TgMAP1c is involved in apicoplast biogenesis in Toxoplasma gondii
Graphical abstract
Introduction
Protist parasites from the phylum Apicomplexa are a global health threat due to their ability to infect humans and animals. Toxoplasmosis, caused by Toxoplasma gondii, affects almost one-third of the world’s population, and is a severe threat to immunocompromised individuals and pregnant women. The prevalence of toxoplasmosis and the emergence of parasitic drug resistance emphasise the need to identify novel chemotherapeutic targets.
Most apicomplexan animals harbour a remnant chloroplast called the apicoplast. Although no longer photosynthetic, this plastid-like organelle still houses important biosynthetic pathways, including type II fatty acid synthesis (Mazumdar et al., 2006), heme synthesis (Obornik and Green, 2005), and isoprenoid biosynthesis (Nair et al., 2011). Apicoplast pathways are essential for cell viability in T. gondii and Plasmodium (Mazumdar et al., 2006, Nair et al., 2011). Because this organelle is unique to these parasites and is not found in the human host, apicoplast proteins and structures are excellent candidates as parasite-specific drug targets.
The T. gondii apicoplast is surrounded by four membranes and contains its own 35 kb genome, which is extremely well conserved and shares similarity with red algae chloroplast genomes (Gibbs, 1981). The proteins encoded by the apicoplast genome mostly function in apicoplast gene expression, e.g., ribosomal proteins and elongation factors (Wilson et al., 1996, Cai et al., 2003). Most of the apicoplast proteins are nuclear-encoded and transported into the organelle. This transport occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence that includes a signal peptide and a transit peptide (Waller et al., 1998). Some of these proteins are located on the apicoplast membrane, between the membranes, or in the interior of the organelle, where they participate in apicoplast biological processes.
Protein synthesis is initiated by either methionine in eukaryotes or formylmethionine in prokaryotes, mitochondria, and chloroplasts. N-terminal methionines can be co-translationally cleaved by the enzyme methionine aminopeptidase (MAP). The Protein Data Bank (PDB; www.ncbi.nlm.nih.gov/protein/) contains more than 100 MAP structures, reflecting the importance of specific targeting of this class of enzyme (Helgren et al., 2016). MAPs contribute to the co-translational control of proteins, as they govern their half-life via the N-terminal end rule and facilitate further modifications such as acetylation (among others). In eukaryotes, the methionine is removed either by cleavage of the N-terminal signal peptide used for secretion (or other processes) or by MAPs. In prokaryotes, the formyl group is first removed by formylmethionine deformylase, yielding an N-terminal methionine that is then processed by a MAP. In humans, MAPs are categorised into two types, MAP1 and MAP2, with the main difference being the insertion of an α-helix of approximately 60 amino acids in the catalytic domain of the MAP2 enzymes (Arfin et al., 1995, Bradshaw et al., 1998). MAP1 enzymes, which are more common in prokaryotes, are further divided into four subtypes: MAP1a, 1b, 1c and 1d (Gonzales and Robert-Baudouy, 1996). MAP1b and 1d are present in humans (Arya et al., 2015), while MAP1a and 1c are additional enzymes found in some bacteria and parasites. Approximately two-thirds of the proteins in any proteome are potential substrates for N-terminal methionine processing, which appears to be essential for cell function. In bacteria and yeast, knockout or inhibition of MAPs is lethal (Chang et al., 1989, Li and Chang, 1995). In humans, MAP1 and MAP2 are essential to control cell proliferation (Bernier et al., 2005). Furthermore, MAPs are overexpressed in cancer cells, and their inhibition is targeted for drug development.
Toxoplasma gondii possesses members of both the MAP1 and MAP2 subfamilies. There are three members of the TgMAP1 subfamily in T. gondii, TgMAP1a–c. Here, we found that these three MAP1 proteins may have distinct substrate preferences, and that all of them are essential for the lytic cycle of parasites. Furthermore, we found that TgMAP1c is required for apicoplast biogenesis.
Section snippets
Bioinformatics analyses and identification of TgMAPs
Human and yeast MAP1 protein sequences were retrieved from the GenBank database, and homologs of these proteins in the Apicomplexa, including T. gondii, were identified by BLASTp queries using EuPathDB (http://EuPathDB.org). The transmembrane helices in TgMAP proteins were predicted using the TMHMM webtool (http://www.cbs.dtu.dk/services/tmhmm/), and the conserved regions of the proteins were identified based on structure predictions in ToxoDB (https://toxodb.org/toxo/).
Phylogenetic analyses
Localization of MAPs encoded in the T. gondii genome
A search of the T. gondii GT1 genome database (http://toxodb.org) for sequences displaying homology to the catalytic domains of human and yeast MAPs identified four candidate members of the MAP family. A phylogenetic tree based on a neighbour-joining analysis revealed that three of the TgMAPs are closely related to human MAP1 (HuMAP1) and Saccharomyces cerevisiae MAP1 (ScMAP1) (Fig. 1A), and have hence been named TgMAP1a–c (Accession numbers in ToxoDB were TGGT1_257730 (TgMAP1a), TGGT1_248850 (
Discussion
The critical roles of MAPs in the maintenance of cellular physiology and the survival of organisms have been demonstrated by several previous studies. Deletion or inhibition of MAPs in bacteria can inhibit their growth (Chai et al., 2011). Inhibition of the MAPs of the microsporidian parasites Enterocytozoon bieneusi, Encephalitozoon hellem and P. falciparum has therapeutic effects both in vitro and in vivo (Molina et al., 2002, Chen et al., 2006, Chen et al., 2009, Arico-Muendel et al., 2009).
Acknowledgements
This work was supported by the National Key Research and Development Program of China (grant number 2017YFD0500400 to H. Jia and C. Du) and Natural Science Foundation of Heilongjiang Province of China (grant number C2015063 to H. Jia ). We thank Dr. Dominique Soldati-Favre for providing the 5’MyoF-TATi1-HX-tetO7S1MycNtMyoF10 plasmid and Dr. Zhigao Bu for providing the pCAGGS plasmid.
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These authors contributed equally.