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Charles University in Prague
Faculty of Science
Drug discovery in Cryptosporidium parvum:
What we can learn from molecular and biochemical analyses
MUDr. RNDr. František Stejskal
Summary of Ph.D. Thesis
Thesis supervisor: Prof. Janet S. Keithly, Ph.D.
Prague, 2004
Department of Parasitology, Faculty of Science, Charles University
Viničná 7, 128 44 Praha 2, Czech Republic
CURRICULUM VITAE
František Stejskal, born March 29, 1963 in Jindřichův Hradec, Czech Republic
1988
Masters degree in Biology - parasitology at the Faculty of Science, Charles
University, Prague
1989
Rerum Naturalium Doctor (RNDr.) degree – State examination in Parasitology at
the Faculty of Science, Charles University, Prague
1993
Medicinae Universae Doctor (MUDr.) degree at the First Faculty of Medicine,
Charles University, Prague
1995
Board examination I in Internal Medicine (Czech Republic)
1995
United States Medical Licencing Examination, Step 1
1996
United States Medical Licencing Examination, Step 2
1998 – 2000 Fogarty Fellow, Division of Infectious Diseases, Wadsworth Center, NY State
Department of Health, Albany, NY
1993 – present: Assistant Professor, 3rd Department of Infectious and Tropical Diseases,
First Faculty of Medicine, Charles University, Prague
1996 – present: Head of Department of Tropical Medicine, First Faculty of Medicine,
Charles University, Prague
This work was supported by NIH-FIRCA grant 1R03 TW01507-01 (J.S. Keithly and F.
Stejskal), by NIH Emerging Infectious Disease Training Grants 5D43 TW00915-04 (D.L.
Morse), 5D43 TW00023-3 (J.A. DeHovitz) and the Czech Ministry of Education grant
FRVŠ (2004) No. 962 (F. Stejskal and V. Čtrnáctá).
Thesis supervisor:
Janet S. Keithly, Ph.D., Associate Professor, Division of Infectious Diseases, Wadsworth
Center, New York State Department of Health, Albany, NY, USA
Thesis reviewers:
Jan Mead, Ph.D., Associate Professor, Medical Research Department,
Atlanta VA Medical Center, Decatur, GA, USA
Doc. RNDr. Petr Folk, CSc., Associate Professor, Department of Animal Physiology and
Developmental Biology, Faculty of Science, Charles University, Praha
Examination board:
Prof. RNDr. Jaroslav Kulda, CSc., Professor, Department of Parasitology and
Hydrobiology, Faculty of Science, Charles University, Praha - Chairmen
Prof. RNDr. Jiří Vávra, DrSc., Professor Emeritus, Faculty of Science, Charles University,
Praha
Prof. MVDr. Břetislav Koudela, CSc., Professor, Institute of Parasitology, Faculty of
Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno
Doc. RNDr. Petr Folk, CSc., Associate Professor, Department of Animal Physiology and
Developmental Biology, Faculty of Science, Charles University, Praha
RNDr. Oleg Ditrich, CSc., Assistant Professor, Institute of Parasitology, Academy of
Science of the Czech Republic, České Budějovice
DISSERTATION ABSTRACT
Introduction
Cryptosporidium parvum is an obligatory intracellular, parasitic protist that
belongs together with other human and animal pathogens like haematozoans Plasmodium
sp., Babesia sp. and coccidians Toxoplasma gondii, Isospora sp., Cyclospora sp. and
Eimeria sp. to the phylum Apicomplexa. Apicomplexans, together with ciliates and
dinoflagellates, form a phylogenetically related, but structurally diverse group of protists
named Alveolata. The most distinctive features of cryptosporidia include the unusual
localization and epicellular attachment to the host epithelial cells, lack of host and organ
specificity, presence of endogenous infective thin-wall oocysts and an unusual broad
resistance to anticoccidial and antimicrobial drugs. Indeed, the recent phylogenetic
analyses based on both rRNA and protein sequences have unequivocally demonstrated
that the cryptosporidia form an early branching group together with the aseptate
archigregarines rather than Eucaccidea and Haematozoea. Both groups, cryptosporidia
and gregarines have a homoxenous life cycle and share extracytoplasmic development
which may be relict of a common ancestral life cycle.
