MSMEG_6382 is essential for growth of <i>M. smegmatis</i> on Middlebrook agar.

<p>The conditional knockout strain 6382CKO was cultured at 30°C on Middlebrook 7H10 agar containing Kn and Sm, then subcultured onto Middlebrook 7H10 agar containing Kn at 30°C and 42°C and examined for growth. Wild-type <i>M. smegmatis</i> mc²155 control strain cultured at 30°C (<b>A</b>) and 42°C (<b>B</b>) on Middlebrook 7H10 agar without antibiotics; 6382CKO strain cultured at 30°C (<b>C</b>) and 42°C (<b>D</b>) on Middlebrook 7H10 agar containing Kn.</p

The Mycobacterial cell wall and pathway for DPA biosynthesis.

<p><b>A</b>. The mycobacterial cell wall is a multilayered structure containing many components unique to these and closely related bacteria, including phosphatidylinositol mannosides (PIM), lipoarabinomannans (LAM), trehalose monomycolates (TMM), trehalose dimycolates (TDM) mycolic acids and arabinogalactan. The biosynthetic pathways for these unique components are a rich source of potential drug targets. Arabinose sugars are shown in orange. <b>B</b>. Decaprenylphosphoryl arabinose (DPA) is formed by the epimerization of decaprenylphosphoryl ribose (DPR) by DprE1 (Rv3790) and DprE2 (Rv3791). DPA serves as an arabinose donor in cell wall biosynthesis, contributing to arabinogalactan and LAM assembly (orange arrows).</p

Conditional disruption of <i>MSMEG_6382</i>.

<p>Southern blot of genomic DNA digested with <i>Hin</i>dIII/<i>Not</i>I/<i>Xba</i>I. Lane 1, DNA molecular weight DNA markers of the sizes indicated (kilobases, kb); lane 2, wild-type <i>M. smegmatis</i> mc²155; lane 3, single crossover strain used to derive the conditional knockout; lane 4, conditional knockout of <i>MSMEG_6382</i>, designated 6382CKO.</p

DprE1 alignment.

<p>Homologs from <i>M. leprae</i> (ML0109), <i>M. tuberculosis</i> (Rv3790) and <i>M. smegmatis</i> (MSMEG_6382) were aligned using CLUSTALW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016869#pone.0016869-Larkin1" target="_blank">[25]</a>. Residues that are completely conserved are reverse shaded while similar residues are indicated in grey. The putative FAD-binding domain is shown by a solid line. The conserved cysteine residue that is altered in BTZ-resistant mycobacteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016869#pone.0016869-Makarov2" target="_blank">[11]</a> is indicated by an asterix.</p

Genetic strategy for conditional disruption of <i>MSMEG_6382</i>.

<p><b>A</b>. The recombination plasmid pPKC72 contained a cloned copy of <i>MSMEG_6382</i> interrupted by a non-polar kanamycin resistance cassette (<i>MSMEG_6382</i>::<i>aphA3</i>), a gentamycin resistance marker (Gm^r), a temperature-sensitive replication origin for <i>M. smegmatis</i> (ori<i>Ms</i> (ts)), a replication origin for <i>E. coli</i> (ori<i>Ec</i>) and a counterselectable marker encoding sucrose sensitivity (<i>sacB</i>). The construct was introduced into <i>M. smegmatis</i> at the permissive temperature (30°C). Integration of the plasmid by a single crossover at the position indicated was detected by growing the cells at the non-permissive temperature (42°C) in the presence of kanamycin. <b>B</b>. Genetic map of the single crossover, showing key restriction sites and fragments. <b>C.</b> Culturing the single crossover strain containing a rescue plasmid encoding <i>MSMEG_6382</i> gave rise to a disrupted copy of <i>MSMEG_6382</i> in the chromosome, producing the conditional knockout (6382CKO). Since the disruption of <i>MSMEG_6382</i> coincided with the loss of the <i>sacB</i> gene, the conditional knockout strain could be selected on sucrose plates. Double lines indicate chromosomal DNA, single lines indicate plasmid DNA. Restriction fragments hybridising to the <i>MSMEG_6382-</i>specific probe are shown in kilobases (kb).</p

Comparative growth patterns of cultures containing curcumin pre-treated and non pre-treated host erythrocytes.

<p>For pre-treatment, erythrocytes were incubated with 5 µM curcumin for 48 h and then mixed with parasite-infected untreated erythrocytes to a final parasitemia of 0.5% and final hematocrit of 3%. In no pre-treatment cultures, erythrocytes were not given any pre-treatment with curcumin. No drug control parasite culture with no drug. Error bars represent standard error of the mean (n = 4).</p

Predictive binding of curcumin, colchicine, paclitaxel and vinblastine to <i>P. falciparum</i> tubulin dimer.

<p>Figures showing bound poses of curcumin diketo form at the interface of tubulin dimer. <i>P. falciparum</i> tubulin is represented as dimer of alpha (yellow) and beta (green) subunit. Panel A shows all the predicted bound poses, mostly at the interface of the dimer. Panel B shows the most probable binding pose according to Autodock (Rank 1) with the curcumin diketo form at the interface of alpha and beta tubulin monomers. Panel C shows binding sites of colchicine (purple), paclitaxel (red) and vinblastine (brown) on parasite tubulin dimer.</p

Proportion of total parasite cells observed with spindle and subpellicular microtubules in control and treated parasite population.

<p>Proportion of total parasite cells observed with spindle and subpellicular microtubules in control and treated parasite population.</p

Controls: Treatment of <i>P. falciparum</i> with antimitotic drugs.

<p>Microtubule stabilizing and destabilizing compounds exert contrasting effects on their target. Panel A shows <i>P. falciparum</i> without any drug treatment after 24 hours. Panel B shows parasites 24 hours after treated with 500 nM paclitaxel. Images in panel C represents parasites treated with 100 nM vinblastine, after 24 hours. Molar concentration of the drugs was chosen on the basis of published IC₅₀.</p

Curcumin uptake by parasitised (IE) and uninfected erythrocytes (E) during first and second cycle of <i>P. falciparum</i> growth.

<p>Error bars represent standard error of the mean (n = 3).</p