Characteristics Of The Toxoplasma-porcine Macrophage Interactions And The Physiological Functions Of FAS? And Phosphate Transport During Parasite Growth

Toxoplasma gondii is a protozoa belonging to the apicomplexan phylum and can infect a wide range of animals.Toxoplasma possesses numerous strategies to adapt to various hosts such as flexible metabolism as well as sophisticated host interaction.Current work on Toxoplasma-host interactions were mainly focused on model animals like mouse.Whereas increasing evidences indicated that different hosts react differently to Toxoplasma infection.Pigs are important agricultural animals and are susceptible to Toxoplasma infection.But little is known about the interactions between Toxoplasma and pigs.In the present,we used porcine alveolar macrophages(PAMs,3D4/21)and the type II strain Me49 to investigate Toxoplasma-pig interaction using dual RNA-seq technology.Our results revealed distinct responses of PAMs to Toxoplasma infection compared to human or mice infection models.From the parasite side,expression of many genes and pathways related to metabolism were found to be increased when the parasites enter host cells.Two such pathways,the type II fatty acid synthesis(FAS?)and phosphate transporters,we studied in detail to examine their roles during parasite growth.(1)Dual transcriptomic analyses of Toxoplasma-PAM interactionsHigh quality data was obtained through Illumina Hiseq platform.The data indicated significant changes of gene expression in both porcine alveolar macrophages and the Toxoplasma parasites.In PAMs,up-regulated genes were mainly related to immune responses,and cell junction related genes was significant down-regulated.This study uncovered unique responses of PAMs upon Toxoplasma infection as compared to mouse macrophages.IL-12 was not induced by Me49 infection in PAMs,but can be strongly induced in mouse macrophages.What's more,we could not detect any different in NO production between infected PAMs and non-infection controls.This implies that NO may not an important effector in PAMs against Toxoplasma infection.From the parasite side,genes related to ribosomal activity,metabolism and molecule synthesis were up-regulated.FAS? and nutrient transporters were analyzed in detail in this work.(2)Functional study of FAS?FAS? related genes were significantly up regulated upon parasite entry into host cells.FAS? does not exist in animal hosts and is unique in apicomplexa parasites.It is located in the apicoplast of parasites.Here,we focused the upregulated gene Fab D to investigate the function of FAS? in Toxoplasma.Our results showed that deletion of Fab D led to attenuated parasite growth in vitro,but had no effects on Toxoplasma invasion,egress or virulence,suggesting important but nonessential role of FAS? during Toxoplasma growth.(3)Functional study on Toxoplasma phosphate transportersAnalyses of our dual transcriptomic data,found that many transmembrane transporters in Toxoplasma were up regulated after the parasite enters the host cell.One of which,TGGT1?235150,showed homology to phosphate transporters in fungi.Further bioinformatic analysis indicated that there were three putative phosphate transporters were found in Toxoplasma: TGGT1?240210,TGGT1?235150 and TGGT1?278990,which were named Pi T1,Pi T2 and MPi T,respectively.Phylogenetic analyses indicated these transporters were classified into different branches.Pi T1 and MPi T were conserved across species.While Pi T2 was only found in coccidial parasites and has limited homology to fungal phosphate transporters.Pi T1 and Pi T2 were localized on parasite plasma membrane,while MPi T was located in mitochondrion.Deletion of Pi T1 and MPi T in RH strain did not alter parasite growth or virulence.while conditional depletion of Pi T2 led to arrest of parasite growth in vitro.Complementation with Pi T2 restored parasites growth.Pi T2 depleted parasites were not able to propagate in mice either.Yeast complementation assays indicated that Pi T2 can improve the growth of a yeast mutant defective in phosphate transport under low phosphate concentrations.These data demonstrate that Pi T2 has phosphate transport activity and it is critical to parasite growth and survival.In summary,this study used a dual transcriptomic approach to assess the interactions between porcine alveolar macrophages and Toxoplasma.The results suggested distinct responses of PAMs to Toxoplasma infection,which expanded our understanding of Toxoplasma interactions with different hosts.We also studied the function of two metabolic pathways in Toxoplasma: deletion of Fab D slowed down parasite growth but did not affect its virulence.Analyses on the three putative phosphate transporters indicated that the coccidial specific Pi T2 was critical to Toxoplasma growth and survival.Together these results provide significant insights into drug design for toxoplasmosis.