Boothroyd Lab
Department of Microbiology & Immunology
Stanford University School of Medicine
This electron micrograph shows the apical end of a tachyzoite with its pronounced conoid that protrudes during invasion. Micrograph by J. Boothroyd and D. Ferguson.
The lab's specific research effort falls under three categories: Developmental Biology, Attachment/Invasion/Egress and Host-Pathogen Interaction. For each, we have several questions that we wish to answer. These questions and the approaches we are taking are outlined below. Please note that what follows is obviously not meant to be a comprehensive review and I exclusively focus on what we have published as examples of the questions we ask and the approaches we take (since that is the single goal of this website!). Our field is populated by many talented people and they have contributed much to the questions I outline below. I encourage any reader of this site to look at that any of the many recent reviews that give a more balanced view of what is going on in the field as a whole.
Recent Reviews:
Knoll, L.J. and Boothroyd, J.C. 1998. Molecular biology lessons about Toxoplasma development: stage-specific homologs. Parasitology Today 14:490-493.
As you will soon see, the approaches we take run the gamut from genetics through cell and molecular biology and even population biology. Throughout it all, however, we try to be always anchored in the true and literal "Biology" of the system. That is, when we delve into the molecular basis of a given phenomenon, we try to understand how it plays into the overall evolutionary strategy (if you'll excuse the teleological faux pas) of this extraordinary organism. As a result, we may stop shy of atomic resolution in our answers and instead move on to asking "well, what does this tell us about how Toxoplasma interacts with its host and has evolved to be so incredibly successful".
OK, on to the three areas…
The developmental biology of Toxoplasma has been well described in morphological terms but relatively little is understood about the detailed processes involved. We and others have focused on the asexual developmental cycle, i.e., the interconversion between the tachyzoite and bradyzoite stages. We focus on this rather than the sexual cycle because of the lack of an in vitro system for the latter (to date, the sexual cycle has only been observed in the intestine of cats).
The questions we are asking are:
1. What are the triggers that stimulate the developmental switch between tachyzoites and bradyzoites?
2. What signaling events then ensue and what changes in the parasite result?
3. And, finally, what is the biological or biochemical role of those changes (e.g., in promoting a difference in structure, metabolism or ability to be transmitted)?
One of the first approaches we took was, in collaboration with many investigators, to look at a large library of ESTs and compare their frequency in bradyzoites and tachyzoites (Manger et al. 1998. Expressed Sequence Tag Analysis of the Bradyzoite Stage of Toxoplasma gondii: Identification of Developmentally Regulated Genes. Infection and Immunity 66:1632-1637. We are now chasing down some of the genes identified as bradyzoite-specific by knocking them out and studying the effect of the disruption on development.
The second major approach we have taken is to devise and use genetic screens and selections for mutants that are disrupted in development (e.g., Knoll and Boothroyd, 1998. Isolation of developmentally regulated genes from Toxoplasma gondii using a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. Mol. Cell Biol. 18:807-814.; Knoll et al., 2001. Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions. Mol. Biochem. Parasitol. 116:11-16. ). Most recently, we have used a parental strain expressing GFP only in bradyzoites to isolated mutants that don't switch (i.e., which fail to turn on the GFP; Singh, Brewer and Boothroyd., in preparation). We are currently trying to identify the genes that are affected in these various mutants and the pathways that are disrupted.
The third major strategy to study differentiation has been microarray analysis. We have exploited our proximity to some of the labs that have pioneered this technique to produce microarrays for Toxoplasma. For the first generation arrays, we have used the set of ~4000 bradyzoite ESTs that we published in Manger et al. We have used these to study changes in transcript abundance during development from tachyzoites to bradyzoites under different conditions and using different parasite lines and mutants (Cleary, Singh, Blader, Brewer and Boothroyd, in preparation). The results are revealing several genes that we did not previously know to be developmentally regulated as well as information on which are affected early vs. late during the differentiation process. This is helping us build a pathway or cascade of events that go on during differentiation.
Toxoplasma is unusual in being able to invade virtually any cell from virtually any warm-blooded animal. We are interested in:
4. What molecules mediate attachment on both the host and parasite side?
Since Toxoplasma is an obligate intracellular parasite, a mutant that cannot invade should be nonviable. To get around this problem, we use the classical approach of selecting for conditional mutants. Thus, one approach we have taken is to isolate cold-sensitive mutants that are disrupted in their ability to attach/invade (Uyetake et al., 2001. Isolation and Characterization of a Cold-Sensitive Attachment/Invasion Mutant of Toxoplasma gondii. Experimental Parasitology 97:55-59.).
