Plenary
Lectures
Horizontally
transferred genes as new targets for drug development
in Cryptosporidium parvum
Boris
Striepen
Center for Tropical and Emerging Global Diseases &
Department of Cellular Biology, University of Georgia,
Athens, GA 30602, e-mail: striepen@cb.uga.edu
Cryptosporidium parvum
has emerged as one of the most troublesome waterborne
infections in the industrialized world. A number of
large outbreaks have occurred in the U.S. with the
largest in Milwaukee causing 403,000 cases of acute
gastrointestinal diseases. Cryptosporidiosis is also
an important factor in severe diarrheal disease in
children around the world who lack access to contamination
free drinking water. Crytosporidiosis is a gastrointestinal
disease, characterized by watery diarrhea, abdominal
cramps, nausea and fever. The disease is self-limiting
with symptoms usually subsiding after 2 to 3 weeks.
In contrast, immunosuppressed patients suffer from
prolonged chronic disease resulting in severe dehydration
and weight loss which can become live-threatening.
It is these patients for which effective antimicrobial
therapy is most urgently needed.
A wide variety
of antimicrobial agents has been tested in vitro,
in animal models and in clinical trials. Despite considerable
effort no fully effective therapy has been established
yet. However, two drugs have emerged which show a
consistent albeit modest benefit in placebo controlled
studies: paromomycin and even more promising nitazoxaninde,
which has now received FDA approval for the treatment
of immunocompetent children. The resistance of C.
parvum against drugs, which are highly effective against
related apicomplexan parasites has puzzled and frustrated
researchers and clinicians alike. Two general models
can be developed to explain this resistance. The first
model is the extracytoplasmatic model. It argues that
the peculiar extracytoplasmatic subcellular localization
of the parasite within its host cell severely limits
access of drugs to the parasite. In addition efflux
pumps could further rid the parasite of toxic compounds
protecting susceptible target enzymes. The second
model, the metabolism model, proposes that the metabolism
of Cryptosporidium differs from other Apicomlexa much
more than initially appreciated and that drugs active
against other apicomplexa fail because their targets
are absent or divergent in C. parvum. Obviously, these
models are not mutually exclusive and both mechanisms
could act synergistically.
The metabolic hypothesis put forward in this talk
explains C. parvum’s resistance to typical anti-apicomplexan
drugs as a reflection of its phylogenetic and metabolic
uniqueness. One of the most striking differences between
C. parvum and other apicomplexan pathogens is the
absence of fully functional mitochondria and plastids.
An experimental study by Zhu and colleagues had predicted
the absence of an apicoplast in C. parvum based on
PCR and hybridization experiments, which failed to
detect the presence of sequences conserved among all
plastid genomes. Indeed the completed and now published
C. parvum and hominis genome sequences do neither
contain the organellar genome nor the extensive set
of nuclear encoded plastid targeted genes described
for P. falciparum and T. gondii. The secondary endosymbiosis
that let to the presence of the apicoplast is generally
viewed as and early event in the evolution of Apicomplexa
and Alveolata. The observation that several genes
in the C. parvum genome show strong phylogentic relationships
to plants and algae support this view, and suggest
that C. parvum is derived from a lineage which once
harbored an algal endosymbiont which was lost later.
The lack of a plastid has important metabolic and
pharmacological consequences as it e.g. explains C.
parvum’s resistance to macrolide antibiotics
like clindamycin which specifically target protein
synthesis in the plastid and which are quite effective
in T. gondii.
C. parvum is a challenging experimental system, and
the lack of continuous culture models and transfection
technology has posed limitations on the molecular
analysis of this pathogen. However, C. parvum has
one of the Apicomplexa’s most accessible genome.
The genome is small and introns are rare making gene
prediction straightforward. The analysis of the genome
sequence has just begun, but it already uncovered
a surprising number of metabolic differences between
C. parvum and other apicomplexans.
Our work has been focused on nucleotide biosynthesis.
Nucleotide biosynthesis has been a main stay of antiprotozoal
treatment. Antifolates are highly active against T.
gondii and P. falciparum. C. parvum on the other hand
is resistant to pyrimethamine/sulfadiazine. DHFR-TS
is one of the few potential C. parvum drug targets
that has been studied in detail. Structural as well
as kinetic analysis recently published by other investigators
suggests that the Cryptosporidium enzyme is quite
different from previously characterized fused enzymes
from kinetoplastids and apicomplexans. In addition
to differences in DHRF-TS the general pattern of pyrimidine
nucleotide synthesis and salvage could equally modulate
the efficiency of antifolates. Recent genomic and
experimental work has uncovered a surprising diversity
of pyrimidine biosynthetic pathways within the Apicomplexa.
P. falciparum is entirely dependent on de novo synthesis
of pyrimidines making DHRF-TS an essential enzyme.
T. gondii posses the ability to salvage uracil using
uracil-phosphoribosyltransferase. However as recently
reported by Fox and Bzik, this salvage pathway is
not sufficient to sustain the parasite in the absence
of de novo synthesis . C. parvum finally, has lost
all six genes for the enzymes in this pathway indicating
that it is unable to synthesize pyrimidines de novo.
