Boothroyd
Lab
Department of Microbiology & Immunology
Stanford University School of Medicine
Welcome to the Boothroyd Lab Description!
The primary purpose of this page is to enable you to 'visit' the lab to better understand who we are, what we do, how we do it and where we do it! If all this is more detail than you need, you can also just go to John Boothroyd's Home Page for a very brief summary of our work with links to a more-or-less complete list of publications. Happy Browsing!
Contents:
I. Who we are...
II. Lab Alumni.
III. Lab Philosophy
IV. Toxoplasma gondii:
A Brief Introduction.
V. Our Research Program.
A. Developmental Biology
B. Attachment/Invasion/Egress.
C. Host/Pathogen Interaction
VI. The Practical Details
VII. Where we do it...
I. Who we are...
Group photo (January 2001) celebrating the successful thesis defense of Manel
Camps. He's the happy one front center in the green sweater. Back row: Jeremy Brewer,
Michael Grigg, Gregg Furie, Michael Cleary, Ira Blader, Peter Bradley. Front
row: Laura Knoll (sitting), Joe Dan Dunn, Upi Singh, Manel Camps, Chris
Lekutis, John Boothroyd.
Our address is:
Department of Microbiology
and Immunology
Fairchild Science Building, Room D305
Stanford University School of Medicine
Stanford, CA 94305-5124
Phone: 650-723-7296 (lab phone)
650-725-4753 (John's administrative assistant)
650-723-7984 (John's office)
Fax: 650-723-6853
email addresses are below.
The group was started in 1982 and has a
steady-state size of 10-12 researchers plus support personnel. The typical
breakdown is two-three graduate students working on their Ph.D., a medical
student, six post-docs (typically, five Ph.D. and one M.D.) and one-two research
associates. The three key support staff (shared with 1-2 other labs) are: one
tissue culture and media assistant, one administrative assistant and one glass
washer. The current roster and their projects are:
Principal Investigator:
John Boothroyd: does this and that ... talks, listens, writes, edits and generally makes a nuisance of himself. (john.boothroyd@stanford.edu)
Graduate Students:
Matt Anderson: What genes are key to differentiation of tachyzoites to bradyzoites? (matthewz@stanford.edu)
Joe Dan Dunn: What changes occur at the host cell surface upon infection? (jddunn@leland.stanford.edu)
Ashley Fouts: How do bradyzoites co-opt host cell function to establish the persistent state (afouts@stanford.edu)
Sandeep Ravindran: How are rhoptry proteins injected into the host cell during invasion? (sandeepr@stanford.edu)
Post-doctoral Fellows:
John Boyle: What is the genealogy of modern strains and what is responsible for their differences in virulence? (boylej@stanford.edu)
Susan Coller: What role do parasite kinases play
in invasion and co-opting of host cell functions? (scoller@stanford.edu)
Seon Kim: How does the parasite evade immunity during persistence?
(seonkim@stanford.edu)
Jeroen Saeij: How do the parasites co-opt host-cell gene expression and how does this differ between strains? (jsaeij@stanford.edu)
Gus Zeiner: How are genes regulated during tachyzoite to bradyzoite differentiation and in the host cell during infection? (gusti@stanford.edu)
Research Associate:
Lidia Barouh: Developmental biology.
(lbarouh@stanford.edu)
Senior Research Associate:
Dave Alexander: How is the moving junction formed and operated during invasion? (dlalexan@stanford.edu)
Tissue Culture Assistant:
Inna Billis
Adminisitrative Assistant:
Bonda Lewis (llewys@stanford.edu)
Glass-washer:
Ella Doyle
II. Lab
Alumni.
A listing of former lab members and what they have gone on to do can be found
at Lab Alumni
III. Lab
Philosophy.
The lab's philosophy is to learn as much as we can about protozoan parasites
using the most powerful tools available to us ... and to enjoy the process.
Initially, we put our major effort into studying the causative agent of African
Sleeping Sickness, Trypanosoma brucei.
