Topoisomerase I (Topo ICS) occupancy on <i>Mtb</i> genome. UCSC genome browser view of Topo I, RNAP and DNA gyrase occupancy across the <i>Mtb</i> genome (0.2 Mb-4.1 Mb representative region of <i>Mtb</i> genome). The peak height correspond to the signal intensity (IP/Mock ratio of RNAP, Gyrase and Topo I IPs) of protein binding on the particular site.</p

Transcription induction recruits topoisomerases to TU.

<p>Determination of the occupancy of RNAP, Topo I and DNA gyrase on activated TU. <b>(A)</b> Schematic of the experimental set up. Induction of transcription introduces supercoils on the template which recruits topoisomerases. Construct pJam-Rv3852 was electroporated into <i>M</i>. <i>smegmatis</i> and the transformants were grown up to the exponential phase. Cultures were induced with acetamide for 6 h to activate the transcription of <i>rv3852</i> cloned under the acetamidase promoter. Arrows (red) indicate the position of primers for the amplification of target region <b>(B)</b> Induced (I) and uninduced (UI) cultures were processed for ChIP and enrichment of RNAP, Topo I and DNA gyrase was monitored by the qPCR using <i>rv3852</i> specific primers. Unrelated IgG antibody was used as a negative control. <b>(C)</b> Depiction of the architecture of TU and the positions of the primers used for monitoring the gyrase binding on promoter region (P) and gene body of <i>Rv1303</i> and <i>rrS</i> TUs. <i>Mtb</i> cells were treated with Moxifloxacin (Moxi) and gyrase-DNA clevage complexes around promoter and gene body were detected by qPCR. <b>(D)</b> Effect of transcription inhibition on topoisomerase activity. <i>Mtb</i> cells were treated with or without Rifampicin (Rif) followed by treatment with Moxifloxacin to induce gyrase-DNA cleavage complex formation. Promoter regions of <i>Rv1303</i> and <i>rrS</i> were monitored for the formation of gyrase-DNA cleavage complex in the presence and absence of Rif. Error bars represent the SD obtained from three independent experiments. Significance of the observations was assessed by applying unpaired t-test (* = P<0.05; *** = P<0.001; **< P<0.01; ns = not significant).</p

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase

<div><p>Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the <i>in vivo</i> interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in <i>Mycobacterium tuberculosis</i> taking advantage of minimal topoisomerase representation in the organism. The study unveils the first <i>in vivo</i> genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in <i>Mtb</i>, near the Ter domain of the <i>Mtb</i> chromosome validates its function as a decatenase.</p></div

Topoisomerase occupancy varies on genes with different expression levels.

<p>The genes were segregated based on the available RNA-Seq data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006754#pgen.1006754.ref022" target="_blank">22</a>] under highly expressed (HE) and lowly expressed (LE) category (N = 342). The HE and LE class of gene co-ordinates were analyzed for the occupancy of topoisomerases. TSS (0) was taken as a reference point and mean read count was plotted around it to generate occupancy profile. Mean read counts corresponding to gene co-ordinates were plotted at a single nucleotide resolution.</p

Topo I, RNAP and DNA gyrase co-localize on TUs.

<p><b>(A)</b> TopoI, gyrase and RNAP association. Enrichment ratio (ER) for Topo I, Gyrase and RNAP was calculated on protein coding genes as described earlier [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006754#pgen.1006754.ref022" target="_blank">22</a>] and in Materials and Methods. RNAP and topoisomerase enriched genes (ER>2) were monitored for the enrichment of Topo I/DNA gyrase and RNAP respectively. The number of genes enriched with both RNAP and topoisomerases are represented at the intesection of venn diagram <b>(B)</b> Distribution profile of Topo I, RNAP and DNA gyrase across the TUs. Read counts from transcriptionally active (RPM>1) protein-coding TUs >1 kb in length (N = 541) were taken with reference to the TSS and mean read counts were calculated in 50 bp sized bins. Data were normalized to the input samples followed by removal of background using the read counts associated with the genes with no expression (RPM<1). Data were normalized with the maxima to generate the pattern of distribution on TUs.</p