cell biology
and molecular genetics of protozoan parasites: Toxoplasma and Plasmodium (malaria); eukaryotic
evolution; designing and mining genome databases; computational
biology
Studies in the Roos laboratory employ a variety of modern techniques
in cell biology, molecular genetics, biochemistry, and genomics
to study protozoan parasites, eukaryotic evolution, and host-pathogen
interactions. At present, our primary focus is on the phylum
Apicomplexa, a group of protozoan parasites that typically replicate
within specialized vacuoles inside the cells of infected animals.
Plasmodium parasites cause malaria, afflicting hundreds
of millions of people each year and killing millions of children,
primarily
in sub-Saharan Africa. Toxoplasma gondii is even more
widespread, chronically infecting ~30% of the US population;
this parasite
is a leading source of congenital neurological birth defects
in humans and farm animals, a prominent opportunistic infection
associated with immunosuppressive treatments and diseases (including
AIDS), and a waterborne pathogen of some concern from a biodefense
standpoint. By virtue of its evolutionary position, molecular
genetic accessibility, and subcellular architecture, T. gondii has
proved useful for studying central features of eukaryotic evolution.
The availability of effectively complete genome sequences
for these parasites also opens up new realms to experimental
analysis – at the lab bench, and at the computer. Ongoing
projects include:
Genetic analysis of parasite biology
The ability to saturate the T. gondii genome by insertional
mutagenesis (and clone the tagged loci), target defined loci
for genetic
deletion or allelic replacement, and control the expression of
recombinant proteins makes powerful genetic approaches feasible.
Successful expression of fluorescent reporters facilitates analysis
of transgenic parasites in living cells and tissues. These tools
have been exploited to isolate mutants elucidating the temporal
and developmental controls that that regulate differentiation
through the complex parasite life cycle, and to devise strategies
for examining host/parasite interac-tions and the host immune
response.
Mechanisms of drug action and resistance
Gene replacement studies at the DHFR-TS locus have defined the
molecular basis of resistance to antifolates in malaria, and
the fitness costs of drug-resistance mutations. Studies on the
surprising efficacy of certain classical prokaryotic inhibitors
against apicomplexan parasites led to the identification of
a novel organelle — the apicoplast — a nonphotosynthetic
plastid acquired by lateral genetic transfer of a chloroplast
from a green alga (secondary endosymbiosis). Related studies
have elucidated the remarkable mechanism used to target proteins
to this organelle, and identified a variety of novel targets
in parasite metabolic and differentiation pathways.
Structure-function
studies on basic processes
Genetic studies have identified key enzymes involved in nucleoside
metabolism, and functional expression and crystallization of
these proteins opens the way to structure-based drug design.
We have also used T. gondii to develop a model for examining
the minimum essential elements of eukaryotic design, focusing
on organization of the secretory pathway. Further cell biological
studies reveal a remarkable array of subcellular structures,
including novel cytoskeletal elements likely to play a role
in parasite assembly, invasion, and motility. Proteomic analysis
of these elements is now underway.
Computational biology research
Ongoing genome and EST projects have led to the development of
a variety of bioinformatics resources, including the malaria
parasite genome database <http://PlasmoDB.org>. Current
research interests include developing new algorithms for comparative
genomic analysis, and databases enabling the integration and
mining of diverse large-scale post-genomics datasets. Coupling
computational database mining with laboratory analysis provides
new insights into eukaryotic biology and evolution, and facilitates
the identification of targets for drug/vaccine/diagnostic development.
We are also actively engaged in bioinformatics training programs
around the world.
Evolutionary studies
Because diversity among the protozoa dwarfs the distances separating
animals, plants and fungi, many of our studies have interesting
evol-utionary implications. Areas of research interest include
the development of eukaryotic transcrip-tional control mechanisms,
the origin and function of subcellular organelles, the nature
of the host-pathogen relationship, the role of lateral genetic
transfer in the phylogenetic history of “higher” eukaryotes,
and comparative genomics/genome evolution.
selected
publications
Li, L, CJ Stoeckert & DS Roos. 2003. OrthoMCL: Identification
of ortholog groups for eukaryotic genomes. Genome Res, in press.
Drozdowicz
et al. 2003. Isolation and functional characterization of TgVP1,
a type I vacuolar H+-translocating pyrophosphatase from
T. gondii. J Biol Chem 278:1075-1085.
Li, L et al. 2003. Gene
discovery in the Apicomplexa as revealed by EST sequencing and
assembly of a comparative gene database.
Genome Res 13:443-454.
Foth, BJ et al. 2003. Dissecting apicoplast
targeting in the malaria parasite Plasmodium falciparum. Science
299:705-708.
Bahl, A et al. 2003. PlasmoDB: The Plasmodium genome
resource. Nucl Acids Res 31:212-215.
Kissinger, JC et al. 2002.
The Plasmodium genome database: Designing and mining a eukaryotic
genomics resource. Nature 419:490-492.
Gardner, MJ et al. 2002.
The genome sequence of the human malaria parasite Plasmodium
falciparum. Nature 419:498-511.
Pelletier, L et al. 2002. Golgi
biogenesis in Toxoplasma gondii. Nature 418:548-552.
Joiner, KA & DS
Roos. 2002. Secretory traffic in Toxoplasma gondii: Less is more.
J Cell Biol 156:1039-1050.
Matrajt, M, RGK Donald, U Singh & DS
Roos. 2002. Identification and characterization of T. gondii
differentiation mutants. Molec
Microbiol 44:735-747.
Hu, K, DS Roos & JM Murray. 2002. A
novel polymer of tubulin forms the conoid in Toxoplasma gondii.
J Cell Biol 156:1039-1050.
Swedlow, JR et al. 2002. Measurement
of tubulin content in the conoid and spindle pole of the parasite
Toxoplasma gondii: A
comparison of laser scanning confocal and wide field fluorescence
microscopy
for quantitative analysis in living cells. Proc Natl Acad Sci
USA 99:2014-2019.
Hu, K et al. 2002 Daughter cell assembly in
the protozoan parasite Toxoplasma gondii. Molec Biol Cell 13:593-606.
Roos,
DS. 2001. Bioinformatics – trying to swim in a sea
of data. Science 291:1260-1261.
He, CY et al. 2001. A plastid
segregation defect in the protozoan parasite Toxoplasma gondii.
EMBO J 20:330-339.
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