Principal Investigator Jeroen Saeij
Toxoplasma has a haploid genome with 14 chromosomes that total 65 Mbp in size, representing ~7900 genes (http://www.toxodb.org). Classical genetic crosses can be performed in Toxoplasma and several experimental crosses have been used to generate genetic linkage maps. To identify the Toxoplasma loci involved in virulence, we mapped virulence in F1 progeny derived from crosses between type II and type III strains. Five virulence loci were thus identified, and for three of these, genetic complementation showed that protein kinases (ROP18 and ROP16, respectively) and pseudokinases (ROP5) are the key molecules. These are hypervariable rhoptryproteins that are secreted into the host cell upon invasion.
We have also used linkage mapping to identify Toxoplasma loci involved in modulation of host gene expression. Our initial analysis of the data identified that most of the strain-specific differences in the modulation of host cell transcription are mediated by two genes ROP16 and GRA15. Upon invasion by the parasite, ROP16 is injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signaling pathways. GRA15, encodes a dense granule protein, which affects activation of NF-kB, a transcription factor involved in the activation of the inflammatory response. Different Toxoplasma strains (e.g. type I, II and III) have different alleles of ROP16 and GRA15. Our results showed that the precise allelic combination of ROP16 and GRA15 determines how distinct Toxoplasma strains modulate the host immune response. For example, type II strains can kill susceptible mice because they induce a hyper-inflammatory response that damages host tissue. We are now combining genetic and biochemical approaches to identify host factors that interact with GRA15. Furthermore, we are continuing to investigate the host genes and pathways that mediate ROP16’s strong anti-inflammatory effects.
Overall, these results suggest that analogous to bacterial pathogens and their secretion system, it seems that Toxoplasma can secrete effectors into host cells to subvert host-cell signaling pathways. ROP16, ROP18 and ROP5 are members of a large protein family suggesting that Toxoplasma has a wide arsenal of effectors to modulate diverse host cell signaling pathways. The detailed characterization of these effectors represents a major focus of the laboratory.