Transcriptomic Profiling and Domain Searches for Functional Characterization of Novel Virulence Determinants of Toxoplasma Pathogenesis
Principle Investigators: Pascale Guiton, Ph.D., Biological Sciences, CSU East Bay
Abstract: Toxoplasma gondii is a unicellular parasite responsible for toxoplasmosis, a disease afflictingapproximately one third of the world human population. There presently exists no cure forchronic toxoplasmosis. During infection, usually following ingestion of contaminated food or water, the parasite invades the host cell and resides within a parasitophorous vacuole (PV). Toxoplasma in its host will interconvert between many developmental forms, each biochemically and functionally distinct. Toxoplasma possess stage-specific transcriptomes. They encompass a plethora of genes, including pep1, gra9, and rop23, that encode hypothetical proteins predicted to be secreted inside the PV and/or directly into the host cell. Preliminary evidence from my laboratory indicate that pep1-; gra9-, and rop23-defective mutant parasites are severely attenuated in virulence in mice, suggesting that PEP1, GRA9 and
ROP23 may be novel virulence determinants of Toxoplasma. We postulate that Toxoplasma secretes these three developmentally regulated proteins at specific phases in the pathogenic process to modulate host processes.To address this hypothesis, my students will use standard molecular cloning techniques to engineer parasite mutants expressing a C-terminal-tagged version of each protein. This tag will be used as proxy in immunofluorescence assays to determine the subcellular locations of PEP1 and ROP23 in Toxoplasma and during infection of human intestinal epithelial cells (hIECs). Furthermore, we will infect hIECs with either parental and knockout mutant strains and perform RNA sequencing, in collaboration with Dr. Ana Almeida, to assess whether PEP1, GRA9, and/or ROP23 modulate host processes during Toxoplasma infection. Furthermore, Dr. Almeida’s group will conduct a variety of bioinformatic analyses to identify functional domains in these proteins that may provide clues as to their mechanisms of action during infection. Once completed, this work will expand our understanding of the pathogenicity of Toxoplasma and the therapeutic potentials offered by developmentally regulated virulence factors in the fight against toxoplasmosis.