The role of NTR2 in resistance to (<i>R</i>)-PA-824 in <i>Leishmania</i>.

<p>(A) Plot of the proteomics data from (<i>R</i>)-PA-824 resistant clone RES III. Each protein identification is represented by a point plotted as the Log₂ of their heavy-to-light isotope ratio (x axis) versus the Log₁₀ value of the intensities of the peptides belonging to each protein (y axis). Proteins plotted in light blue were determined to have significantly different expression levels compared to those in WT parasites, with the most significantly changed protein shown in red (LinJ.12.0730; NADH:flavin oxidoreductase/NADH oxidase, putative). The identity of the proteins found to be consistently over- or under-expressed are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s004" target="_blank">S1 Table</a>. (B) Dose-response curve of WT promastigotes (black circles) and promastigotes overexpressing NTR2 (red circles) to (<i>R</i>)-PA-824. EC₅₀ values of 140 ± 4.6 and 3.5 ± 0.2 nM were determined for WT and NTR2-overexpressing parasites, respectively. Overexpression of NTR2 was confirmed by western blotting (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s003" target="_blank">S2 Fig</a>). (C) The susceptibility of RES III parasites (red circles) and RES III parasites overexpressing NTR2 to (<i>R</i>)-PA-824 (black circles). RES III parasites were insensitive to (<i>R</i>)-PA-824 at concentrations up to and including 100 μM while these promastigotes overexpressing NTR2 returned an EC₅₀ value of 1 ± 0.02 nM. Data are the mean ± SD of triplicate cultures. (D) Representation of the frame shift and premature termination of NTR2 translation that results from deletion of a cytosine at genomic position 483544 in (<i>R</i>)-PA-824-resistant clones. The frame shift results in a shortened open reading frame and corresponding amino acid sequence (highlighted in bold), with red indicating the frame-shifted part of this sequence.</p

(<i>R</i>)-PA-824 resistance <i>in vitro</i>.

<p>(A) Schematic representation of the generation of a (<i>R</i>)-PA-824 resistant cell line in <i>Leishmania donovani</i>. Each passage of cells in culture (black circles) is indicated. (B) Dose-response curves for WT (black circles) and RES III resistant cells (red circles). The curves are the non-linear regression fits using a two-parameter EC₅₀ equation, yielding EC₅₀ values of 262 ± 14 nM and > 100 μM for (<i>R</i>)-PA-824 against WT and RES III cells, respectively. Data are the mean ± standard deviation of triplicate cultures in a single experiment.</p

Sensitivity of WT, (<i>R</i>)-PA-824 resistant and transgenic <i>L</i>. <i>donovani</i> promastigotes to nitroheterocyclic compounds.

<p>Sensitivity of WT, (<i>R</i>)-PA-824 resistant and transgenic <i>L</i>. <i>donovani</i> promastigotes to nitroheterocyclic compounds.</p

Characterisation of LdNTR2.

<p>(A) Purification of recombinant LdNTR2 from <i>E</i>. <i>coli</i> BL21(DE3)pLysS [pET15b-LdNTR2]. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, pooled fractions from Ni²⁺-affinity chromatography; lane 4, soluble protein following removal of histidine tag; and lane 5, pooled fractions from size-exclusion chromatography. MALDI analysis confirmed that the minor bands represent NTR2 degradation products. (B) Gel filtration profile of the LdNTR2. The inset shows a plot of elution volume against the log molecular mass (MW) of a standard protein mixture (black circles). The red circle represents the elution volume of NTR2. (C) Metabolism of nitroheterocyclic compounds by recombinant NTR2. Initial rates of metabolism were measured in assays containing 100 μM nitro-compound, 100 μM NADPH and 500 nM NTR2. Rates of metabolism with NADPH and NADH alone were 0.0124 ± 0.001 and 0.0084 ± 0.0006 μmol min^-1 mg^-1, respectively. Rates represent the mean ± SD of triplicate measurements. (D) Immunoblots of whole cell extracts (equivalent of 5×10⁶ parasites in each lane) from <i>L</i>. <i>donovani</i> mid-log promastigotes (L), metacyclic promastigotes (M) and axenic amastigotes (A) were probed with LdNTR2-specific polyclonal antiserum. Known amounts of purified recombinant LdNTR2 were loaded as standards for the quantification of the cellular levels of NTR2.</p

Chemical structures of the bicyclic and monocyclic compounds used in this study.