We have also taken a biochemical approach to identify the nature of the molecules involved and found that polysaccharides are key (which might be expected given the incredible range of cell types that can be infected; Ortega-Barria, E.O. and Boothroyd, J.C. 1999. A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection. J. Biol. Chem. 274:1267-1276.).
5. What signaling events trigger subsequent invasion and which molecules physically mediate this process?
We have pursued leads that emerged from our EST project, including an intriguing homologue of a protein that was implicated in invasion by Plasmodium, the so-called AMA1 protein (Hehl et al., 2000. A Toxoplasma gondii homologue of the Plasmodium Apical Membrane Antigen 1 is involved in invasion of host cells. Infection and Immunity 68:7078-7086. ). Having shown that this protein is released onto the surface of the parasite upon invasion, we are now trying to identify what it interacts with on the host cell side and, since it is a transmembrane protein, what it binds to within the cytosol of the parasite. It is possible that this protein represents the critical molecule that connects the actin/myosin motors of the parasite (which drive the invasion process) with the host cell surface allowing the parasite to pull itself in.
6. What mediates egress from the infected host cell?
We believe that egress is not a "passive" process whereby a host cell full of parasites is lysed by the pressure of so many parasites within. Instead, we believe this is a specific process and are interested in the signals that may trigger it. We also believe that egress and invasion are closely related phenomena, perhaps employing many of the same molecules and pathways. Thus, we have exploited the fact that calcium ionophores induces egress and isolated mutants that fail to respond to such stimuli (Black et al., 2000. Ionophore-resistant mutants of Toxoplasma gondii reveal host-cell permeabilization as an early event in egress. Mol. Cell Biol. 20:9399-9408. ). We are now expanding these selections and screens and trying to identify the genes that are affected in the mutants. The mutants also helped us see that permeabilization of the host cell is normally a precursor to egress (i.e., the host plasma membrane becomes permeable to large molecules and only then do the parasites become motile and undergo egress). We are trying to identify the factor(s) produced by the parasite that mediate the permeabilization.
7. What are the targeting signals that cause a protein to be efficiently sent to the various compartments or organelles that are dedicated to the attachment/invasion/egress processes?
For this question, we are focusing on soluble proteins in the rhoptries, especially ROP1 which is secreted into the parasitophorous vacuole during the invasion process. This molecule is processed as it wends its way to the rhoptries (Soldati et al., 1998. Processing of Toxplasma ROP1 protein late in secretion. Mol. Biochem. Parasitol. 96:37-48.). We have identified the precise processing site as a prelude to determining the role of the "pro" region in trafficking (Bradley and Boothroyd, 1999. Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry. Mol. Biochem. Parasitol.100:101-103.). Interestingly, the pro region can target a fusion protein to the rhoptries but so too can a region within the mature portion of the protein (Bradley and Boothroyd, 2001. The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal. Intl. J. Parasitol. (in press)).
Targeting of PRO-ROP1-VSG to the rhoptry necks.
We are interested in several issues that fall under this broad category. Questions include:
8. Which parasite molecules are recognized by the host and allow the infection to be controlled?
Two surface antigens, SAG1 and SAG2, are immunodominant in the acute stages of infection. But these two proteins are part of an extensive gene family whose function remains largely mysterious and many of whose members are expressed simultaneously on the surface of the asexual forms (Manger et al., 1998. The surface of Toxoplasma tachyzoites is dominated by a family of GPI-anchored antigens related to SAG1. Infection and Immunity 66:2237-2244.; Lekutis et al., 2000. Identification and characterization of multiple SAG2-related-sequences of Toxoplasma gondii, with homology to the SAG1 family. Experimental Parasitol. 96:89-96). Interestingly, these molecules are often highly stage-specific with different members abundantly but exclusively present on either the tachyzoite of bradyzoite stage (Lekutis et al., 2001. Surface Antigens of Toxoplasma gondii: Variations on a Theme. Intl. J. Parasitol. (in press).). We are taking a molecular genetic approach to understand the function of these molecules through deletion of the genes that encode them and analysis of the resulting mutant. Through such studies, and in collaboration with Lloyd Kasper's group at Dartmouth, we have found that the immune response to one of these, SAG1, mediates much of the gut pathology associated with the early stages of infection (Dutta et al., Pathogen-induced acute ileitis in mice is dependent upon the expression of a single parasite membrane antigen (SAG1) of Toxoplasma (submitted)). We now want to understand how this immunodominance occurs and, more intriguingly, how such fits into the survival strategy of the parasite. Ironically, it may be that establishing a chronic infection (by bradyzoites) is dependent on clearing the acute stages (tachyzoites) after they have done their job of disseminating the infection throughout a given host. A failure to clear the virulent tachyzoites might result in death of the host and thus no transmission since tachyzoites in tissue do not survive the digestive process in a host that ingests them.