C. parvum infects mammalian gastrointestinal epithelium and can produce a selflimited diarrhea in healthy adults and children, but potentially life-threatening infection in
immunocompromised persons, especially those with the acquired immunodeficiency
syndrome (AIDS). Cryptosporidiosis is one of the most common enteric infections in
humans and domestic animals, accounting up to 6 percent of all diarrheal diseases. It was
recognized as a cause of diarrhea with significant weight loss among young children in
developing countries and serious, difficult-to-treat cause of waterborne and foodborne
outbreaks worldwide. Together with other waterborne microorganisms, C. parvum has
recently been classified as a category B bioterrorism agent by the Centers for Disease
Control and Prevention (CDC). Cryptosporidiosis is one of a few infections for which no
curative treatment is known, especially in immunosupressed patients. Despite the
importance and severity of this infection, the metabolism of Cryptosporidium is poorly
understood.
The unique intracellular, extracytoplasmic localization within the host intestinal
epithelial cells is the defining characteristic of all cryptosporidia. They share with
microaerophilic diplomonads, parabasalids, entamoebids and microsporidians unique
structural and metabolic adaptations for life in the intestinal tract. The energy metabolism
of C. parvum is fermentative, and its glycolysis resembles that of coccidia and other
microaerophilic protists. C. parvum relies primarily upon the transport of monosaccharides,
amino acids, and purine or pyrimidine nucleotides from the host intestine. In contrast to the
coccidia, C. parvum appears to lack a functional mannitol cycle. In addition, a relict
mitochondrion, to which both chaperonin Hsp60 and Hsp70 can be localized, was
described. Although the C. parvum mitochondrion is incapable of generating ATP by
oxidative phosphorylation (Krebs cycle and respiratory chain enzymes are not present)
evidence is accumulating that it may serve for iron-sulfur cluster biosynthesis and
assembly.
C. parvum polyamine metabolism resembles that of certain bacteria and plants
which use arginine decarboxylase (ADC) and agmatine iminohydrolase to produce
putrescine. The ADC activity and inhibition of this enzyme by difluoromethylarginine was
reported. C. parvum also has a very active back-conversion pathway from spermine to
spermidine via action of spermidine/spermine N1–acetyltransferase and polyamine
oxidase. The activity of these enzymes is 20-fold greater than activity of ADC. Sadenosylmethionine (AdoMet) is synthesized from methionine and ATP through the action
of S-adenosylmethionine synthetase (SAMS). S-adenosyl-homocysteine (SAHH) regulates
the entire methionine cycle and AdoMet production. AdoMet is a major donor of methyl
groups for DNA, RNA and protein methylation, and decarboxylated AdoMet is a donor of
an aminopropyl group for the polyamine synthesis.
A giant 25 kb multifunctional Type I fatty acid synthase (FAS1) gene was
identified and characterized in C. parvum. It differs from the Type II fatty acid synthases
(FAS) characterized in T. gondii and P. falciparum. These FAS consist of discrete
monofunctional proteins for fatty acid synthesis and are targeted to the apicoplast. C.
parvum lacks any Type II FAS which is in agreement with the absence of an apicoplast in
this apicomplexan. The in vitro analysis the C. parvum FAS1 enzymatic activities and
substrate preference confirmed that this parasite may be able to use C12 – C24 saturated
fatty acids as loading units for synthesis of long chain fatty acids.
The aims of the work

Characterisation of the C. parvum pyruvate:ferredoxin oxidoreductase (PFO)cytochrome P450 reductase (CPR) fusion and other proteins for energy
metabolism.

Characterisation of the C. parvum S-adenosylmethionine cycle enzymes,
especially
S-adenosylmethionine
synthetase
(SAMS)
and
S-adenosyl-
homocysteine hydrolase (SAHH), and testing of their specific inhibitors.

Cloning and characterisation of the fatty acid/polyketide synthase (Type I) gene
(FAS/PKS1) in C. parvum.
