PROJECT SUMMARYToxoplasma gondii is an intracellular parasite that chronically infects many hosts including humans. Chronicinfection requires T. gondii switching from a fast growing form to a slower encysted form. In humans this life-long persistence primarily occurs in the brain where T. gondii can reactivate in the setting of acquired immunedeficiencies. In AIDS patients toxoplasmic encephalitis is the most common cause of focal brain lesions. Re-cent studies also suggest that persistent T. gondii infection may adversely affect cognition and global immuneresponses even in HIV+ patients on effective antiretroviral therapy. Currently we have no drugs that target theencysted form of T. gondii. The goal of this grant is to develop new options for cyst-targeted anti-microbials bybuilding upon our novel finding that ROP16 a parasite protein that is secreted into host cells early during inva-sion and that varies between T. gondii strains affects encystment in a strain-specific manner. Among the ca-nonical encysting T. gondii strains type II and III the type III allele of ROP16 (ROP16III) phosphorylatesSTAT3 and 6 and possibly STAT5 while the type II allele (ROP16II) does not. In two different in vitro encyst-ment assays we determined that a type III strain that lack ROP16 (IIIrop16) has a 65% decrease in encyst-ment while a type II strain that lacks ROP16 (IIrop16) has a mild increase in encystment. In host cells co-infected with a wild-type type III strain but not a type II strain the IIIrop16 strain now shows normal encyst-ment. As there is no evidence that parasites re-uptake secreted effector proteins this trans complementationsuggests that ROP16-dependent encystment depends upon host cell manipulations. We hypothesize there-fore that efficient type III encystment depends upon ROP16III manipulations of host cell signaling. To test thishypothesis we will determine which properties of ROP16III are essential for ROP16-dependent encystment bycomplementing the IIIrop16 strain with rop16III variants that lack nuclear localization kinase activity or STATbinding (Aim 1). To identify the host cell genes and pathways pertinent to ROP16-dependent encystment wewill use RNA-seq to perform transcriptional analysis of fibroblasts infected with the parental type III IIIrop16or the complemented strain and exposed to encystment conditions (Aim 2). Top differentially expressed tran-scripts/genes will be validated by Q-PCR and when possible immunofluorescent assays or western blots.With the completion of these aims we will have determined which functions of ROP16III are essential for typeIII encystment and identified the host transcripts and pathways specifically affected by ROP16III in the contextof encystment. These outcomes will establish a foundation on which to build long-term mechanistic studies todefine strain-specific mechanisms for T. gondii encystment. The work proposed here represents an importantfirst step toward developing strain-specific treatments for the persistent form of T. gondii.