A Systematic Screen to Discover and Analyze Apicoplast Proteins Identifies a Conserved and Essential Protein Import Factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes – a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle

Cell Division in Apicomplexan Parasites Is Organized by a Homolog of the Striated Rootlet Fiber of Algal Flagella

<div><p>Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in <em>Toxoplasma gondii</em> demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.</p> </div

Daughter microtubules depend on SFA fiber, but the fiber does not depend on microtubules.

<p>(A and B) Immunofluorescence of SFA2-HA parasites treated with oryzalin, a microtubule-disrupting agent that prevents formation of new microtubules in daughter cells, and that has a more moderate effect on existing stable microtubules in the mother <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Morrissette3" target="_blank">[34]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Morrissette4" target="_blank">[35]</a>. Note that after 12 h of treatment parasite cells fail to assemble buds (B) and compare to the matched control (A). SFA2-HA fibers are nonetheless detected. Note that in untreated parasites, daughter cells are normally detectable when similar fibers are observed. (C) SFA3-YFP parasites were treated with oryzalin and scored for the presence of SFA3-YFP after 0, 12, or 24 h of treatment. Note that treated parasites accumulate fibers. (D) Fiber length was measured in control parasites and parasites treated with oryzalin for 24 h. Data points reflect the mean fiber length per field of view scored (<i>n</i> = 18–81 fiber/field, four fields for each of three independent repeats). Fibers of treated parasites are overall shorter and more uniformly distributed in size. iΔSFA2 parasites were grown in the absence (E) or presence of ATc (F) and stained for acetylated tubulin (red), centrin (green), and DAPI (blue). The anti-acetylated tubulin antibody labels daughter buds strongly (as do antibodies to unmodified tubulin) in untreated mutants. ATc-treated iΔSFA2 parasites exhibit acetyl-tubulin staining exclusively in the mother cell cytoskeleton (note that two mutant cells are shown with microtubules encaging each entire cell; the cells are abnormally large due to the block in budding). No daughter microtubular-skeletons are discernible in these cells despite the fact that each cell shown has two nuclei, both of which are entering mitosis as indicated by the duplicated centrosomes. Scale bars = 5 µm.</p

Parasite lacking SFA go through mitosis normally but fail to form daughter cells.

<p>(A) iΔSFA2 parasites grown in the presence or absence of ATc for 24 h were labeled with anti-IMC1 (green) and DAPI (blue) anti-centrin (red, centrosomes, upper panels) or anti-CenH3 (red, centromeres, lower panels). Representative examples are shown for cells in interphase or division. Interphase nuclei associate with a single centrosome, while larger 2N nuclei associate with two. This is unchanged by ATc treatment. Interphase nuclei contain a haploid genome and exhibit one CenH3 dot representing a cluster of the centromeres of all 14 chromosomes bundled in the nucleus in close proximity to the centrosome (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Brooks1" target="_blank">[9]</a>). Dividing nuclei exhibit two CenH3 dots representing duplicated chromosomes. Again, this labeling pattern is not affected in the mutant when judged on a per nucleus basis. Note though that in both cases the ATc-treated cell is already tetraploid with no sign of cytokinesis. (B) The number of CenH3 dots per nucleus was quantified in IFA experiments for untreated or ATc-treated iΔSFA2 parasites. The number of CenH3 dots, representing the number of chromosome sets per nucleus, is graphed, error bars show standard deviation (>50 parasite vacuole per experiment counted, <i>n</i> = 3). (C) Immunofluorescence of iΔSFA2 parasites after 48 h ATc treatment showing IMC1 (green), DAPI (blue), and anti-ISP1 (red). ISP1 labels the apical cap of the IMC of both mother and daughter cells and is an early marker of budding <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Beck1" target="_blank">[27]</a>. ISP1 staining for daughter cells is absent in the ATc-treated mutant. Note that the nuclei have completed mitosis in these cells and compare to a similar stage shown for untreated parasite.</p

A fiber links the centrosome and the apical complex of the daughter bud.

<p>Transmission electron micrographs of serial sections of dividing wild-type parasites. (A and B) Two consecutive sections through a daughter cell are shown. An electron dense structure is highlighted in (A) (arrowheads, note some striation in this section, in particular toward the conoid end) emanating from close to one centriole (Ce; B) and reaching up to the conoid of the daughter cell (Cn; A and B). The structure ends in close proximity of the central pair of microtubules within the conoid (arrow; A and B). (C–E) Three consecutive sections perpendicular to the conoid and the orientation of the parasite depicted in (A and B) are shown. A bent electron dense structure (arrowheads) runs within the conoid (Cn) towards the apical ring. The end of this structure appears in contact with the conoid-associated microtubule pair (arrow, also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio-1001444-g010" target="_blank">Figure 10</a> for a schematic outline). (F–I) Four consecutive sections through an early daughter cell. Both parallel centrioles (Ce) of the centrosome can be seen in (F). A fiber is visible reaching into the conoid and touching the apical ring (Cn; F–H). The fiber emerges between the centrioles (arrow; F and G). The last section shows intra-nuclear microtubules as part of the centrocone (CC, a nuclear envelope structure associated with centromere organization in Apicomplexa); these link the kinetochores of the chromosomes to the centrosome. Scale bars = 250 nM.</p

<i>T. gondii</i> tachyzoites express two SFAs that localize to a structure close to the nucleus.

