<i>Tg</i>ATrx1 and <i>Tg</i>ATrx2 are essential and their function requires their CXXC motifs.

<p>A. Scheme of the manipulation performed to each of the <i>Tg</i>ATrxs loci to replace their promoters with the tetracycline-regulatable promoter. The gene model in the scheme is based on <i>Tg</i>ATrx1’s gene model, however the promoter replacement occurred via the same strategy for both <i>Tg</i>ATrxs. Black boxes–exons; asterisk–stop codon. B. Western blot analysis of <i>Tg</i>ATrx1 expression using anti-<i>Tg</i>ATrx1 (i), and endogenously HA-tagged <i>Tg</i>ATrx2 using anti-HA (ii), upon ATc treatment. C. Fluorescent microscopy showing <i>Tg</i>ATrx1 (anti-<i>Tg</i>ATrx1, bottom, green) and <i>Tg</i>ATrx2 (anti-HA, top, green) depletion at 48 hours of ATc treatment (+ATc) compared to non-treated control (-ATc). D. Plaque assays performed with TATiΔKu80_PIATrx1 (top) and TATiΔKu80_PIATrx2-3HA (bottom) with (+) or without (-) ATc. E. Plaque assays performed with TATiΔKu80_PIATrx1 constitutively expressing a copy of <i>Tg</i>ATrx1^CXXC (i) or <i>Tg</i>ATrx1^CXXA (ii) and with TATiΔKu80_PIATrx2-3HA constitutively expressing a copy of <i>Tg</i>ATrx2^CXXC (iii) or <i>Tg</i>ATrx2^CXXA (iv). F. Fluorescent microscopy of the localization of <i>Tg</i>ATrx1^CXXC (i); <i>Tg</i>ATrx1^CXXA (ii); <i>Tg</i>ATrx2^CXXC (iii) and <i>Tg</i>ATrx2^CXXA (iv), all in green, co-stained with Streptavidin (SA) which labels the apicoplast acetyl CoA carboxylase (i, ii) or CPN60 (iii, iv) both in red. White broken line shows parasites’ shapes. Scale bar, 1 μm.</p

Phylogenetic analysis of ATrx1 sequences.

<p>ATrx1 phylogeny and conservation of the CXXC motif. Unrooted <i>maximum likelihood</i> phylogeny of ATrx1 homologues defines a monophyletic clade of predicted plastid-targeted proteins with the apicomplexan/chromerid clade branching with sequences from a selected group of heterokont lineages. Outside of this clade are cytosolic proteins that include heterokont paralogues and dinoflagellate proteins. Branch support values for well supported major nodes are bootstraps then Bayesian posterior probabilities. The CXXC motif is highlighted.</p

<i>Tg</i>ATrx1 and <i>Tg</i>ATrx2 show different localization patterns.

<p>Fluorescent microscopy of <i>Tg</i>ATrx1 (red) co-stained with (i) the PPC marker PPP1, (iii) the luminal marker CPN60 and (v) <i>Tg</i>ATrx2 (green); and of <i>Tg</i>ATrx2 (red) co-stained with (ii) PPP1 and (iv) CPN60 (green). The images on the right are blow-ups of the regions marked by a white empty square, and their scale bars are 1 μm. The schemes on the left depict the four apicoplast compartments and depict the sub-cellular localization of the markers used for co-staining (PPP1 or CPN60). The illustration in (v) shows the putative sub-compartment localization of <i>Tg</i>ATrx1 and <i>Tg</i>ATrx2 based on the microscopy and the phylogenetic distribution data, though further work is needed to determine this with certainty. Scale bar, 1 μm.</p

roGFP1 probe suggests apicoplast oxidation upon <i>TgATrx2</i> depletion.

<p>A. Cytosolic roGFP1 localized in <i>T</i>. <i>gondii</i>. B/C. Measures of emission ratios (385/470 nm, a higher ratio indicates a more oxidizing environment) of cytosolic roGFPiL (B) and roGFP1 (C) in TATiΔKu80_PIATrx2-3HA (B, Ci) or in RH (Cii). D. FNR-roGFP localized to the apicoplast. E/F. Measures of emission ratios of apicoplast roGFPiL (E) and roGFP1 (F) in TATiΔKu80_PIATrx2-3HA. X and Y axis show time and ratio of emissions. 10 mM Diamid and 10 mM DTT were added at min 3 and 6. +ATc data is from 72 hours time point except for F, which is 48 hours.</p

Inducible overexpression of <i>TgATrx2</i>^CXXC/A and substrate trap identifies gene expression components.

