Toxoplasma is unusual in being able to invade virtually any cell from virtually any warm-blooded animal. Once inside, it resides within a parasitophorous vacuole comprised largely of invaginated plasma membrane of the host cell. Recently, we have found that many Toxoplasma proteins are actually injected into the host cell where they reach the host nucleus and have a major impact on host gene expression. Most of these proteins are initially present in the bulb-shaped, anterior rhoptries.

Questions related to these various cell biology issues are:

1. What molecules mediate attachment on both the host and parasite side?
2. What signaling events trigger subsequent invasion and which molecules physically mediate this process
3. How are proteins introduced into the host cell?
4. By what means and for what purpose do such injected proteins then impact host cell functions?

On the attachment side of things, we have determined the structure of a major surface antigen, SAG1 (He et al., 2003; in collaboration with Chris Garcia’s lab here, at Stanford). SAG1 is part of an extensive family of related proteins (Manger et al., 1998). Our results suggest that these proteins use a conserved basic groove that binds a negatively charged, extended molecule. The data fit a model involving binding of one or more host proteoglycans (e.g., heparin) but this has yet to be convincingly demonstrated as biologically important to the parasite.

The next phase of invasion (after initial attachment mediated by the SAG1 family) may involve one of the “finds” in the early EST project, an intriguing homologue of a protein that was implicated in invasion by Plasmodium, called “apical membrane antigen 1” or AMA1 (Hehl et al., 2000). We showed that this protein is released from a set of anterior organelles called micronemes.

Given its conservation across the phylum and our finding that antibodies to the Toxoplasma version of AMA1 inhibit invasion, we hypothesized that it played a central role in this process. To investigate this, we went after interacting partners and found, very surprisingly, that this micronemal protein forms a complex with several proteins emanating from the most anterior portion of a completely different set of secretory organelles, the rhoptries! The resulting complex is specifically localized to the ring-shaped moving junction that forms at the point of contact between invading parasite and the host cell surface (Alexander et al., 2005). We are now trying to understand how this complex functions during invasion, including the very speculative (but fun) idea that the parasite inserts proteins into the host plasma membrane to get a purchase on the host cytoskeleton during invasion. This would certainly nicely explain how they will invade virtually anything (i.e., if they are providing their own receptor/ligand pairing)!

How Toxoplasma proteins are introduced into the host cell during infection is surely one of the most glaring gaps in our knowledge of Toxoplasma cell biology. As for so many of our projects, we are taking a combination of biochemical and genetic tacks to addressing this. We are hoping that mutants that are resistant to drugs that block injection will reveal the targets of those drugs and, hence, a part of the machinery that does the injecting. We are also pursuing some candidate genes that came out of the genome and EST sequencing efforts of others.

As to what the injected proteins do once inside, their sequence indicates that many of them are protein kinases or protein phosphatases (e.g.,Gilbert et al., 2007; Saeij et al., 2006, 2007; see discussion of ROP16, above). But this is just a tease since, as mentioned above, it leaves unanswered the most interesting question: what proteins are they (de)phosphorylating?!? We presume these targets are primarily host but cannot exclude that some are parasite in origin. We hope answers will come from expression of these proteins in host cells on their own and analysis of parasites engineered to express different forms (or no form at all) followed by careful analysis of host responses. We are also taking a biochemical approach using pull-down experiments (followed by mass spectrometry) as well as methods pioneered in the Shokat lab. The latter allows a kinase’s substrate to be specifically identified using a combination of a modified form of the kinase together with a reciprocally modified ATP substrate that only that kinase can use for phosphorylation.