The parasite depends entirely on salvage, and three
pyrimidine salvage enzymes have been identified, two
of them uridine kinase-uracil phosphoribosyltransferase
(UK-UPRT) and thymidine kinase (TK) are not found
in any other apicomplexan. Phylogenetic analysis suggests
that both enzymes were obtained from other organisms
by horizontal gene transfer, UK-UPRT from an algal
endosymbiont which has since been lost, and TK from
a proteobacterium. The presence of TK in C. parvum
provides an additional and potentially alternative
source of dTMP for this parasite and could reduce
its sensitivity to inhibition of DHFR and the subsequent
starvation of the thymidylate synthase reaction. Equally
the finding of UK-UPRT explains the difference in
susceptibility to cytosine arabinoside between C.
parvum and T. gondii. While T. gondii is highly resistant
to this prodrug, which has to be activated by cytosine
or uridine kinase, C. parvum was surprisingly susceptible
in a large drug screen conducted by Woods and Upton.
Both UK-UPRT and TK could be new targets to pursue
for C. parvum. Especially TK might hold promise based
on the successful exploitation of this target in the
therapy of Herpes viruses. A large variety of compounds
subverting the viral TK has been generated, and the
relationship between enzyme structure and drug sensitivity
and resistance is well understood. The divergent phylogenetic
origin of the parasite enzyme from a proteobacterium
might allow for the identification of compounds with
selective specificity for the parasite versus the
human enzyme. But will nucleoside analogs known to
subvert this enzyme be able to reach their target
within the parasites? Experiments using 5-deoxybromo-uridine
in C. parvum infected tissue cultures indeed suggest
that this class of compounds gains access to the parasite.
Horizontal gene transfers into the C. parvum nucleotide
metabolism are not limited to the pyrimidine pathway.
C. parvum has obtained its inosine 5’-monophosphate
dehydrogenase (IMPDH), an enzyme central to the purine
salvage pathway, from an e-proteobacterium whereas
P. falciparum and T. gondii harbor enzymes of clear
eukaryotic phylogeny. As TK, IMPDH is a well established
target of antiviral and immunosuppressive therapy.
Furthermore prokaryotic and eukaryotic IMPDH differ
in structure and mechanism which should facilitate
the identification of C. parvum specific inhibitors.
Kinetic analysis of recombinant C. parvum IMPDH has
shown pronounced differences between parasite and
human enzyme, most importantly a 1000 fold difference
in susceptibility to mycophenolic acid. This protein
has also been crystallized recently and the results
from the ongoing structural study should greatly enhance
the ability to design parasite specific inhibitors.
Genomic and experimental studies show a highly streamlined
salvage pathway for C. parvum which relies on adenosine
as sole source of purine (earlier biochemical studies
on crude parasite lysates had also predicted adenine,
hypoxanthine, xanthine and guanine salvage, however
the genes for these enzymes seem not to be present
in the genome. IMPDH is at the center of this streamlined
pathway and an essential enzyme of the multi-step
conversion of AMP to GMP. Treatment of infected tissue
cultures with the IMPDH inhibitors mycophenolic acid
and ribavirin consequently results in dose dependent
inhibition of C. parvum development.
One of these drugs, ribavirin, has also been tested
in a neonatal mouse model of cryptosporidiosis and
treatment with 50 mg/kg for one week resulted in a
90% reduction of parasite load when compared to untreated
controls. Interestingly, in these experiments the
drug was injected into the peritoneum, rather than
given orally suggesting that uptake from the intestinal
lumen through the apical membrane of the host cell
might not be necessary for this compound. Ribavirin,
like the aforementioned drug targeting TK, is a nucleoside
analog. This class of drugs does not freely diffuse
across membranes, but subverts the nucleoside transporters
of their target cell to get access. But where is the
parasite nucleoside transporter localized? Several
nucleoside transporters have been characterized in
related intracellular parasites and most seem to localized
over the entire surface of the parasite. A potential
nucleoside transporter with similarity to the T. gondii
adenosine transporter has been identified in C. parvum
and could provide an important molecular reagent to
further address nutrient and drug transport in C.
parvum.
A series of
recent genomic, biochemical and cell biological studies
has produced considerable support for the metabolism
hypothesis for C. parvum’s drug resistance.
C. parvum’s metabolism differs dramatically
from its better studied cousins P. falciparum and
T. gondii. In all cases where the target of a widely
used anti-apicomplexan drug has been characterized
in molecular detail, it was either absent in C. parvum
(e.g. clindamycin and atovaquone) or the enzyme was
highly divergent and resistant (pyrimethamine). Limited
drug access might still remain as an important challenge
to treatment, but the data warrants a fresh look at
a new and metabolically more appropriate set of drugs
and targets. The divergence of C. parvum from the
generic eukaryotic metabolism might after all present
an Achilles heel, and the presence of numerous bacterial
enzymes provides an exciting set of candidate targets
for parasite specific inhibition. Transgenic models
might provide urgently needed assay systems to validate
targets identified by genome mining and screen compounds.
Acknowledgements:
The abstract format prevented me from citing published
work by colleagues appropriately for which I apologize.
Work in the author’s laboratory is currently
funded by grants from the National Institutes of Health
(AI 48475 & AI 55268), the American Heart Association,
and Merck Research Laboratories. I would like to thank
Liz Hedstrom, Jessica Kissinger and Jan Mead for many
discussions.