Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum

Molecular and Biochemical Parasitology 105 (2000) 253 – 260 www.elsevier.com/locate/parasitology

Molecular analysis of a Type I fatty acid synthase in Cryptosporidium par6um  Guan Zhu a,*, Mary J. Marchewka a, Keith M. Woods b, Steve J. Upton b, Janet S. Keithly a,1 a

New York State Department of Health, Wadsworth Center, PO Box 22002, Albany, NY 12201 -2002, USA b Di6ision of Biology, Kansas State Uni6ersity, Manhattan, KS 66506 -4901, USA Received 14 July 1999; received in revised form 29 September 1999; accepted 30 September 1999

Abstract We report here the molecular analysis of a Type I fatty acid synthase in the apicomplexan Cryptosporidium par6um (CpFAS1). The CpFAS1 gene encodes a multifunctional polypeptide of 8243 amino acids that contains 21 enzymatic domains. This CpFAS1 structure is distinct from that of mammalian Type I FAS, which contains only seven enzymatic domains. The CpFAS1 domains are organized into: (i) a starter unit consisting of a fatty acid ligase and an acyl carrier protein; (ii) three modules, each containing a complete set of six enzymes (acyl transferase, ketoacyl synthase, ketoacyl reductase, dehydrase, enoyl reductase, and acyl carrier protein) for the elongation of fatty acid C2-units; and (iii) a terminating domain whose function is as yet unknown. The CpFAS1 gene is expressed throughout the life cycle of C. par6um, since its transcripts and protein were detected by RT-PCR and immunofluorescent localization, respectively. This cytosolic Type I CpFAS1 differs from the organellar Type II FAS enzymes identified from Toxoplasma gondii and Plasmodium falciparum which are targetted to the apicoplast, and are sensitive to inhibition by thiolactomycin. That the discovery of CpFAS1 may provide a new biosynthetic pathway for drug development against cryptosporidiosis, is indicated by the efficacy of the FAS inhibitor cerulenin on the growth of C. par6um in vitro. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Molecular analysis; Type I fatty acid synthase; Cryptosporidium par6um

Abbre6iations: FAS, fatty acid synthase; ACP, acyl carrier protein; AT, acyl transferase; KS, ketoacyl synthase; DH, dehydrase; ER, enoyl reductase; KR, ketoacyl reductase; TE, thioesterase; RT-PCR, reverse transcriptase-polymerase chain reaction; GST, glutathione-S-transferase.  Note: Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession number AF082993. * Corresponding author. Tel.: + 1-518-4742187; fax: + 1-518-4736150. E-mail addresses: [email protected] (G. Zhu), [email protected] (J.S. Keithly) 1 2nd Corresponding author. 0166-6851/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 1 8 3 - 8

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1. Introduction

2. Materials and methods

Fatty acids are essential to all organisms because they function as a source of energy, components of biomembranes, and metabolic regulators. The biosynthesis of fatty acids and their derivatives typically consists of several two-carbon (C2) elongation cycles. In each cycle, an acetyl primer is condensed from malonyl-CoA into a fatty acid precursor, reduced, dehydrated and reduced again to form a saturated fatty acid chain longer by two carbons than the precursor [1]. There are at least seven fatty acid synthetic enzymes, including acyl carrier protein (ACP), acyl transferase (AT), ketoacyl synthase (KS), ketoacyl reductase (KR), dehydrase (DH), enoyl reductase (ER) and thioesterase (TE). In animals and some fungi, these FAS enzymes are fused into one or two large multifunctional polypeptides, referred to as Type I fatty acid synthases (FAS). In bacteria and plants (chloroplast), these enzymes and their isoforms are discrete and monofunctional, i.e. Type II FAS [1–3]. Little was known about fatty acid synthesis in members of the Phylum Apicomplexa, until the recent discovery of several fatty acid synthetic enzymes including ACP (acpP), KS III ( fabH) and DH ( fabZ) in T. gondii and P. falciparum [4,5]. Like plant Type II FAS, these enzymes are nucleus-encoded, but targetted to the apicoplast. This finding not only suggested a vital function for the apicoplast, but also provided the first molecular evidence that some parasitic protists may synthesize fatty acids de novo. In the present study, we report the identification and molecular analysis of a FAS gene from C. par6um (CpFAS1). However, this CpFAS1 gene contains a large, 25 kb open reading frame (ORF) that encodes a cytosolic Type I FAS consisting of 21 enzymatic domains, rather than an organellar Type II FAS as observed for T. gondii and P. falciparum. We have also investigated CpFAS1 gene expression and protein localization in sporozoites of C. par6um, and have tested the efficacy of FAS inhibitors against the in vitro growth of C. par6um.

