This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.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. Currently we have 10 mutants under characterization. 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. Shouthern blot analysis shows that different loci have been disrupted in these mutants. In the past year we focused our research on dissecting the expression profiles of 7 of these mutants using whole genome microarray analysis. RNA was harvested from freshly lysed out tachyzoites from wild type or the 7 mutant parasites. RNA was also harvested from wild type and mutants cultivated for 3 days under bradyzoite differentiation conditions. All these samples were hybridized in triplicates to a whole genome Toxoplasma gondii Affymetrix microarray. In addition, for the wild type parasites only, we performed a bradyzoite induction time course: we harvested RNA 24 hours, 36 hours, 48 hours and 72 hours post bradyzoite induction, and hybridized these samples to the microarray as well. Mutants were analyzed for whole genome expression differences compared to wild type parasites using differential expression plots (differential gene expression under bradyzoite conditions compared to tachyzoite conditions) in order to identify gene(s) that contribute to the mutant phenotype. The differential expression plots for each mutant versus wild type show a unique whole genome expression profile for each of the seven different mutants. In addition, these plots allow us to visualize which genes behave like wild type and which genes differ in expression levels, and we can see: genes that respond similar to wild type parasites under bradyzoite induction conditions; genes that respond to a lesser extent in the mutant parasites under bradyzoite conditions; and genes that do not respond at all in the mutant parasites under bradyzoite conditions. The bradyzoite induction time course with wild type parasites showed that 788 genes were upregulated more than 2 fold under bradyzoite differentiation conditions. These genes clustered into three distinct subsets: early genes (n = 356), middle genes (n = 183), and late genes (n = 249). The first subset of genes (early gene cluster) are expressed at 24h post bradyzoite induction and significantly induce from 0h to 24h, remain highly expressed 24h-48h, and then begin to decrease at 72h post bradyzoite induction. The second subset of genes (middle gene cluster) are moderately induced from 0h to 24h, remain steady 24h-48h, and then increase expression at 72h post-induction. The third subset of genes (late cluster) are induced between 48h and 72h post bradyzoite induction and are repressed from 0h to 48h. When we look at these gene clusters in the mutant parasites we can see that the 7 mutants fail to induce most of the middle and late genes. The most variation among the 7 mutants can be seem in the expression of the middle genes. Regarding the early genes, interestingly, 5 out of the 7 mutants have these genes induced under tachyzoite growth conditions, and the other 2 mutants have their expression similar to wild type parasites. We are still in the process of analyzing in more detail these results. Mentoring Summaries: Gary WardDr. Ward meets bimonthly with Dr. Matrajt to discuss experimental plans, data interpretation, and the overall direction of her work. Dr. Ward critically reads and provides feedback on Dr. Matrajt's manuscripts and grant applications. He has provided ongoing advice on both funding opportunities and approaches to the revision and resubmission of her RO1 application. Dr. Ward and Dr. Rincon meet together annually with Dr. Matrajt to discuss the current status of her research and funding situation, and brainstorm with her on ways to move her research program forward. Less formally, Dr. Ward and Dr. Matrajt participate in a joint, biweekly lab meeting, which also includes one of the other junior investigator on the COBRE grant, Dr. Huston. This data-centered meeting is highly interactive, and an excellent way for Dr. Matrajt and her students and postdocs to receive regular feedback on the course of their research. The Matrajt and Ward labs also have a joint monthly journal club that focuses on the latest papers in the field of Toxoplasma and toxoplasmosis. The discussions at these meetings are frequently far-ranging and highly stimulating; it is a very interactive and critical group, and Dr. Matrajt benefits from and contributes to these discussions. Dr. Ward serves on the dissertation committee of each of Dr. Matrajt's PhD students, and provides her with regular advice on the mentoring of her students and postdocs. Dr. Ward also provides every opportunity to Dr. Matrajt to meet with visiting scientists and seminar speakers who come to UVM.Mercedes RinconDr. Rincon is the primary mentor for Dr. Matrajt. Dr. Rincon meets Dr. Matrajt in regular bases through the joint monthly Immunobiology/Microbiology lab meetings where she has the opportunity to present the progress in her group at least twice per year. Dr. Rincon is accessible to Dr. Matrajt any day and time for relative urgent questions or issues. They also discuss the research progress after she presents her work in lab meetings or retreats. In addition, Drs. Rincon and Matrajt have long endless meetings (3-4 h) every 3-4 months to discuss not only research progress, but also problems faces during the last few months, and future directions with clear dead lines regarding grant and manuscript submissions. Another common theme of discussion in these meetings is how to manage the lab and the lab personnel, how to encourage students and postdoct to increase their interest in the research, and how make the group more effective. Dr. Rincon meets with Dr. Matrajt prior to a grant submission, and provides suggestions for the preparation. She then reads the full proposal and provides Dr. Matrajt the feedback with potential changes to improve the application. Because her expertise as a member of an NIH Study Section, she has also teaching Dr. Matrajt how the review process works and what are the key things reviewers will request. She also reads the summary statements of the reviewed applications and helps Dr. Matrajt with her response to the reviewers. Dr. Rincon has also be of great influence for Dr. Matrajt to initiate studies in in vivo models, and helped her with the writing of animal protocols. In addition, Dr. Rincon has greatly influenced Dr. Matrajt interest in moving towards the immune response to Toxoplasma. This will broad the research scope of Dr. Matrajt and will help her to be more competitive for NIH funding.
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