VERMONT COBRE: PROJECT 4: SUBVERSION OF HOST CELL SIGNALING BY TOXOPLASMA GONDII

University Of Vermont & St Agric College, Burlington VT

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Abstract

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 the Tables and Figures mentioned below would not reproduce in this format. Please see attachments sent with the paper copy.*** 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. Shouthern blot analysis shows that different loci have been disrupted in these mutants. Using a combination of plasmid rescue and inverse PCR, we identified the disrupted locus in 5 out of these 7 mutants (Table 1). In the previous report we described the microarray analysis carried out with these seven mutants. In the past year we continued with the analysis of the microarray data. For example, we have examined the gene induction level of bradyzoite specific genes in each of the mutant parasite lines. Among 418 bradyzoite specific genes induced in wild-type parasites, only 0.2%-4.7% of them were up-regulated to the wild-type levels in the bradyzoite mutants (Fig. 1). Another small fraction of bradyzoite specific genes were up-regulated in the mutants but the induction levels substantially lower than in wild-type parasites (blue bars, Fig.1). These results indicate the genes disrupted in the mutants, are likely involved in the early steps of bradyzoite differentiation and, thereby, their loss prevents the expression of a large number of downstream genes. In order to gain a better understanding of the key events required for T. gondii parasites to differentiate from the tachyzoite stage to the bradyzoite stage, we have ordered the mutants along the differentiation pathway. Principle Components Analyisis (PCA) is a statistical method that finds patterns among samples given data of high dimension. For example, in our current microarray analysis, we have 87 samples (including replicates) and ~8000 gene expression values for each sample;data of high dimension. For this example, PCA will take into account the level of variation in expression across all samples (87 components) and then plot the samples using the top two components that consist of the most variation. In short, the PCA plot is useful for visualizing which samples behave most like each other, and most different from each other. We performed principle component analysis and imposed a trend line for the wild type samples only, in order to visualize the trend of the transition from tachyzoite to bradyzoite and where each mutant lies in relation to the WT path. The PCA plot is a two-dimensional view that shows that after exposure to bradyzoite induction conditions for 72h each mutant is blocked at a different point along the transition pathway. ***Please see attachment for figures and tables*** Publications De Miguel N, Lebrun M, Heaslip A, Hu K, Beckers CJ, Matrajt M, Dubremetz JF, Angel SO. 2008. Toxoplasma gondii Hsp20 is a stripe-arranged chaperone like protein associated with the outer leaflet of the inner membrane complex. Biol Cell. 100(8):479-89. Mentoring Summaries: Dr. Mercedes Rincon Dr. Rincon is the primary mentor for Dr. Matrajt. Dr. Rincon is accessible to Dr. Matrajt any day and time for relative urgent questions or issues, and she follows Dr. Matrajt progress by different avenues. Both Dr. Rincon and Dr. Matrajt attend the joint monthly Immunobiology/Microbiology lab meetings where Dr. Matrajt has the opportunity to present the progress in her group, and the same time she can also provides feed back to other investigators on her area of expertise. Dr. Rincon encourages Dr. Matrajt to use her presentation in lab meetings as a mechanism to obtain criticisms and suggestions for upcoming papers and grants before submission. Thus, during the last year Dr. Matrajt has presented twice. At the first presentation, she talked about the data her group has compiled with the goal of writing a manuscript. The feed back Dr. Matrajt obtained was key for helping her to outline the manuscript. At the second presentation, she discussed her plan for a grant resubmission. We discussed potential preliminary data and research design. 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 encountered during the last few months, and future directions with clear dead lines regarding grant and manuscript submissions. Dr. Rincon has provided substantial help to Dr. Matrajt for the writing of the grant application she just resubmitted, by reading the proposal and the summary statement, and providing suggestions, primarily on the description of preliminary studies and proposed experiments. Dr. Rincon is also providing her expertise in writing manuscripts since Dr. Matrajt is currently planning to submit another of her manuscripts. Dr. Rincon expertise as a member of an NIH Study Section and member of editorial board of different journal will be strongly beneficial for Dr. Matrajt. Dr. Gary Ward Dr. 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 investigators 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.