Stage-specific essentiality predictions of experimentally validated druggable targets and single-gene deletion experiments.

<p>Comprehensive map of experimentally tested treatment targets for <i>P</i>. <i>falciparum</i> with stage-specific model predictions projected (in color) projected on top of the map. Colored reaction pathways correspond to drug inhibition studies (Table C in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s002" target="_blank">S1 Tables</a>) and colored reaction names in rectangles correspond to single gene deletion experiments (Table B in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s002" target="_blank">S1 Tables</a>). The color legend inset corresponds to iAM-Pf480 predictions. Validated drug targets that are also predictive to reduce growth in proliferative as well as late gametocyte stages are of particular interest. This comprehensive assessment of the model and experimental results enables stratification of existing drugs, new drugs to target, as well as new areas of metabolism warranting further investigation. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s004" target="_blank">S2 Fig</a> for the high resolution version of the figure.</p

Species-specific models provide mechanistic explanation for differences in drug response between human- and rodent-infecting malaria species.

<p><b>(a)</b> The core and pan metabolic content of 5 malaria species was identified based on the respective species-specific reconstructions. The core content, illustrated by the intersection of the Venn diagram, is shared by all species. The pan content represents the union of the content across all of the multi-species reconstructions. <b>(b)</b> 14 metabolic reactions differed in their presence across the 5 reconstructed Plasmodium species. <b>(c)</b> Thiamine pyrophosphokinase (TPK) and <b>(d)</b> Choline kinase (CK) were predicted by the models to be essential for the growth of the rodent-infecting species (<i>P</i>. <i>berghei</i>) while their deletion had no effect on the growth of human and non-human primate species. Differential essentiality of TPK is due to absence of phosphomethylpyrimidine kinase and thiamine-phosphate pyrophosphorylase the rodent-infecting species. In the case of CK, the differential essentiality is due to the absence of phosphoethanolamine N-methyltransferase. (See Table A in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s002" target="_blank">S1 Tables</a> for reactions abbreviations and gene-protein-reaction associations). <b>(e)</b> Pantothenate metabolism showed differences in essentiality between stage- and species-specific models. Tables indicate percentage in growth reduction compared to the WT upon deletion of the respective gene. ‘X’ indicates absence of a reaction from the respective reconstruction, ‘—‘ indicates no effect on growth upon deletion of the corresponding reaction and ‘%’ indicates the growth reduction percentage resulting from deletion of the corresponding gene. T: trophozoite, GII: early gametocyte stage, GV: late gametocyte stage, Ook: ookinete. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s005" target="_blank">S3 Fig</a> for the high resolution version of the figure.</p

Life cycle stage specific models of <i>P</i>. <i>falciparum</i> predict gene targets that are essential for asexual, sexual and mosquito stages.

<p><b>(a)</b> The core and pan metabolic content of the genome-scale life cycle stage specific models of <i>P</i>. <i>falciparum</i> are 720 and 1002 reactions, respectively. <b>(b-c)</b> Parameters and AUC plots for performance evaluation of stage-specific pairwise differential gene expression comparisons following a similar approach to Nam, et al.[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.ref052" target="_blank">52</a>] (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s004" target="_blank">S2 Fig</a>). AUROC: area under the receiver operator curve (ROC), DEG: differential expression analysis, T: trophozoite, GII: early gametocyte stage, GV: late gametocyte stage, Ook: ookinete.</p

Stage-specific central metabolic flux patterns in malaria.

