Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family

Abstract

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Keywords: BMR1_03g00960; Babesia microti; BmIPA48; human babesiosis; piroplasmid rhoptry-associated protein-1 (pRAP-1).

Figure 1

Representative figure of the comparisons performed with the pRAP-1 Cys-rich motif among babesial rRAP-1 proteins and the newly identified B. microti putative pRAP-1. The comparisons include the Cys-rich regions of B. bovis RRA, B. bovis RAP-1, B. bigemina RAP-1c, and the B. microti RAP-1- like proteins BmIPA48 and Bm960.

Figure 2

Synteny map of the rap-1 locus of T. equi, putative rap-1 B. microti, and typical s.s. B. bovis. (A) Structure of the prap-1 and rra genes localized in the chromosome 4 of B. bovis. (B) Conserved synteny among the rap-1 loci of B. bovis and T. equi and the BMR1_03g00960 (BmIPA48) and BMR1_03g00947 (Bm960) genes of B. microti, that encode for proteins containing the typical Cys-rich region of the pRAP-1 proteins. (C) Conserved synteny among the B. bovis rra and the B. microti BMR1_03g00960 and BMR1_03g00947 genes.

Figure 3

Schematic representation of the locus encoding for the B. microti RAP-1 putative proteins BmIPA48 and Bm960. A ~300 bp region upstream the two ORFs is repeated (yellow boxes).

Figure 4

Predicted secondary structure of the pRAP-1-like proteins BmIPA48 and Bm960 using the Program TMpred. Predicted location of signal peptide (SP) is marked with a red bar. A dashed red line marks the boundary between predicted hydrophilic and hydrophobic transmembrane regions of the proteins.

Figure 5

Phylogenetic neighbor joining tree inferred using amino acid sequences of pRAP-1 from the reference genomes of s.s. Babesia (Clade VI), s.s. Theileria (Clade V), T. equi (Clade IV), C. felis (Clade III), and B. duncani (Clade II) and the B. microti RAP-1 proteins BmIPA48 and Bm960 (bold fonts, red boxes). Bootstrap values of 1000 replicates are shown next to the branches. BmIPA48 is used as outgroup. The scale gives the evolutionary distance used to construct the tree.

Figure 6

Immunogenicity of BmIPA48 and Bm960 in B. microti-infected humans. Expression of the recombinant BmIPA48 and Bm960 containing a HIS-tag in the immunoblots was demonstrated using an anti-HIS monoclonal antibody (panels (A,B), respectively). A control lysate of cells not expressing the recombinant protein was included as a negative control (HEK 293). Immunoblots were incubated with human B. microti positive and negative sera. ELISAs were performed with four positive (Pos 1, Pos 2, Pos 3, and Pos4) and one negative (Neg) human serum samples were tested. A control sample incubated only with secondary anti-human IgG serum (secondary only) was also included in the ELISA analysis. ** p < 0.001. * p < 0.01.