Summary of results
Several unique, Cryptosporidium specific genes and novel metabolic pathways
were investigated during the work on this thesis. This effort has led to a better
understanding of the biology of C. parvum. In addition, several enzymes that significantly
differ from the mammalian host and may serve as potential drug targets were identified:
1. An unusual fusion pyruvate:NADP+ oxidoreductase (PNO) gene which
consists of the N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and C-terminal
cytochrome P450 reductase (CPR) domains was cloned and characterized. PNO is
expressed in both sporozoites and intracellular stages. It is cytosolic in contract to a similar
fusion protein identified in the mitochondrion of the photosynthetic protist Euglena gracilis.
Phylogenetic analyses support the monophyly of PFO in eukaryotes and agree with the
endosymbiotic origin of eukaryotic mitochondria and their derivatives. It has been
suggested that PNO might be involved in the activation of nitazoxanide, a recently
recognized promising drug against cryptosporidiosis.
2. The gene for the C. parvum Narf-like protein, homologue of [Fe]-hydrogenase,
has been cloned and characterized. It resembles Narf-like proteins of aerobic protists and
higher eukaryotes rather than functional [Fe]-hydrogenases of microaerophilic protists and
anaerobic bacteria. The C. parvum enzyme which in silico analysis indicates cytosolic
localization does not appear to have retained any [Fe]-hydrogenase activity. Therefore, the
function of the Narf-like protein will need to be further elucidated.
3. A unique 40 kb gene for polyketide synthase (PKS1) was identified, cloned
and characterized in C. parvum. The analyses of PKS1 and FAS1 indicate that these C.
parvum enzymes may be able to elongate, from medium-chain fatty acids, saturated and
unsaturated long-chain fatty acids and polyketides using acetyl-CoA as an elongation unit.
Although the exact function is not known for either PKS1 or FAS1, one or both may
represent attractive drug targets which should be explored.
4. The enzymes of the S-adenosylmethionine cycle and polyamine metabolism
may also be potential drug targets. Both C. parvum SAMS and SAHH genes were
characterized and display significant differences from mammalian homologues. Together
with the fact that C. parvum polyamine biosynthesis differs significantly from that of its host
(using arginine- instead of ornithine decarboxylase to produce putrescine), and that the
back-conversion pathways are active, combination chemotherapy targeting the Sadenosylmethionine and polyamine metabolism may offer an alternative treatment of
cryptosporidiosis.
5. The analyses of the C. parvum genome have revealed that the parasite
contains a set of genes for iron-sulfur cluster assembly, and that these enzymes may
operate in the novel, recently characterized relict mitochondrion. Hence, another drug
target may have been identified by this research.
PUBLICATIONS:
Rotte, C., Stejskal, F., Zhu, G., Keithly, J.S. & Martin, W. 2001. Pyruvate:NADP+
oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan
Cryptosporidium
parvum:
A
biochemical
relic
linking
pyruvate
metabolism
in
mitochondriate and amitochondriate protists. Mol. Biol. Evol. 18:710-720.
Stejskal, F., Šlapeta, J., Čtrnáctá, V. & Keithly, J.S.. 2003. A Narf-like gene from
Cryptosporidium parvum resembles homologues observed in aerobic protists and higher
eukaryotes. FEMS Microbiol. Lett. 229:91-96.
LaGier, M.J., Tachezy, J., Stejskal, F., Kutišová, K. and Keithly, J.S. 2003. Mitochondrialtype iron-sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan
Cryptosporidium parvum. Microbiology 149:3519-3530.
Šlapeta, J., Stejskal, F., & Keithly, J.S.. 2003. Characterization of S-adenosylmethionine
synthetase in Cryptosporidium parvum (Apicomplexa). FEMS Microbiol. Lett. 225:271-277.
Zhu, G., LaGier, M.J., Stejskal, F., Millership, J.J., Cai, X. and Keithly, J.S. 2002.
Cryptosporidium parvum: The first protist known to encode a polyketide synthase. Gene
298:79-89.
ABSTRACTS AND PRESENTATIONS:
Stejskal, F., Zhu, G. & Keithly, J.S. Characterization of two genes for polyamine synthesis
and methylation in Cryptosporidium parvum. Molecular Parasitology Meeting X. Woods
Hole, MA, USA, September 12 - 16, 1999. Abstract 242A. Poster presentation.