<p><b>(a)</b> Correlated reaction sets for iAM-Pf480 were used to define stage and model specific pathways, which were analyzed and compared across different stages. Modularity indices (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#sec012" target="_blank">methods</a>) were 0.023, 0.024, 0.026, 0.145, 0.022 for the T, Schizont, GII, GV, and Ook stages, respectively. In proliferative versus non-proliferative stages of malaria, there were changes in the patterns of central carbon metabolism, notably the non-oxidative PPP and glycolysis. <b>(b)</b> The direction of flux in the non-oxidative branch of PPP goes towards production of glycolytic intermediates in the Trophozoite, Schizont, GII, and GV stages but not the Ookinete stage. Reversal of non-oxidative pentose phosphate pathway fluxes in the Ook enables provision of ribose 5 phosphate (r5p) needed for the synthesis of nucleotide precursors of DNA. The non-oxidative branch in the schizont is colored in red indicating its coupling to growth rate in this stage (Table G in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s002" target="_blank">S1 Tables</a>). Both the oxidative and non-oxidative PPP branches were correlated in GII. Glycolysis was split into upper and lower branches in all stages except GV where the non-oxidative PPP branch was correlated with inositol metabolism. Arrows are omitted from the schizont pathway map to account for reduced flux values relative the other 4 stages. <b>(c)</b> Predicted sampled flux distribution are shown in the non-oxidative branch of PPP (Transketolase; TKT1) and inositol metabolism (myo-inositol-3-phosphate lyase; MI3PS) across all the stages showing increased involvement of inositol metabolism in the GV stage (see supplementary material for discussion and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005895#pcbi.1005895.s003" target="_blank">S1 Fig</a> for the high resolution version of the figure).</p

Role of NBDs domains of EccC5 in ESX-5 dependent secretion and membrane complex assembly.

<p>A) Predicted transmembrane domains (dark grey), and NBD (light grey) of EccC₅ are indicated. The positions of relevant residues are depicted with a black bar. The numbers represent the position in amino acids. B) Secretion of PE_PGRS proteins in the different EccC₅ mutant strains was analyzed by immunoblot of supernatants and cell pellets of wild-type (WT) <i>M</i>. <i>marinum</i> and the <i>eccC5</i> deletion strain <i>(</i>Δ<i>eccC5</i>) complemented with various <i>eccC5</i> mutated genes. GroEL2 staining was used as a control for lysis and equal loading. C) Immunoblot analysis of EccC₅ expression in isolated membranes of indicated strains. D) Blue native PAGE and immunoblot analysis using an anti-EccD₅ antibody of the ESX-5 membrane of <i>M</i>. <i>marinum</i> Δ<i>eccC5</i>::<i>mspA</i>, complemented either with an empty vector (-) or with various <i>eccC5</i> mutated genes. For all samples that contained EccC₅ variants the characteristic pattern of ESX-5 membrane complexes was observed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.ref028" target="_blank">28</a>], consisting of the largest ~1.5 MDa complex and two additional smaller subcomplexes (indicated by the three arrowheads).</p

Secretion analysis of ESX-5 mutant strains.

<p>A) A schematic representation of the ESX-5 region of <i>M</i>. <i>marinum</i> with the different ESX-5 mutations used in this study. Bars above the gene cluster indicate regions deleted by targeted knock-out mutagenesis. Arrows below indicate position and orientation of transposons (named LA1 to LA12) in mutants of the parental strain <i>M</i>. <i>marinum</i>::<i>mspA</i> defective in ESX-5 dependent secretion. B) Secretion analysis of <i>M</i>.<i>marinum</i>::<i>mspA (</i>WT::<i>mspA)</i>, a <i>mycP5</i> transposon mutant (<i>mycP5</i>::<i>tn</i>, corresponding to LA9 in (A)) and the complemented version of this strain (<i>mycP5</i>::<i>tn</i>-C). Secreted proteins (S) were separated from bacterial cells (P) by centrifugation. In addition, surface-associated proteins were enriched from the bacterial cells by extraction with 0.5% Genapol X-080 (GS) and separated from non-solubilized proteins (GP) by centrifugation. All fractions were analyzed for the presence of PE_PGRS proteins by immunoblotting. GroEL2 staining was used as a loading and lysis control. C) Expression of EccB₅ and EspG₅ was analyzed by immunoblotting of total cell lysates of wild-type <i>M</i>. <i>marinum</i> (WT), the <i>Δesx-5</i>::<i>mspA</i> mutant and the complemented <i>Δesx-5</i>::<i>esx-5tub</i> strain. D) The same strains as under (C) were analyzed for their ability to express and secrete PE_PGRS proteins following the same procedure as under (B).</p

Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.