2.1. Parasites Fresh oocysts of two laboratory strains (KSU-1 and Iowa) of C. par6um were collected from the feces of infected calves and were purified by several steps of sucrose and CsCl gradient centrifugation [6]. Before excystation (Iowa) or infection of monolayers (KSU-1), oocysts were further sterilized for 5 min in 10% Clorox® on ice, and washed 5–8× by centrifugation in sterile water. Free sporozoites were obtained by in vitro excystation at 37°C for 1 h in Hanks’ balanced salt solution (HBSS) containing 0.25% trypsin and 0.75% taurodeoxycholic acid [7]. After washing 3–5× with HBSS or PBS, oocyst shells and other debris were removed by a Percoll® gradient method [8].

2.2. Gene manipulation A 294 bp amplicon encoding a peptide highly homologous in sequence to animal Type I FAS was discovered as part of our search for novel drug targets in C. par6um. Because this appeared to be the first FAS gene observed from a protist, we decided to isolate and characterize this gene. Both a HindIII and EcoRI genomic DNA (gDNA) library of C. par6um KSU-1 strain were screened to obtain overlapping clones by genewalking. Large inserts from these libraries were subcloned into pBluescript II SK(+ ) vector. All clones were sequenced twice by the Wadsworth Molecular Genetics Core Facility. Sequences were analyzed using the GCG Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI) within the unix cluster at the Wadsworth Center. DNA and RNA were isolated both from free sporozoites (Iowa) and HCT-8 cells infected with C. par6um (KSU-1) using a Puregene™ DNA isolation (Gentra Systems) and Qiagen RNeasy kit (Qiagen), respectively. A 1 kb [32P]-dATP labelled DNA fragment released by EcoRI and HindIII digestion (Fig. 1) was used for Southern blot analysis. Expression of CpFAS1 in sporozoites and intracellular stages was determined by RT-PCR. A 50 ml reaction used 5 mM each AMV

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reverse transcriptase and Tfl DNA polymerase, 100 ng C. par6um total RNA, and one mM each of sense 5% GTC-GCA-GAC-TTT-CTA-TTT-TCC 3% and antisense 5% GCA-TGG-AAC-TTA-CACAAC-ATC 3% primer and other appropriate reagents specified by Access RT-PCR (Promega, Madison, WI). Two negative controls lacking either total RNA or reverse transcriptase were always included. The first strand cDNA synthesis was performed at 48°C for 45 min, followed by inactivation of reverse transcriptase at 94°C for 2 min and 33 PCR cycles. The RT-PCR product was analyzed using 2.0% agarose gels.

