Babesia Genome Sequencing And Mapping And A High-Abundance Antigen 03g00237 Identification And Charactrization

Babesiosis is a type of blood protozoal disease caused by Babesia.The symptoms of this disease are mainly fever,hemoglobinuria,hemolytic anemia and even death.and more than 100 types have been reported.Babesia gibsoni（B.gibsoni）,Babesia canis（B.canis）,Babesia canis vogeli（B.canis vogeli）and Babesia canis rossi（B.canis rossi）are the main causes of babesiosis in dogs,and the main pathogen in China is B.gibsoni.At present,there is no commercial detection kit for Babesiosis,no effective vaccine to promote and no effective treatment,so the comprehensive prevention and control of babesiosis is urgent.After infection,Babesia spp.reproduce asexually in the host by fission reproduction.The released merozoites invade more red blood cells,resulting in continuous rupture of host red blood cells and damage to the host circulatory system.During this process,Babesia spp.secretes antigens that allow the insect to specifically recognize,adhere to,or invade host red blood cells.In theory,this type of secreted antigen is exposed to the host immune system and can cause the host immune protection mechanism.Therefore,they have been considered as an excellent diagnostic marker or vaccine candidate,which is of great significance for the diagnosis and prevention of Babesiosis.Therefore,screening high-quality secreted proteins is of great significance to explore the invasion and pathogenesis of Babesia.With the development of bioinformatics,high-throughput screening of proteins with certain characteristics based on genomic information,such as secreted proteins with typical secretion characteristics or GPI proteins with Glycosylphosphatidy-linositol-anchored,etc.Complete genomic information is the prerequisite for screening secreted antigen.In this study,the whole genome（extranuclear genome and nuclear genome）of B.gibsoni was sequenced to complete the mapping and analysis of its genome,and the secreted antigen with typical secreted characteristics on the genome was successfully screened.Combined with the protein mass spectrometry analysis of the parasite in vitro culture supernatant,a highly abundant secreted antigen was finally screened and identified.The main results are as follows:（1）Mapping the genome of the extranuclear（apicoplast）: The extranuclear genomes of B.gibsoni include mitochondrial genome and apicoplast genome,of which mitochondrial genome have been reported.In this study,the apicoplast genome of B.gibsoni was studied,and the results showed that the apicoplast genome of B.gibsoni is composed of28386 bp（28.4 Kb）circular DNA molecules,with A+T content as high as 86.33%.The number of t RNA was less than that of other Babesia（23/24）,and the gene structure of the four gene clusters was relatively conserved.Phylogenetic analysis showed that the apicoplast genome of B.gibsoni and B.orientalis are highly similar.In the process of evolution,the apicoplast genome of B.gibsoni tends to be simplified,leaving only some genes with important functions,a few genes are transferred to nuclear genes,and finally targeted to the apicoplast to function.The results of this study will contribute to the design of novel drug targets against Babesiosis based on the apicoplast.（2）Mapping nuclear genome: In this paper,the collection of pure B.gibsoni without host contamination was overcome by an improved method.Complete mapping,annotation and analysis of B.gibsoni nuclear genome based on whole genome sequencing technology.The results showed that the nuclear genome size was about 7937104 bp（7.94 Mb）,containing 4 chromosomes and about 3552 genes.Genomic collinearity analysis showed that B.gibsoni had 2903 collinearity genes with Babesia bovis（B.bovis）,which was higher than that with Babesia microti（B.microti）.Species time tree analysis showed that B.gibsoni had an earlier divergence time than other Babesia.Furthermore,the comparative genome-Venn diagram showed that B.gibsoni had 22 unique COGs,which were mainly composed of transporter secreted proteins and parasite merozoite proteins,revealing the potential species-specificity of B.gibsoni.KEGG analysis and GO analysis showed that about 73.9%（2641/3572）genes were enriched into 316 KEGG pathways and 608 GO functions,respectively.（3）Screening and identifying 03g00237 as a high abundance secreted antigen: In this study,376 secreted proteins were selected based on the characteristics of signal peptides and transmembrane regions of all proteins predicted by genomic information.Combined with the results of protein mass spectrometry analysis of the supernatant of babesia in vitro,the high abundance secreted protein Bg WH₀3g00237（03g00237）was selected as candidate antigen.The candidate antigen was identified as GPI1,a member of the GPI protein family（GPI1-9）.The results showed that the protein was a high abundance protein with good immunogenicity and reactivity.It was mainly located in parasite cytoplasm and could also be secreted into the parasite.（4）C onstruction of single gene knockout strain Δ03g00237: Two monoclonal strains of Δ03g00237 were screened out and identified by conventional PCR,Southern blot,q PCR,Western blot and IFAT,respectively.The single gene knockout strain Δ03g00237 was successfully constructed.The protein 03g00237 was identified to affect the growth ofparasite,and it was speculated that it might be a protein related to parasite adhesion/invasion of red blood cells.In summary,this study mapped the genome of B.gibsoni and filled in the gap of B.gibsoni’s genome information.A high abundance protein 03g00237 associated with parasite growth was screened and identified.This protein is highly similar to 8 other members of the GPI protein family（Bg GPI1-9）,indicating that this protein is a potential diagnostic marker or drug target.The results of this study laid a foundation for the exploration of biological functions of B.gibsoni,and also provided a theoretical basis and scientific basis for the study of diagnostic identification and drug targets of babesiosis.