Analysis Of Evolutionary Characteristics And Comparative Genomics Analyses Of Cryptosporidium Species With Different Host Ranges

Cryptosporidium spp.are important apicomplexan parasites,causing gastrointestinal diseases in humans and other vertebrates,which greatly threaten public health and animal husbandry.Thus far,there are 40 recognized Cryptosporidium species,most of the species/genotypes are host-specific,but few of them could spread across different hosts.Among them,five species are common in humans,but novel human-pathogenic species have been reported continuously.To date,the genomes of seven Cryptosporidium species have been sequenced,and genomes of the two major species infecting humans have been analyzed thoroughly.However,the evolutionary characteristics of Cryptosporidium spp.and the genetic determinants of host adaptations are not yet clear.To address these issues,we conducted the following studies.(1)Cryptosporidium chipmunk genotype I(a novel human-pathogenic species),Cryptosporidium bovis,and Cryptosporidium ryanae(two bovine-specific species)were selected for isolation of the oocysts using immunomagnetic separation.After extracting genomic DNA from purified oocysts,we conducted whole-genome amplification(WGA)and assessed the purity of WGA products using qPCR analysis.The whole genome sequences of the three species were obtained using paired-end Illumina sequencing,which lays the foundation for revealing the genetic mechanism of the difference in host range among the species infecting the same host.(2)Through the evolution analysis of protein families among 10 Cryptosporidium species and related apicomplexans,we found that the protein families have undergone various of losses and gains during the evolution of Cryptosporidium species.Compared with other apicomplexans,the genomes of Cryptosporidium spp.are highly condensed and most of the gene families are single-copied.Only a few protein families have experienced significant duplications,suggesting that they could play an important role in the invasion and growth of Cryptosporidium species.A comparative analysis between intestinal and gastric Cryptosporidium species revealed that the major divergence between them is in the number of metabolism and invasion-related protein families.(3)To further understand the genetic characteristics among human-pathogenic Cryptosporidium species,we.compared the genome of a-novel zoonotic.pathogen,Cryptosporidium chipmunk genotype I,with genomes of Cryptosporidium parvum,Cryptosporidium hominis,and Cryptosporidium meleagridis,which are recognized as the common human-pathogenic Cryptosporidium species.Cryptosporidium chipmunk genotype I possessed genome organization and metabolic pathways similar to the three common humanpathogenic Cryptosporidium species,indicating that the genetic characteristics are similar among human-pathogenic species.The comparative genomics analysis further revealed that the number of copies in two subtelomeric gene families(those encoding insulinase-like proteases and MEDLE secreted proteins)is higher in Cryptosporidium species with a broad host range.(4)To confirm some of the above conclusions,three common intestinal Cryptosporidium species in cattle were selected for comparative genomics analysis.It was found that the bovinespecific species have similar gene organization and metabolic pathways.Compared with bovine-specific species,in addition to the increased numbers of genes encoding insulinase-like proteases and MEDLE proteins,the copy numbers of several other multi-copy genes(including those for mucin-type glycoproteins,FLGN,SKSR,and NFDQ proteins)are also higher in C.parvum,which has a broad host range.Furthermore,sequence polymorphism was found in mucin-type glycoproteins,hydrolases,and oocyst wall proteins among the three bovineinfecting species.(5)To verify the differences in the number of multi-copy genes and the sequence polymorphisms among Cryptosporidium species,two representative genes(MEDLE and gp60 genes)were selected for PCR and sequence analyses.We found that more MEDLE genes were detected in C.parvum(with a broad host range),but only one or two MEDLE genes were detected in other Cryptosporidium species with narrow host ranges.In addition,by PCR amplification and DNA sequencing of the gp60 gene in different C.bovis isolates,we found that the sequences obtained could be divided into six subtypes,reaffirming the polymorphic nature of this gene.In conclusion,during the evolution of Cryptosporidium spp.,two major protein families have undergone duplication;intestinal Cryptosporidium species infecting the same host possess similar genome organization and metabolic patways;the differences among Ceyporidium species with different host ranges are not only in the number of multi-copied genes but also in the sequence polymorphism of homologous proteins.These observations improved our understanding of genetic determinants for biologic differences among Cryptosporidium species,which also help to promote research on the invasion and host adaptation in Ceyptosporidium species.