This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ***Please note Dr. Mariana Matrajt's funding was terminated as of 8/31/09.*** The human pathogen Toxoplasma gondii is one of the most widely distributed protozoan parasites, infecting approximately one-third of the world's population. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing 'tachyzoites'and latent 'bradyzoite'tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. The interconversion between tachyzoites and bradyzoites, at the heart of parasite survival and pathogenicity, is poorly understood at a genetic and molecular level, which makes understanding this process an important goal. We are interested in identifying genes involved in the bradyzoite differentiation process in order to better understand the biology of the conversion between tachyzoites and bradyzoites. To this end we have successfully developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites under differentiation conditions. Seven of these mutants were selected for further characterization and microarray analysis. All these mutants show significantly increased replication rates and reduced expression of bradyzoite markers which are features that confirm that indeed these mutants have defects forming bradyzoites. In the previous report we described the microarray analysis carried out with these seven mutants. In the past year we finished the analysis of the microarray data and recently submitted this work for publication. The summary of this work is as follows: Background: Toxoplasma gondii pathogenesis includes invasion of host cells by extracellular parasites in the tachyzoite stage, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. Results: Here, we present the analysis of several novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant samples in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. We identified three stages based on gene expression patterns in wild-type parasites: extracellular tachyzoites;intracellular tachyzoites;and bradyzoites. Conclusions: Strikingly, some of the mutant samples appear to exhibit high proportions of the intracellular tachyzoite stage regardless of whether they are intracellular or extracellular, suggesting that bradyzoite differentiation and switching to the extracellular stage are linked at the molecular level. In addition to the genes identified by the insertional mutagenesis screen, statistical analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite classes [unreadable]genes that may play a mechanistic role in switching. In addition, in the past year further studies were carried out with one of the mutants, mutant P11. Microarray hybridizations revealed that the P11 mutant had a global deficiency of bradyzoite gene induction strongly suggesting that the mutation involves a very early step in the crucial cascade that triggers differentiation. We analyzed the locus disrupted in the P11 mutant and found that it included a transcript that encodes a predicted serine/arginine rich-4 (SR) splicing factor as well as an antisense transcript with no obvious open reading frame. Importantly, complementation of this mutant with the wild-type locus restores the ability of the parasites to differentiate. In the mutant parasites the insertion disrupted both the sense and antisense transcripts which partially overlap. In order to begin dissecting whether the putative splicing factor or the antisense transcript or both genes are responsible for the differentiation defect, we made a construct with the antisense transcript disrupted but containing the complete splicing factor gene. When wild-type parasites were transfected with this construct, these parasites formed bradyzoites under tachyzoite growth conditions, strongly suggesting that the presumed splicing factor plays an important role triggering bradyzoite differentiation and that the antisense transcript present in this locus regulates the expression of the splicing factor. We hypothesize that this mutant cannot correctly splice one or more bradyzoite-specific transcripts involved in the regulation of bradyzoite differentiation. We are currently doing experiments to test that the antisense regulates the expression of the SR splicing factor and this regulation is essential for correct stage conversion. Genome-wide analysis has revealed extensive antisense transcription in most species studied to date. The function of these natural antisense transcripts (NATs) is largely unknown, however, their regulatory importance is becoming apparent since a growing number of studies are showing that the interplay between sense/antisense expression has critical roles in developmental, physiological and pathophysiological processes. Natural antisense transcription has been reported in T. gondii parasites but very little is known about NATs function or mechanism of action.