<p>A. Scheme of the inducible system. Triangles—LoxP sites. Arrow–promoter. B. Fluorescent microscopy analysis of <i>TgATrx2</i>^CXXA-Myc inducible expression in the presence of 50 nM Rapamycin for 48 hours, showing the switch from killer-red (red) to <i>TgATrx2</i>^CXXA-Myc (green). Scale bar, 5 μm. C/D. Western blot analyses showing the rapamycin induction of <i>TgATrx2</i>^CXXA-Myc (C) and its immunoprecipitation fractions (D) analyzed with anti-Myc antibody. E. Western blot analysis of the co-IP of HA-tagged <i>TgATrx2</i> with Myc-tagged <i>TgATrx2</i> captured with Myc-Trap beads. F. Fluorescent microscopy of the HA-endogenously tagged TGME49_292320 (green); CPN60 –red; DAPI–blue; Arrowheads highlight apicoplast staining. G. Western blot analysis of the co-IP of HA-tagged TGME49_292320 with Myc-tagged <i>TgATrx2</i> captured with Myc-Trap beads (left) and of Myc-tagged <i>TgATrx2</i> with HA-tagged TGME49_292320 captured with HA-agarose beads. In D and F, DTT elutes the disulphide bonded partners via reduction and DTT at 96°C treatment elutes all the parasite proteins bound to the beads.</p

A proposed model of how <i>TgATrx2</i> may mediate apicoplast gene expression via disulphide exchange with translation and transcription components as they travel through the PPC.

<p><i>Tg</i>ATrx1 and <i>Tg</i>ATrx2 are illustrated using dark grey shapes with their thiols shown and are positioned at their putative localization based on previous studies and findings reported herein. The translation and transcription factors whose translocation is proposed to be controlled by <i>Tg</i>ATrx2 are represented by green lines. Endomembranes—light grey; Cell cytoplasm–light orange; periplastid compartment–yellow; inner-most compartment–light red; and stromal compartment–red.</p

Illustrations of the two main processes studied in this work.

<p><b>(A)</b> Complex plastid evolution and the resulting architecture and protein trafficking starting from the co-translational (mRNA in blue, new polypeptide in green) translocation into the ER (light grey) through the four apicoplast compartments (grey, yellow, light-red, red) via their corresponding translocons (depicted as dark grey split ovals): Sec61, SELMA (symbiont-specific ERAD (endoplasmic reticulum-associated degradation)-like machinery), TOC/TIC (translocon of the outer/inner chloroplast membrane) <b>(B)</b> Disulphide exchange between Trx (light-grey) and its substrate (dark-grey oval), and of its dependence on the CXXC motif.</p

Apicoplast protein import and gene expression are the first biogenesis pathways to reach peak reduction for <i>TgATrx1</i> and <i>TgATrx2</i> depletion respectively.

<p>Summary graphs of apicoplast numbers, LytB protein import, relative genome copy number and mRNA levels measured in both knockdown lines at 24, 48 and 72 hours with ATc. The control (no ATc) data is normalized to 100%. Error-bars are SEM. The corresponding individual graphs are found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006836#ppat.1006836.s007" target="_blank">S5 Fig</a>.</p

Libyan map showing the collection points Libyan Propolis samples P1 (Alagoria), P2 (Gaminis), P3 (Byda), P4 (Quba), P5,P6, P7 (Kufra), P8(Ghadames), P9 (Tripoli), P10 (Khasr Khiar), P11, P12 (Khumas).

<p>Libyan map showing the collection points Libyan Propolis samples P1 (Alagoria), P2 (Gaminis), P3 (Byda), P4 (Quba), P5,P6, P7 (Kufra), P8(Ghadames), P9 (Tripoli), P10 (Khasr Khiar), P11, P12 (Khumas).</p

OPLS plot of observed against predicted activity against <i>P</i>. <i>falciparum</i> based on five compounds (A-E).

<p>OPLS plot of observed against predicted activity against <i>P</i>. <i>falciparum</i> based on five compounds (A-E).</p