Transcriptomic And Proteomic Studies Of Feline Intestines During Toxoplasma Early Infection

Toxoplasma gondii is an obligate intracellular protozoon,which can cause the zoonotic toxoplasmosis.The disease is widely prevalent in swine,sheep,horses,dogs,chickens,cats and human beings.Cat,including members of the family Felidea,is the only definitive host of Toxoplasma,and feline intestine is the only tissue that exclusively supports its sexual reproduction.Previous studies have showed that the development of gametogenesis begins at 8 h post T.gondii infection in cats,which lasts for 4-6 d.Humans can be infected via accidental ingestion of water contaminated with oocysts,or ingestion of oocysts in contaminated food sources.For infants,pregnant women and immunocompromised individuals,toxoplasmosis is able to result in severe clinical symptoms or even death.However,no effective measures are available to prevent and control cat toxoplasmosis until now due to limited information on the formation of oocysts.Here,we collected the feline intestines during the development of T.gondii gametogenesis,and analyzed the transcriptome and proteome of feline intestines from each time point during early infection.The study will provide the base-data for further study of molecular mechanisms of the development of T.gondii gametogenesis.The Chinese Li Hua cats(Felis catus)were inoculated with T.gondii PRU strain.At 6,12,18,24,72,and 96 h post-infection(hpi),cats from each group were humanely sacrificed to collect the intestines.Then the transcriptome and proteome of cat intestines during T.gondii early infection were sequenced by RNA-seq and i TRAQ techniques,respectively.1.The transcriptome of cat intestines during T.gondii early infectionThe in-depth transcriptome analysis revealed a total of 2363 different expression genes(DEGs).Our analysis also revealed 56,184,404,508,400 and 811 DEGs in infected intestines compared to uninfected control at 6,12,18,24,72 and 96 hpi,respectively.The chromosome location of DEGs between the different time points after infection showed more upregulated gene expression on chromosomes D2,D3 and E2.However,a striking pattern of biased/downregulated gene expression on chromosomes X was associated with T.gondii infection.Gene ontology(GO)analysis revealed catalytic activity and metabolic process as the most prominent processes involved in infection.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that transcriptional changes in the intestines of infected cats evolve over the course of infection,with the greatest number of differences in enriched pathways was observed at 96 hpi.The main response of cat intestine to infection was mediated by specific pathways,such as the MHC I presentation,HSPs binding,proteasome responses and FABPs release.The infection-related alterations in gene expression have also been involved in xenobiotic metabolism and the development of Alzheimer's disease.2.The proteome of cat intestines during T.gondii early infectionThe in-depth proteome analysis revealed a total of 3401 proteins,thereinto,649 different expression proteins.Our analysis also revealed 76,102,85,177,262 and 320 different expression proteins in infected intestines compared to uninfected control at 6,12,18,24,72 and 96 hpi,respectively.KEGG pathway analysis revealed that the expression of protein changes in the intestines of infected cats are involved in glycometabolism,lipid metabolism and amine acid metabolism,which suggested that during Toxoplasma early infection in cats,numerous nutrient and metabolites should be utilized.The interaction of different expression proteins was also analyzed by String database.The quality of proteomic sequencing was verified by parallel reaction monitoring(PRM).Six proteins were identified by PRM with the same sequences,which indicated the high sequence quality of proteome.3.The reactionogenicity of proteins from T.gondii during early infection in catsSeven T.gondii proteins were selected from proteomic data of feline guts during T.gondii early infection.Thereinto,only 6 genes could be examined by q RT-PCR.After bioinformatics analysis,5 proteins of T.gondii were cloned and expressed by prokaryotic expression system.The reactogenicity of the 5 proteins was identified by T.gondii positive sera from cats using western-blot.The results showed that a strong reaction was detected in r Tg Pr4 protein,but no reaction in r Tg Pr6 protein.Then,the possibility of using r Tg Pr4 protein to develop diagnostic method for cat toxoplasmosis was evaluated based on ELISA.The results showed that r Tg Pr4 would be a potential candidate to develop ELISA diagnostic method for cat toxoplasmosis.