<p>Procedures for the synthesis of DNDI-VL-2098 and CGI-17341 are described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005971#ppat.1005971.s001" target="_blank">S1 Text</a>.</p

Metabolism of nitroheterocyclics in cultures of WT and NTR2-overexpressing promastigotes.

<p>Metabolism of 160 nM (<i>R</i>)-PA-824 (A), 15 nM delamanid (B) and 20 nM DNDI-VL-2098 (C) in media alone (black circles), wild type <i>L</i>. <i>donovani</i> promastigotes (blue circles) and NTR2-overexpressing <i>L</i>. <i>donovani</i> promastigotes (red circles). The half-life of (<i>R</i>)-PA-824 in media alone, in cultures of WT promastigotes and cultures of promastigotes overexpressing NTR2 were >24 h, 14 h and 0.5 h, respectively. DNDI-VL-2098 incubated in media alone had a half-life of 3.1 h and half-lives of 0.83 h and 0.13 h in cultures of WT and NTR2-overexpressing parasites, respectively. The half-life of delamanid was 12 h in media alone and 1.6 h in WT promastigotes. As the disappearance of delamanid in NTR2-overexpressing parasites was plotted to a double exponential decay, two half-lives were calculated as 0.096 h for <i>k</i>₁ and 0.64 h for <i>k</i>₂.</p

Modulation of NTR2 levels and its effect on (<i>R</i>)-PA-824 potency, compound metabolism and infectivity.

<p>(A) (i) Schematic representation of the stepwise generation of the <i>NTR2</i> DKO cell line in <i>L</i>. <i>donovani</i>. One allele of <i>NTR2</i> was replaced with the puromycin resistance gene (PAC) by homologous recombination; the remaining allele was replaced with a hygromycin resistance gene (HYG) by homologous recombination resulting in a NTR2 null cell line. (ii) Southern-blot analysis of XhoI-digested genomic DNA (∼5 μg) from wild-type <i>L</i>. <i>donovani</i> (LdBOB) cells (lane 1), NTR2-double knockout clone 1 (DKO1) cells (lane 2) and NTR2-double knockout clone 2 (DKO2) cells (lane 3). The <i>NTR2</i> ORF probe shows allelic <i>LdNTR2</i> at 10 kb. (B) EC₅₀ values were determined for (<i>R</i>)-PA-824 against WT (black), DKO1 (red), DKO2 parasites (blue) and DKO1 parasites with an NTR2 add-back (green). An EC₅₀ value of 115 ± 3 nM was determined for (<i>R</i>)-PA-824 against WT parasites, DKO1 and DKO2 were unaffected by the drug at concentrations up to and including 10 μM, while an EC₅₀ value of 5.5 ± 0.03 nM was determined for DKO1 parasites expressing an NTR2 add-back. (C) Metabolism of 160 nM (<i>R</i>)-PA-824 in media alone (black circles), wild type <i>L</i>. <i>donovani</i> promastigotes (blue circles), DKO parasites (red circles) and DKO parasites plus NTR2 add-back (green circles). The half-life of (<i>R</i>)-PA-824 metabolism in cultures of WT parasites is 12.5 h, while the half-life of DKO parasites with an NTR2 add-back is 0.5 h. (D) Mean numbers of WT, DKO (clone 1) and RES (clone RES III) amastigotes infecting mouse peritoneal macrophages. Grey bars, invasion after 24 h; black bars, replication after 72h. Differences in the replication of WT versus RES and DKO amastigotes in macrophages were confirmed as highly significant (P values equal to 0.0057 and 0.0087, respectively) using an un-paired student t-test (**).</p