Over the last decade, however, our emphasis has shifted to another
parasitic protozoan, the opportunistic pathogen Toxoplasma gondii. This transition has been driven by the
success of this lab and others at developing tools to study Toxoplasma, by the
growing importance of this parasite as an AIDS pathogen and by the enormous
number of questions that were crying out to be answered about the intriguing
biology of this intracellular protozoan. In the end, I decided to commit the
entire lab's effort to Toxoplasma as of the beginning of 1998. So, with tremendously
mixed emotions, we no longer work on what I still consider one of the most
intriguing organisms on earth and we turn our full attention to a system that
has very different challenges and where the biology of the host-parasite
interaction is rich with complexity.
Our ultimate hope (and already it's worked out this way with a few projects) is
to learn something that others can then develop into a product or procedure
that proves useful in alleviating suffering from the diseases these parasites
cause. What follows is a brief description of the issues we have been studying
in recent years.
IV. Toxoplasma
gondii: A Brief Introduction.
Toxoplasma is an Apicomplexan parasite that is related to the
organisms that cause chicken coccidiosis (Eimeria), malaria (Plasmodium) and a number of
other important diseases. It is an obligate, intracellular parasite and, as
such, has evolved a very different survival strategy from the extracellular
trypanosomes. The life cycle of Toxoplasma is very complex but
breaks down into two parts, one sexual and the other asexual.
The sexual cycle occurs exclusively in cats and is initiated when a cat eats an
infected prey or accidentally ingests feces contaminated with oocysts.
Following a typical process of gamete formation and fusion in the intestinal
epithelium, the zygote is formed. This ultimately develops into an immature
oocyst which, after being shed in the feces will mature into an extraordinarily
resistant entity containing 8 sporozoites (representing, we assume, all of the progeny
of meiosis).
The oocysts are highly infectious not only to other cats (in which case a new
sexual cycle is initiated) but to virtually any warm-blooded animal.
Herbivorous grazing animals, of course, will be particularly susceptible but
it's also found in strict carnivorous animals, as well. How? Well, once in the
herbivore, the sporozoites are released from the oocyst in the intestine and
invade the intestinal epithelium. There they differentiate into the rapidly
dividing tachyzoite form (tachy from the greek for fast (dividing)) which is capable
of indefinite replication in vivo and in vitro. These diseminate
through the host, infecting virtually any cell in any tissue. As the host's
immune response rises to the challenge, the parasites encyst and differentiate
to the very slowly dividing form, the bradyzoite (brady = slow in greek).
These are very stable and infectious if tissue from the animal is eaten.
Toxoplasma bradyzoites (left) and tachyzoites (right).
The scanning electron micrograph shows a tissue cyst with its many bradyzoites within the brain of an infected mouse. Photo courtesy of David Ferguson, Oxford University. The Giemsa stain is of tachyzoites in a human foreskin fibroblast (HFF) grown in culture. Photo by Manel Camps.
Thus, carnivores that eat herbivores become infected and carnivores and
scavengers that eat these carnivores become infected and ... you get the idea.
The end result is the parasite is found throughout the food chain, througout
the world. It is also found at very high prevalence. In fact, one can make a
pretty good argument that Toxoplasma is the most successful parasite of
warm-blooded animals.
Humans are infected through either accidental (one hopes) ingestion of cat
feces or eating under-cooked meat (especially lamb since pork is well known to
be a danger if not well-cooked (perhaps because of toxoplasmosis as well as
trichinellosis???) and beef tends not to be heavily infected). Based on
serology, infection rates range from 15-85% world-wide with the U.S.A. being
~15-25%. Note that, because of the chronic tissue cyst stage, once infected
always infected and so these serologies probably translate to current
infection. In healthy adults, disease is not life-threatening although it can
range from virtually asymptomatic to a rather acute, flu-like illness and
sometimes serious blindness, depending on the strain infecting (e.g., Grigg et
al., 2001 Unusual abundance of atypical strains associated with ocular
toxoplasmosis in humans. J.
Inf. Dis. (in press). ).