2.3. Immunological detection of CpFAS1 protein A 454 bp ACP gene fragment from the ACP domain of Module 3 (Fig. 1; FAS1E4) was ligated into the pGEX-4T-3 vector (Pharmacia Biotech) to produce a glutathione-S-transferase fusion protein (GST-FAS1E4), which was used to transform competent Escherichia coli BL21 cells. Colonies expressing GST-FAS1E4 and with the correct orientation were identified by sequencing the plasmid through the cloning site. GSTFAS1E4 was purified using a beaded-Sepharose4B column linked with glutathione, and was analyzed on a 10% SDS-PAGE gel under reducing conditions. Purified GST-FAS1E4 was concentrated to 1.0 mg ml − 1 using an Ultrafree®

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Centrifugal filter containing a BioMax-10K NMWL membrane (Millipore), and then used to immunize a 2 kg female rabbit. Serum was obtained prior to immunization and 2 weeks after the second and third boosting doses, respectively. Responses were tested by western blot analysis and the rabbit was bled after the fourth boost. Antiserum was affinity-purified over GST-linked Sepharose-4B to remove antibodies against GST. After excystation, purified C. par6um sporozoites were fixed in 10% formalin, washed in PBS, and affixed onto poly-L-lysine-coated glass slides for protein localization by immuno-fluorescence. Air-dried slides were extracted with ice cold methanol for 10 min, incubated with a 1:100 dilution of either polyclonal antibody or pre-immune serum in 5% BSA-PBS at RT for 2 h, rinsed 2× with BSA-PBS, and treated for 1 h at room temperature with goat anti-rabbit IgG-FITC conjugate (Bio-Rad). FITC-labeled sporozoites were rinsed 2× with BSA-PBS, sealed with glycerol/ PBS (9:1), and examined with an Olympus BH2 phase/fluorescence microscope.

2.4. In 6itro drug assays Cerulenin (Sigma Chemical) is a non-competitive inhibitor of Type I and II FAS [9]; thiolactomycin (kindly provided by Dr G. Besra [4]) only inhibits Type II FAS [10]. The ability of both to

Fig. 1. Diagram of the CpFAS1 gene. (A) Sequenced overlapping clones identified from C. par6um libraries. (B) Gene map of the CpFAS1 and adjacent regions. (C) Domain organization of deduced protein complex. Both nucleotides and amino acids are to scale. Repeating sequences and regions used for RT-PCR and Southern blot analysis are indicated in the gene map. Abbreviations: ACP, acyl carrier protein; KS, ketoacyl synthase; AT, acyl transferase; DH, dehydrase; ER, enoyl reductase; KR, ketoacyl reductase. Question mark indicates the terminating domain without apparent FAS enzyme homologues except the yeast a-aminoadipate reductase.

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inhibit intracellular growth of C. par6um in vitro was tested by an ELISA assay as described elsewhere [11–13]. Briefly, four replicate 96-well plates were seeded with 5×104 HCT-8 cells (ATCC cCCL244) 18 h prior to infection with 3×104 sterilized, fresh C. par6um oocysts and incubated for 1.5 h at 37°C. Both inhibitors were dissolved in dimethylsulfoxide (DMSO) and added to the culture medium; 200 mg ml − 1 paromomycin served as the positive anti-Cryptosporidium control. Host cell cytotoxicity was evaluated colorimetrically by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [14,15]. Infected cells were fixed in 8% formalin after 48 h incubation, blocked with 1% BSA/ 0.002% Tween-20 in PBS, and labelled with a 1:1000 dilution of rat antiserum against C. par6um membrane proteins. Goat anti-rat IgG conjugated to 1:800 horseradish peroxidase (Sigma) was added, and absorbance detected with a BioTek EL311S ELISA-plate reader (lem, 630 nm). Inhibition was calculated as percent difference between experimental and negative controls.

3. Results Using a 294 bp PCR product of the CpFAS1 gene, seven overlapping gene fragments, each representing several independent plasmid clones, were isolated from the two C. par6um gDNA libraries (Fig. 1). These clones span more than 30 kb of the parasite genome, and like other C. par6um genes are AT-rich in coding (66%) and non-coding (75%) regions, respectively. Four ORFs were identified within the 30-kb region, and the first 24 732-bp ORF appears to encode a multifunctional Type I FAS (CpFAS1) of 8243 amino acids (aa), with a predicted Mr =920.7 kDa. Since the size of the C. par6um genome is approximately 10.6 megabases [16], this gene represents  0.25% of it. The ORFs downstream of CpFAS1 have sequence similarity to proteosomes, or the large subunit of replication protein A [17], respectively. Twenty-one enzymatic domains were identified within CpFAS1 based upon a sequence comparison of its deduced amino acids with that of other

