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 self-limited 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. S-adenosylmethionine (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 S-adenosylmethionine 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. Mitochondrial-type 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.