The most serious problems occur in either of two situations. First, if a woman
becomes infected for the first time during pregnancy, her fetus is at risk of
severe neurological problems, even death. Second, if a chronically infected
person develops AIDS or is immuno-suppressed for any other reason, the disease
can reactivate with the quasi-latent tissue cysts releasing the bradyzoites which
rapidly differentiate back to tachyzoites. The resulting disseminated infection
is of greatest concern when it enters the brain where an encephalitis can
ensue. Current drugs are fairly toxic, especially to the developing fetus and
especially if treatment must be sustained for a long period, as in AIDS
patients (the drugs don_t have much effect on the encysted bradyzoites and thus
the infection is never completely cleared; stop treating and it will rapidly
flare up again).
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.
This electron micrograph shows a tachyzoite within a macrophage of an infected mouse.
Note the mitochondria that completely surround the parasitophorous vacuole (as
yet, for unknown reasons). Micrograph by J. Boothroyd and D. Ferguson.
V. Our Research Program.
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.
McFadden, G.I. and Roos, D.S. 1999. Apicomplexan plastids as drug targets. Trends
Microbiol 7:328-33
Bonhomme, A., Bouchot, A., Pezzella, N., Gomez, J., Le Moal, H., Pinon, J.M.
1999. Signaling during the invasion of host cells by Toxoplasma gondii. FEMS
Microbiol Rev 23:551-61
Black, M.W. and Boothroyd, J.C. 2000. The lytic cycle of Toxoplasma
gondii. Microbiology
and Molecular Biology Reviews 64:607-623.
Kaasch, A.J. and Joiner, K.A. 2000. Protein-targeting determinants in the
secretory pathway of apicomplexan parasites. Curr
Opin Microbiol 3:422-8
McFadden, D.W., Camps, M. and Boothroyd, J.C. 2001. Resistance as a tool in the
study of old and new drug targets in Toxoplasma. Drug
Resistance Updates 4:79-84.
Lekutis, C., Ferguson, D.J.P., Grigg, M.E., Camps, M. and Boothroyd, J.C. 2001.
Surface Antigens of Toxoplasma gondii: Variations on a Theme. Intl.
J. Parasitol. 31:1285-1292.
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’Ķ
A. Developmental Biology.
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.
Mutant B43 does not switch to bradyzoites when grown at high pH in vitro.
The parental line was transfected with GFP under control of the bradyzoite-specific promoter from the LDH2 gene. Mutants were then selected by FACS for those which fail to turn on GFP under high pH conditions (a technique developed by Dubremetz and colleagues for inducing differentiation from tachyzoites to bradyzoites in vitro). This image shows the parent and a mutant ("Clone B43") using either phase optics, staining for the cyst wall polysaccharide ("CWPS" - a marker for differentiation to bradyzoites) or fluorescence of GFP. The mutant fails to make a cyst wall or turn on GFP.
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.
Microarray analysis of transcript abundance in wild type and non-switching
mutant parasites after differentiation.
Wild type and mutant (B43) parasites were grown under bradyzoite conditions in vitro (using high pH to induce differentiation). RNA was isolated from the two cultures and labeled cDNA was prepared. cDNA from wild type parasites was labeled green while red corresponds to cDNA from mutant parasites. Yellow spots, therefore, represent genes whose transcripts are equally abundant in the parental and mutant parasites. Green spots correspond to transcripts that are more abundant in the parental vs. the mutant parasites. Since these arrays are from a bradyzoite cDNA library, many of the spots correspond to bradyzoite-specific genes. Thus, it is not surprising that many of the spots are green indicating that the relevant gene is on in the wild type but not in the mutants, as predicted for parasites that fail to switch. Many of the green spots are known bradyzoite-specific genes such as BAG1 while others are novel genes that are now being pursued.
B. Attachment/Invasion/Egress.
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.
31:1177-1186.).
Targeting of PRO-ROP1-VSG to the rhoptry necks.
Parasites were transfected with a construct containing the pro-region of ROP1 fused to trypanosome VSG (a great marker for the secretory pathway from an organism that has no rhoptries and for which we have excellent antibodies). The VSG was detected with texas red-conjugated antibodies while ROP2/3/4 was detected with FITC-conjugated antibodies (courtesy of Jean-Francois Dubremetz). The VSG localizes to the rhoptries although, interestingly, it is more in the anterior "necks" than in the bulbous base where most of the ROP2/3/4 is localized.
