In-depth Analysis Of The Genome Of Trypanosoma Evansi And Preliminary Study On Inhibitors Targeting Key Enzymes

Trypanosoma evansi is a unicellular flagellated pathogen that proliferates extracellularly in the blood of a wide diversity of mammals.T.evansi is transmitted by vectors,or directly infected by eating raw meat of the infecting animals through damaged mucosal tissue.Trypanosomiasis caused by T.evansi is known as Surra,which is a serious economic burden in tropical and subtropical regions around the world.The susceptible animals of T.evansi are buffalo,horse,camel,cow and dog in China.In recent years,there have also been reports of human infected by T.evansi,suggesting that T.evansi is likely to be a new pathogen of zoonotic parasitic diseases.So far,there are limited treatments and preventions deal to trypanosomiasis.T.evansi relies on the variant surface glycoprotein(VSG)to avoid elimination by the immune system,but the potential factors for regulating its gene expression are still unclear.In-depth analysis of the molecular biological characteristics of this pathogen is the theoretical basis for establishing effective control measures.The availability of its closely related parasite T.evansi genome allows us to compare their genetic and evolutionarily shared and distinct biological features.20 S proteasome and phosphatidylinositol 3-kinase(PI3K)are two key enzymes of eukaryotes,which are involved in the protein degradation pathway and PI3K-AKT signaling pathway of T.evansi,respectively.The stability of these two proteins plays an important role in survival,growth and apoptosis in T.evansi.Firstly,a complete genomic sequence of T.evansi YNB strain was obtained in this study using a combination of genomic and transcriptomic sequencing,de novo assembly,bioinformatic analysis.Secondly,large-scale of synteny analysis,regulation of(post-)transcriptional gene expression and identification of genes associated with pathogenesis were carried out by in-depth analysis of omics.Lastly,based on omic results,we explored the two inhibitors targeting to key enzymes 20 S proteasome and PI3 K and their effects on survival,development and apoptosis.As the results,the genome size of T.evansi YNB strain was 35.2 megabases,with 96.59% similarity in sequence and 88.97% in scaffold alignment to that of T.brucei.A total of 8,617 protein coding genes,accounting for 31% of the genome,were predicted and 1,641 alternative splicing(AS)events of 820 genes were identified with a majority mediated by intron retention,which represented a major difference in posttranscriptional regulation between T.evansi and T.brucei.Disparities in copy number and in the variant surface glycoprotein(VSG)genes,expression site-associated genes,micro RNAs and RNA-binding protein(RBP)genes were clearly observed between the two parasites,while the glycosylphosphatidylinositol anchor(GPI)and transcriptional factors were consistent among the two species.Bioinformatics analysis of the two enzymes revealed that 20 S Proteasome of T.evansi participates in the Proteasome pathway with its relatively conserved amino acid sequence,and it contains a conserved proteasome recognition marker and functional domain.PI3 K participates in PI3K-Akt signal pathway,and there has no transmembrane region or signal peptide in the amino acid sequence.PI3 K has a typical PI3 K C2 domain conserved functional domain.The functions of both proteins are related to the survival,growth,development and apoptosis.The 20 S proteasome inhibitor PS-341 and the PI3K? inhibitor AS-605240 were added to T.evansi in vitro.It was found that the viability of T.evansi was significantly inhibited at 48 h,and T.evansi was almost dead after 72 h.Gene expressions of downstream genes of both enzymes in the signal pathway were reduced.Both of the two inhibitors could induce apoptosis especially the early apoptosis by the method of flow cytometry.In conclusion,we resequenced the genome of T.evansi,and the biological determinants of the parasite were analyzed in-deep by comparing with that of T.brucei.The results revealed the genomic determinants of T.evansi,which encoded specific biological characteristics that distinguished from other related trypanosomal species.Depended on omic analysis,we identified two key enzymes and explored its targeting function by inhibitors.Our results provide a molecular basis for searching the pathogenic mechanism in T.evansi and a new insight for developing other drugs and therapies for trypanosomiasis.