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.