Why there are two signals and how they actually mediate the trafficking (i.e.,
what they interact with as the proteins traffic through the secretory pathway)
are not known. By further analysis of these proteins and identification of
other soluble rhoptry proteins, we hope to shed light on these issues. We are
taking a proteomics (in collaboration with J. Wastling in Glasgow) and genetic
approach to identifying such proteins.
C. Host/Pathogen Interaction.
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).
Human foreskin fibroblasts were infected with Toxoplasma and at various times thereafter RNA was isolated, labeled and compared to uninfected control cultures. The level of induction (green) or repression (red) of several thousand human genes in response to the infection is represented by a bar - the more intense the color, the more dramatic the change. In the paper, we give the identities for the many genes that are affected by infection and the conclusions that can be drawn based on the trends. We are currently trying to determine the parasite factors and the signaling pathways in the host that mediate these changes.
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. 2001. Success
and virulence in Toxoplasma
as the result of sexual recombination between two distinct ancestries. Science.
294:161-165. 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. 184:633-639.). 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, M.E. and Boothroyd, J.C. 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. Interestingly, the apicoplast
appears to be almost dispensable in vitro but extremely important for virulence
in vivo.
VI. The practical (and fun)
details...
Of course, we need money to do what we do and the lab currently has four
grants: three major NIH grants and usually one small grant from a foundation or
other agency. Most of the people in the lab have their own or institutional
fellowship support.
The lab meets weekly, with an in depth presentation by one person on his/her
recent results and future plans each week (2 hours). We also use this
opportunity for people to mention new techniques that they've come across and
pleas for help with things that are not working well.
On the social side, people in the lab enjoy indoor and outdoor sports and we
have formidable skills on the volleyball court (with relatively occasional
rotator cuff injuries). The department softball team has actually WON THE
LEAGUE CHAMPIONSHIP some years with a heavy representation from our lab
although the injury rate about equals the number of runs [why is it always the
pipetting hand that gets broken?!?]. And, who would want to play a dumb game like softball, anyway -
some of us think hockey is far safer and far more fun. Snow-shoeing and skiing are also
popular favorites (the mountains are ~3.5 hours away with more snow than you
can imagine) and biking is a year-round favorite in the hills around us for many of us (road and mountain biking; we never get snow around here except on the very tops of the highest hills and then maybe just once or twice a winter).
To sum up, I think it's fair to say that we try to do our work (and our play)
with gusto and, most of all, we try to enjoy the process!
VII. Where we do it...
Answer: at Stanford University ...
The lab is ~1400 square feet (~140 square meters to the rest of the
world) and is located in a single, contiguous room on the third floor of the
Fairchild Building (~1980 vintage). This is the home of the Department of Microbiology and
Immunology in the Stanford University School of Medicine on the main
campus of Stanford University. You
should link to any of these last three places to gain more information on them.
The lab faces north-west on its long axis which is ceiling-to-floor windows and
sliding glass doors opening onto balconies. Each person has his/her own bench,
including integrated desk space. (By the way, Northern Californian climate is
actually on the cool side (apart from occasional heat waves) and so we really
don't spend much time on the balconies... honest!) We are 35 miles (~55
kilometers) south of San Francisco and 25 miles (~40 kilometers) from the
Pacific Ocean. If you don't know this area, you may be surprised to know that
90% of the coast here is pristine and completely undeveloped - the water may be
freezing but at least that has kept the hordes away. We are ~200 miles (~350
kilometers) from the 14000 foot (~4500 meter) high Sierra Nevada mountains.
We're about 1 mile (you don't want to know that in metric) from the San Andreas
Fault but nowhere is perfect, right? There are a huge number of "open
space" areas less than an hour away by car including redwood forests,
wide-open beaches and rolling hills (~2500 feet (~800 meters) high). Most of
these are not parks in the usual sense, just very large chunks of land
(totaling several hundred square miles) where you can hike in quiet; one
weekend (mid-October), my daughter and I and a couple of friends camped at one
and hiked two half-days in redwood forests. We were less than 50 minutes from
the lab yet saw not one other person on the trails either day ... enough said.
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