Abstract:

Objective To analyze the soluble proteins of Babesia microti by using proteomics and bioinformatics methods. Methods BALB/c mice were intraperitoneally injected with 100 μl of blood containing B. microti parasites to establish the mouse model of B. microti infection. Red blood cells were separated from the B. microti-containing blood of the infected mice at the peak of infection. The B. microti parasites were enriched by Percoll density gradient centrifugation, freeze-thawed and sonicated to obtain soluble proteins of B. microti. SDS-PAGE was used to identify the molecular weight distribution of proteins. The gel containing soluble proteins was divided into 2 samples by molecular weight and identified by the electrospray ionization mass spectrometry (ESI-MS) method. The ESI-MS data were collected. Protein data for Babesia spp., B. microti (strain RI) and Plasmodium falciparum were searched on the Uniprot KB database. The identified proteins were aligned with the protein sequences in the NCBI nr database, and Gene Ontology(GO) function associated with the aligned sequences of all identified proteins were extracted with the Mapping function in Blast2GO (Version 2.8.0). The GO annotations were used for analysis of biological processes, molecular functions, and cellular components of the identified proteins. The target protein sequence was aligned with the Babesia protein sequence in the KEGG GENES database using KAAS, and the relevant KEGG pathway was annotated with the KO number of the homologous/similar protein.  Results The B. microti parasites were enriched by Percoll gradient centrifugation and soluble proteins were obtained by freeze-thawing and sonication. SDS-PAGE showed that there were 5 major bands and 7 minor bands. After ESI-MS and alignment with proteins of Babesia spp., B. microti (strain RI) and P. falciparum, 757, 600 and 138 proteins were identified, respectively, of which 368, 375 and 12 had more than 2 unique peptide segments, respectively. Further analysis revealed that those with more unique peptide segments were surface antigens or secreted antigenic proteins, proteases, heat shock family proteins and rod-like proteins. Bioinformatics analysis resulted in 876 annotations for the soluble proteins according to the biological processes in which they are involved, 219 annotations by molecular function and 146 annotations by cell components. By KEGG annotation of homologous/similar proteins, a total of 172 KEGG signal/metabolic pathways related to the soluble protein sequences were extracted.  Conclusion The soluble proteins of B. microti are mainly composed of secretory proteins, proteases, and HSP family member proteins, as revealed by ESI-MS identification and bioinformatics analysis.

Key words: Babesiosis, Babesia microti, Mass spectrometry, Bioinformatics analysis