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. 2008;3(10):e3611.
doi: 10.1371/journal.pone.0003611. Epub 2008 Oct 31.

In silico identification of specialized secretory-organelle proteins in apicomplexan parasites and in vivo validation in Toxoplasma gondii

Zhongqiang Chen  1 , Omar S Harb, David S Roos
Affiliations

In silico identification of specialized secretory-organelle proteins in apicomplexan parasites and in vivo validation in Toxoplasma gondii

Zhongqiang Chen et al. PLoS One. 2008.

Abstract

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pfam domains and domain patterns in the microneme proteins of apicomplexan parasites.
Microneme proteins are shown for representative species in each parasite genus: Tp, Theileria parva; Bb, Babesia bovis; Pf, Plasmodium falciparum; Cp, Cryptosporidium parvum; Et, Eimeria tenella; Sm, Sarcocystis muris; Nc, Neospora caninum; Tg, Toxoplasma gondii. The tree provided at top indicates phylogenetic relationships (branch lengths not proportional to phylogenetic distance). Known microneme proteins are indicated by green circles, and Pfam domains by colored boxes (lengths proportional to the actual domain length, except for AMA-1). Subscripts (m,n) mark domains that may be repeated. Domains include: VWA, von Willebrand factor type A domain; TSP_1, thrombospondin type 1 domain; EGF, epidermal growth factor-like domain; EGF-CA, calcium binding EGF domain; PAN_1, PAN domain; GETHR, GETHR pentapeptide repeat; FAINT, frequently associated in Theileria; AMA-1, apical membrane antigen 1; Etmic-2, microneme protein Etmic-2; Duffy_binding, Duffy binding domain; Rhomboid, rhomboid family; Peptidase_S8, subtilase family. Accession numbers (where available): Tp: VWA: EAN31658, FAINT: AAA18217, AMA-1: XP_766171; Bb, VWA: AAS58046; AMA-1: AAS58045; Pf: VWAmTSP_1n: CAD52497 AAC46961; TSP_1n: AAN36262; AMA-1: AAN35928; Duffy_binding: AAK49521 AAS46319 P19214; Cp: TSP_1n: AAC48311; TSP_1mEGFn: AAC16621; Et: VWAmTSP_1n: AAD03350; (EGF or EGF_CA)n: CAC34726; PAN_1n: CAB52368; Etmic-2: AAD05566; Sm: PAN_1n: AAF36512 Q08668 Q26539; GETHR: CAA81555; Nc: VWAmTSP_1n: AAF01565; (EGF or EGF_CA)n: AAF19184; PAN_1n: DAA05464; AMA-1: BAF45372; Peptidase_S8: AAF04257; Tg, VWAmTSP_1n: AAB63303; (EGF or EGF_CA)n: AAD28185 CAB56644 AAK35070 AAK19757; PAN_1n: CAJ20618 AAD33906; AMA-1: AAB65410;Peptidase_S8: AAK94670; Rhomboid: AAT29065. This figure does not include the following validated microneme proteins, which lack Pfam domains: Pf: AAM47192 AAN35478 CAD49152; Cp: AAC98153; Et: AAR87666 AAR87667; Sm: AAK35069; Nc: AAL37729 AAG32025 AAN16380 AAK74070; Tg: CAA96466 CAA70921 AAK19758 AAG32024 AAN16379 AAK58479 AAK51546.
Figure 2
Figure 2. Subcellular localization of eight candidate adhesive domain-containing proteins in T. gondii.
Left: Direct fluorescence of YFP in T. gondii tachyzoites expressing fusion constructs of eight candidate adhesive domain-containing proteins (numbers indicate gene IDs). Right: Fixed parasites transfected with YFP- or HA-tagged transgenes were stained with anti-HA (76.m01642 only) or anti-YFP (green), anti-TgMIC10 (red), and anti-TgROP2/3/4 (blue). Open and filled arrowheads in 8.m00177 indicate rhoptry and microneme staining, respectively; arrows in 44.m04666 indicate dense granules.
Figure 3
Figure 3. Interactomic predictions of human host partners for parasite adhesive domain-containing proteins.
A: Venn diagram depicting parasite-host iPfam (Phifam) and the parasite-host interactome (Phint) datasets (see Supplemental Table S15 for complete list). B: Graphical representation of the percentage of secretory signal peptide (SP) and signal anchor (SA)/transmembrane (TM) containing proteins within Phifam, Phint and their intersection, demonstrating SP/SA/TM enrichment in the dataset of predicted interacting partners (compare with the entire human proteome, as indicated by background color). C: List of host factors known to interact with T. gondii and/or P. falciparum found in either the Phifam or Phint datasets (Supplemental Table S18). D: A selected list of host proteins predicted to interact with PAN domain-containing proteins.
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