Stejskal, F., Zhu, G. & Keithly, J.S. Implication for chemotherapy of two genes for
polyamine synthesis and methylation reactions in Cryptosporidium parvum. 48th American
Society of Tropical Medicine and Hygiene Annual Meeting, Washington, DC, USA,
November 28 – December 2, 1999. Abstract 370. Am. J. Trop. Med. Hyg. 61(Suppl.3):
301-302. Poster presentation.
Stejskal, F., Zhu, G. & Keithly, J.S. Pyruvate:ferredoxin oxidoreductase/NAD(P)H
cytochrome P450 reductase fusion in Cryptosporidium parvum - a novel target for drug
design? 9th annual ICTDR meeting. NIH, Bethesda, MD, USA, April 10 - 12, 2000 and
International Training and ResearchProgram in Emerging Infectious Diseases (ITREID).
NIH, Rockville, MD, USA, April 12 - 13, 2000. Poster presentation.
Stejskal, F., Zhu, G. & Keithly, J.S. Implication for chemotherapy of two genes for
polyamine synthesis and methylation reactions in Cryptosporidium parvum. 9th annual
meeting ICTDR. NIH, Bethesda, MD, USA, April 10 - 12, 2000 and International Training
and Research Program in Emerging Infectious Diseases (ITREID). NIH, Rockville, MD,
USA, April, 12 - 13, 2000. Poster presentation.
Stejskal, F., Zhu, G. & Keithly, J.S. Cloning and characterization of a pyruvate:ferredoxin
oxido-reductase/NADPH cytochrome P450 reductase fusion gene from the Apicomplexan
Cryptosporidiumparvum. Joint Meeting of The American Society of Parasitologists & The
Society of Protozoologists, San Juan, Puerto Rico, June 24 - 28, 2000. Abstract 151. Oral
presentation.
Stejskal, F. A pyruvate:ferredoxin oxidoreductase and cytochrome P450 reductase fusion
from Cryptosporidium parvum: Implication for evolution of lower eukaryotes. XIII Meeting of
the International Society for Evolutionary Protistology. České Budějovice, Czech Republic,
July 31 – August 3, 2000. Oral presentation.
Stejskal, F., Zhu, G. & Keithly, J.S. Characterization of two genes for the methionine cycle
in Cryptosporidium parvum. COST-B9 expert Meeting and Satellite Symposium of the XI.
International Congress of Protozoology – Anaerobic Protozoan Parasites: From Basic
Science to Drug Targets. Prague, Czech Republic, July 21 - 24, 2001. Abstract p.39. Oral
presentation.
Stejskal, F. Cryptosporidium parvum energy metabolism. Czech-American Fogarty
International Conference. Prague, Czech Republic, April 23, 2002. Oral presentation.
Stejskal, F., Šlapeta J., Čtrnactá, V. & Keithly, J.S.: Molecular characterisation of the
hydrogenase
and
ferredoxin
oxidoreductase
homologues
from
apicomplexan
Cryptosporidium parvum. International Society for Evolutionary Protistology 14th Meeting.
Vancouver, Canada, July 19 - 24, 2002. Oral presentation.
Stejskal, F., Čtrnactá, V., Hrdý, I., Šlapeta, J. & Keithly, J.S.: Iron-sulfur cluster
biosynthesis components in apicomplexan Cryptosporidium parvum. 3rd European
Congress on Tropical Medicine and International Health. Lisboa, Portugal, September 8 11, 2002. Abstract MOPS 009. Acta Tropica. 83(Suppl.1):S34. Oral presentation.
Stejskal, F., Hrdý, I., Čtrnáctá, V. & Keithly, J.S.: NADPH:ferredoxin oxidoreductase from
Apicomplexan Cryptosporidium parvum . VIII International Workshop on Opportunistic
Protists (IWOP-8) and International Conference on Anaerobic Protists. Hilo, Hawaii, USA,
July 25 - 29, 2003, Abstract A 48. Oral presentation.
Stejskal, F., Čtrnactá, V., Hrdý, I. & Keithly, J.S.: Energy metabolism in Cryptosporidium
parvum. International Symposium Thread of Infection. July 25 - 28, 2004, Würzburg,
Germany. Abstract 31. Poster presentation.