To search for the presence of MOB genes in the parasite T. gondii, we performed a bioinformatics analysis by searching the ToxoDB.org database using as queries the deduced amino acid sequences of the best-characterized MOB1 proteins, the H. sapiens HsMOB1A and the S. cerevisiae ScMOB1. This search revealed the presence of one gene coding for a MOB1 protein, TgMOB1 (TGME49_304730). TgMOB1 is predicted to have 313 amino acids and a molecular mass of 34.9 kDa. This protein presents 24.5%/15.7% of identity and 38.9%/23.6% of similarity with the HsMOB1A and ScMOB1 sequences, respectively (Figure 1A). Additionally, we extended our search to other Hippo/MEN/SIN core members to assess the putative conservation of these pathways in T. gondii (Figure S1). We identified two NDR kinases that are probable homologs to Dbf/2/20p/LATS1/2 and NDR1/2 kinases, and an STE kinase that could perform a similar function to that of Cdc15p/MST1/2 kinases [49].

Next, we compared the putative MOB1 sequence from T. gondii with MOB1 proteins from another eukaryote previously characterized. This comparison revealed that, as expected, TgMOB1 has the highest similarity with the MOB1 sequence from the related apicomplexan parasite Neospora caninum, and both diverge substantially in the N-terminal and C-terminal regions from the MOB1 sequences of the other eukaryotes assessed. Still, the central region of TgMOB1 contains the conserved residues corresponding to the MOB kinase activator family domain (T. gondii amino acids 69–229, PF03637) (Figure 1B). The zinc-binding cysteine and histidine pairs are conserved in all the species assessed. However, while the T. gondii and N. caninum MOB1 proteins present a CQC motif, the rest of the assessed species present a CXXXXC motif (Figure 1B). The ScMOB1 homodimer, detected in crystallography studies, is mediated by conserved residues that allow the association of a shallow hydrophobic depression and the amphipathic helix H1 (referred to as H0 in Mrkobrada et al. [32]). These residues are moderately conserved in T. gondii (Figure 1B). Of eight ScMOB1 residues identified as participating in the NDR–MOB1 interaction, the four residues located more in the C-terminal are conserved in T. gondii (D88, E91, D99, and L106) [33]. Of the more N-terminal residues, the NDR kinase binding motif LPXGED [33] is minimally conserved in T. gondii that presents a PATCVD motif instead (Figure 1B). To further analyze TgMOB1, we obtained a T. gondii MOB1 theoretical 3D structure model based on the known structure of the ScMOB179-314 region (PDB h2jn) (Figure 1C). The raw alignment included the residues 67–235 (54% coverage), 31% identity, a global model quality estimate (GMQE) of 0.33, and a qualitative model energy analysis distance constraint (QMEANDisCo) global score of 0.55 ± 0.07. The difference in identity score to that reported in Figure 1A occurs because the model alignment uses the ScMOB179-314, lacking the 78 amino acid N-terminal region that is specific to S. cerevisiae. The resulting model presents a core domain with an α helix-based structure organized in a bundle, common to MOB proteins and similar to the structure of the template ScMOB179-314. The results of the 3D model indicate high confidence in homology between proteins.

In order to investigate the evolutionary relationship between MOB1 proteins from different organisms, we performed a phylogenetic analysis, including parasites as well as model organisms (Figure 2). The first node separates animals from two other groups: (i) the group containing plants and unicellular organisms such as microalgae (Chlamydomonas reinhardtii) and ciliates (T. thermophila, Stentor coeruleus); and (ii) the group containing the remaining organisms. Focusing on the unicellular organisms included in the phylogenetic analysis, we observe that free-living and parasitic organisms are in different clades of the tree. Regarding parasitic organisms, a distinction between the Trypanosoma and Leishmania species, which belong to the supergroup Excavata, and parasites belonging to the phylum Apicomplexa (T. gondii, Hammondia hammondi, N. caninum, Eimeria tenella, Eimeria maxima, Cryptosporidium parvum, and Cryptosporidium muris) was detected. However, the MOB1 sequence from Giardia lamblia (Excavata) appears closer to the Apicomplexa than to the Trypanosomatida. The Apicomplexa form a monophyletic group in which cyst-forming coccidia (T. gondii, H. hammondi, N. caninum) appear very closely related and separated from the monoxenous genus Eimeria, while C. muris and C. parvum form a more distant group, mirroring the evolutionary history of these species. To date, no MOB proteins have been identified in hematozoan genomes, namely in Plasmodium spp., Babesia spp., or Theileria spp. [49].