<p>Indicated strains were electroporated with the input DNA shown on the left. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. Valid insertion of input DNA was scored as + or-, similarly as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005190#pgen.1005190.t001" target="_blank">Table 1</a>. Results are representative data of three independent experiments.</p><p>Complementation of <i>mas</i>::<i>tn</i> in the Δ<i>eccC</i>_<i>5</i> mutant by replacement of the integrated pMV vector.</p

Expression of MycP5 is essential for growth of <i>M</i>. <i>bovis</i> BCG.

<p>A, B) The BCG-Pasteur c-<i>mycP5</i>-tet-on (A) and c-<i>mycP5</i>-tet-off (B) mutants were grown for 21 days on Middlebrook 7H10 agar plates containing the indicated ATc concentrations. Full growth of c-<i>mycP5</i>-tet-on was only observed at 10 μg/ml ATc, whereas this concentration of ATc did not completely abolish colony growth of c-<i>mycP5</i>-tet-off. C, D) Resazurin reduction is dependent on ATc-induced expression/repression of <i>mycP5</i>. Cells of the BCG-Pasteur c-<i>mycP5</i>-tet-on (C), or c-<i>mycP5-</i>tet-off (D) mutants were grown as liquid cultures in 96-well microtiter plates for 6 days at 37°C at the indicated ATc concentrations, after which 10% Alamar Blue was added and fluorescence (585 nm) was measured after 16 h incubation to determine metabolic activity as a correlate of growth. Values are means of triplicates; error bars represent the standard deviation.</p

ESX-5 is involved in fatty acid uptake.

<p>A) Growth of indicated <i>M</i>. <i>marinum</i> strains on Tween-80 as a sole carbon source was assessed by measuring optical density at different time points. Depicted is the average of three biological replicates. Error bars indicate standard deviations. B) Uptake of a fluorescently labeled fatty acid after 72 hours of hypoxic growth was measured by FACS analysis. 20.000 events gated for similar size were acquired for WT::<i>mspA</i> (black), Δ<i>mycP5</i>::<i>mspA</i> (light grey) or Δ<i>mycP5-C</i>::<i>mspA</i> (dark grey). C) Quantification of FACS analysis. Mean fluorescent intensity of three experiments per strain was acquired by FACS. Background staining, quantified by adding the fluorescent fatty acid to an unstained culture one hour before washing the cells, was deducted from the measured values. Error bars indicate the standard deviations and One-way ANOVA showed a statistical difference between the samples of <i>p</i> = 0.010. D) Uptake of the fluorescently labeled fatty acid and formation of lipid bodies was confirmed by confocal microscopy.</p

Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.

<p>* The <i>eccC</i>_<i>5</i> NBD mutants appear to have a dominant negative effect on the functioning of endogenous EccC₅.</p><p>^$ Colonies showed a strong growth defect, i.e. colonies were visible only after 17 days, compared to 10 days for the wild-type strain.</p><p>Replacement of pMV-<i>eccBC</i>_<i>5</i> by the input DNA was scored. Input DNA consisted of the pMV-361-<i>hyg</i> plasmid containing the indicated constructs. “+” indicates that more than 100 colonies were detected after electroporation with the indicated vector. “–” indicates between 0–20 colonies were found after electroporation. These latter colonies were shown by PCR to still contain the original vector, indicating illegitimate recombination or spontaneous antibiotic resistance. Results are representative data of three independent experiments.</p><p>Essentiality of <i>eccC</i>_<i>5</i> and analysis of functional domains.</p