FAS and polyketide synthases (Fig. 1). These domains are organized into a starter unit, three FAS modules, and a region at the carboxy terminus, without sequence similarity to any known thioesterases (TE). The 880 aa starter unit contains a loading domain and an ACP. The former is similar in sequence to a number of ATP-dependent CoA ligases from prokaryotes and eukaryotes, as well as plant 4-coumarate- and o-succinylbenzoic acid-CoA ligases. Each of the three modules of CpFAS1 comprises a complete set of the enzyme domains AT, KS, KR, DH, ER and ACP for elongation, reduction and dehydration of C2 units, with the expected conserved, functional motifs (Fig. 2). Using numbers based upon human Type I FAS (P49327), the latter include: (1) Cys161 and His292/330 within all three KS domains [1,18,19]; (2) 578GXSXG, Gly446 and Arg604 in all three AT domains (Figs. 1 and 2) Ala679 and His781 within the AT of modules 2 and 3 [19]. Although DH protein sequences are less conserved between species, both His876 and Pro885 are present in each module (Fig. 2), whereas Gly880 is only present in the DH domain of module 2. With the exception of a few bases, all three modules share identical nucleotide sequences for the ER domain (Fig. 1), and these include the 1667 motif LXHXXXGGVG proposed for NADP(H)-binding [20]. The KR domains of modules 2 and 3 share identical nucleotides and contain an alternate NADP(H)-binding motif, 1885 GGXGXXG (Fig. 2). There are four ACP domains in CpFAS1, one in the starter unit and three that are modular. All of the ACP contain the 2150DSL motif, in which serine serves as the anchor for pantothenic acid and the attachment of growing fatty acid chains [1]. Since the C-terminal domain lacks the TE-specific GXSXG and GDH motifs [20], but contains amino acids with high similarity to a yeast a-aminoadipate reductase, C. par6um may use a different enzyme to release its final product(s), or like yeasts and fungi, use a non-hydrolytic mechanism [21]. Southern analysis of C. par6um gDNA (Iowa) digested with EcoRI and HindIII using a CpFAS1 probe (Fig. 1) shows single bands at 2.5 and 11.5 kb, respectively (data not shown). A 340 bp transcript of CpFAS1 corresponding to nucle-

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Fig. 2. Alignment of CpFAS1 motifs with those of human FAS. Amino acids discussed in the text are shaded. Active sites within the motifs of KS, AT, DH, and ACP are boxed. Residues important to the function of ER and KR are underlined.

otides 8278 through 8618 was detected by RTPCR using total RNA both from sporozoites and intracellular stages of C. par6um. In addition, the CpFAS1E1 fusion protein successfully generated high-titer antibodies in a rabbit after the third boost. Using rabbit polyclonal antibodies, both free sporozoites and intracellular stages of C. par6um were labelled by immunofluorescent, or immunochemical microscopy. This Type I FAS in C. par6um could be observed intracellularly, and primarily within the perinuclear region (data not shown). In three independent trials, growth of intracellular C. par6um was inhibited 96% by 10 mg ml − 1 cerulenin, a non-competitive inhibitor of Type I FAS (Fig. 3A). In contrast, and unlike T. gondii and P. falciparum, 100 mg ml − 1 thiolactomycin, a specific inhibitor of Type II FAS had no effect upon the intracellular growth of C. par6um in

vitro. Furthermore, the ‘gold standard’ paromomycin, and positive control in this assay, inhibited parasite growth only 60% even at the highest concentration tested (200 mg ml − 1; Fig. 3A). MTT assays for viability [14,15] show that thiolactomycin is non-toxic to HCT-8 cells (Fig. 3B), whereas above 20 mg ml − 1 cerulenin was toxic to them (Fig. 3B). This was confirmed microscopically by cell detachment.