9. What happens in the host cell upon infection?
We have explored this with microarrays and discovered some very interesting and unanticipated changes in transcript abundance that suggest major changes in host cell metabolism (Blader et al., 2001. Microarray analysis reveals previously unknown changes in Toxoplasma gondii infected human cells. J. Biol. Chem. 276:24223-24231. ).
Time course showing changes in gene expression in HFF cells upon infection with Toxoplasma (from Blader et al., 2001).
10. What is the relationship between different strains of Toxoplasma?
Toxoplasma population biology is intriguing, especially given that it is so widespread in its geographic distribution and host range. To cut a long story short, three (or maybe four) genotypes massively dominate the strains so far sampled and are clearly reproducing clonally. Whether these clonal types are bypassing the cat or whether cat infections are mostly uniparental (which for a haploid beast like toxo, results in F1 progeny that are genetically identical to the parent) is unknown. Occasional strains are identified that are genetically closely related to the major clonal Types but have a scrambled assortment of the alleles but it is not yet possible to say if these are recombinants from a cross between the major Types, sibs of those Types or cousins.
Remarkably, the total gene pool in all strains sampled consists almost entirely of just two allelic classes at all loci indicating that two very distinct lines intermixed to yield these various strains, some of which are tremendously successful while others are less so (Grigg, M.E., Suzuki, Y. and Boothroyd, J.C. Success and virulence in the AIDS pathogen Toxoplasma as the result of sexual recombination between two distinct ancestries. Science (in press)). Even the most exotic strains, however, appear to be drawing on this extremely limited (i.e., dimorphic) gene pool.
11. Do infections with different strains in humans cause different disease outcomes?
It is well established that Type I strains are highly virulent in mice while Types II and III are relatively avirulent. To begin to address whether this is the case in humans, and in collaboration with Todd Margolis' group at UCSF, we have determined the strain type in the rare cases where otherwise healthy people develop severe disease of the eye. We observed that indeed, strain type appears to be a significant factor with type I strains (which are hypervirulent in mice) and a new strain, dubbed type IV, being found disproportionately often. (Grigg et al., 2001. Unusual abundance of atypical strains associated with ocular toxoplasmosis in humans.J. Inf. Dis. (in press)). The numbers are small but the trend clear and we hope our clinical colleagues will pursue this observation to see if the trend holds up. Such information is very important clinically in deciding on the best treatment - an aggressive parasite may warrant more aggressive therapy, even with drugs that are relatively toxic. Asking the same question in pregnancy will be crucial and we hope facilitated by identification of easily amplified polymorphic genes(as in Grigg and Boothroyd, 2001. Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-RFLP analysis at B1. J. Clin. Micro. 39:398-400.)
12. How do the best drugs work?
Although it may seem a bit of a stretch, I include chemotherapy under the broad category of host/pathogen interaction because a drug is only of use if it affects processes that the parasite depends on for survival in the host. Thus, drugs can give us much useful information about the host/pathogen interaction.
Current, first-line therapy relies on two major drugs - pyrimethamine (which targets dihydrofolate reductase) and sulfadiazine (which targets dihydropteroate synthase), with clindamycin and atovaquone as second line therapies. The targets for the latter two drugs were guessed at based on their action in other organisms but, until recently were not definitively known. We have taken a genetic approach and identified the target for atovaquone as mitochondrial electron transport (McFadden et al., 2000. Characterization of cytochrome b from Toxoplasma gondii and Qo domain mutations as a mechanism of atovaquone-resistance. Mol. Biochem. Parasitol. 108:1-12.).
Similarly, we have shown that clindamycin targets the apicoplast ribosome (Camps, et al. An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii. (in revision for <B>Molecular Microbiology</B>). Interestingly, the apicoplast appears to be almost dispensable in vitro but extremely important for virulence in vivo. We are now trying to understand why this is so.