<p>(A) Electron micrograph and schematic of a dividing <i>T. gondii</i> parasite. Two daughters are assembled within a mother cell. Centrosomes are shown in blue, apicoplast in green. (B) Robust multi-array averages of SFA2 and SFA3 transcripts over two consecutive division cycles based on dataset collected by Behnke and colleagues <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Behnke1" target="_blank">[19]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Gajria1" target="_blank">[20]</a>. (C) Parasites do not express TgSFA2 at all times. Parasites expressing SFA2-HA were scored by IFA using anti-HA antibody in an asynchronous parasite population (<i>n</i> = 250). (D) Western blot analysis shows a 30-kDa band when probed with anti-HA antibody in SFA2-HA transgenics but not the parental strain. Anti-SFA3 antibodies recognize the recombinant protein used for immunization (rSFA3), as well as the native protein in parental parasites. Lower mobility bands of approximately 60 kDa likely correspond to dimers formed both by the recombinant and native SFA3 proteins. Anti-SFA3 antibodies also recognize YFP tagged SFA3 in the endogenously tagged TgSFA3-YFP strain. The expected masses for SFA3 and SFA3-YFP are 36 kDa and 56 kDa, respectively. Anti-GFP antibodies specifically recognize SFA3-YFP in the endogenously tagged strain, but not in the parental strain. (E) IFA of SFA2-HA parasites stained with anti-SFA3 and anti-HA antibody and DAPI. Note that both SFA proteins localize to two fiber-like structures per parasite.</p

Is the apical host cell invasion complex derived from the flagellum of the algal ancestor?

<p>(<b>A</b>) Schematic of the flagellar rootlet system of <i>Chlamydomonas</i> (simplified after <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Harris1" target="_blank">[64]</a>). The two flagellar basal bodies are coordinated by rootlet fibers (only SFA and distal fibers are shown here) and bundles of rootlet microtubules (two or four microtubules each) (<b>B</b>). Schematic outline of cell division in the hypothetical flagellated algal ancestor of Apicomplexa. Basal bodies of the flagella also serve to organize the mitotic spindle (flagella are resorbed or shed during mitosis in some flagellated algae, note that number and behavior of flagella in the apicomplexan ancestor is hypothetical). Rootlet fibers (white) may have additional roles in division <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Dutcher1" target="_blank">[32]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Mittelmeier1" target="_blank">[37]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Ehler1" target="_blank">[38]</a>. (<b>C</b>) Apicoplast, green; nucleus, grey; SFA fiber, white; basal body/centrosome, red; conoid, blue, rhoptries (secretory component of the apical invasion apparatus), light grey. Apicomplexans are intracellular parasites and have lost flagella in most stages. SFA rootlet fiber is only expressed during division and coordinates the centrosome with the MTOC of the daughter bud. This suggests that the system that controlled the positioning and assembly of flagella in the ancestor now organizes the assembly of the apical host cell invasion complex. (<b>D</b>) Schematic of the SFA fiber and its relationship to other cellular structures during <i>T. gondii</i> cell division (only a single daughter bud is shown for simplicity).</p

The SFA fiber extends in the apex of the forming daughter.

<p>IFAs showing SFA2-HA stained with anti-HA (green) in combination with markers for daughter bud. (A) anti-tubulin (red) labels the sub-pellicular microtubules. SFA2 staining extends beyond the sub-pellicular microtubules and into the conoid. (B) Anti-IMC3 (blue) labels the IMC of emerging daughter cells and is shown as a reference for the position of daughter cells. RNG1-YFP was detected using anti-GFP antibodies (red). RNG1 is a marker of the conoid <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Tran1" target="_blank">[29]</a>. The SFA2-HA signal extends into and slightly beyond the ring of RNG1 staining. (C) anti-ISP1 (blue) labels the apical cap of the IMC. Anti-Morn1 (red) labels the basal complex of both the mother's and the daughter's IMC, as well as, a nuclear structure in immediate proximity of the centrosome <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Gubbels1" target="_blank">[30]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Hu4" target="_blank">[31]</a>. The SFA2-HA signal spans from the centrosome region to the apex of the daughter bud. Scale bars = 5 µm.</p