4. Discussion As mentioned before, the fatty acid biosynthetic enzymes are either dissociated individual proteins (Type II) in prokaryotes, plants and some apicomplexans, or fused into enzymatic domains within one or two polypeptides (Type I) in animals and fungi. In eukaryotic cells, Type II en-

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zymes are compartmentalized either into the chloroplast (plants) or apicoplast (apicomplexans). Based upon an assumption that the apicoplast is widely distributed among members of Apicomplexa [22], one might reasonably expect that other apicomplexans would possess Type II FAS. Thus the discovery of cytosolic Type I CpFAS1 in C. par6um was unanticipated. That C. par6um has and may utilize this Type I FAS for synthesis of fatty acids, rather than Type II as do T. gondii and P. falciparum, also agrees with its insensitivity to thiolactomycin (Fig. 3) [4]. These observations are further supported by our recent finding that C. par6um appears to lack a plastid genome [23]. Thus, the apicoplast may be unavailable as a host organelle for Type II FAS proteins [23]. The differentiation between FAS proteins of C. par6um and other apicomplexans is also con-

gruent with other biological differences between C. par6um and its relatives, including the alternative polyamine biosynthetic pathway [24], the insensitivity to most anticoccidial drugs [25], and its apparent early emergence at the base of Apicomplexa based upon phylogenetic reconstruction using SSU [26], fused SSU/LSU rRNA and six proteins (Zhu, G., Keithly, J.S. and Philippe H., unpublished observation). Although several major metabolic pathways have been investigated in parasitic protists, little is known about the acquisition of fatty acids and/or their biosynthesis. Therefore, the discoveries of both a cytosolic Type I CpFAS1 in C. par6um, and organellar Type II enzymes in T. gondii and P. falciparum, suggest that the biosynthesis of fatty acids might be a characteristic of the Apicomplexa. Because fatty acids are the major com-

Fig. 3. Efficacy of FAS inhibitors (A) on the development of C. par6um in vitro, and (B) cytotoxicity to host cells using the MTT assay. Three independent trials are individually plotted, and standard deviations are calculated from four or more replicates.

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ponent of all biomembranes, and are essential for growth and development, these may play an important role for the intracellular survival of apicomplexans within the parasitophorous vacuole (PV), the membranes of which are composed of lipids both from host cells and invading parasites [27]. Therefore, it is likely that an apicomplexan FAS, whether Type I or II, would contribute to the biosynthesis and formation of a PV membrane. Fatty acid synthetic enzymes have also been considered promising drug targets in fungi [28,29], other apicomplexans[4], and a variety of tumors [30,31]. Recent studies have revealed that the bacterial FAS, specifically enoyl reductase ( fabI) is the molecular target both for diazaborines, including the front-line antituberculosis drug isoniazid [32,33], and for triclosan and other 2hydroxydiphenyl ethers [34 – 36]. In this study, we tested whether fatty acid biosynthesis was essential to C. par6um by examining the effect of FAS-specific drugs against its intracellular development in vitro. These data show that the FAS inhibitor cerulenin can inhibit the growth of C. par6um at concentrations non-toxic to host cells (Fig. 3). Therefore, the discovery of CpFAS1 not only provides additional molecular evidence for the divergence of C. par6um from other apicomplexans, but also indicates a potential for the development of drugs against this opportunistic pathogen for which no treatment is yet known.

Acknowledgements We thank Wadsworth Center Molecular Genetics Core Facility for the synthesis of oligonucleotides and automatic sequencing; the Division of Animal Health for assisting in the preparation of rabbit antiserum. We also thank Dr. G. Besra at the Medical School, University of Newcastle upon Tyne for providing thiolactomycin. This work was supported in part by funds from the National Institutes of Health, NCDDG, AI40320.

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