Molecular characterization of a coccidian parasite cGMP dependent protein kinase

https://doi.org/10.1016/S0166-6851(01)00451-0Get rights and content

Abstract

The cGMP-dependent protein kinase (PKG) of Eimeria tenella and Toxoplasma gondii is the target of a novel coccidiostat that is effective against coccidiosis and toxoplasmosis in animal models. Preparations of native PKG enzyme from Toxoplasma and Eimeria contain a membrane-associated polypeptide (isoform-I) of about 110 kDa and a slightly smaller soluble polypeptide (isoform-II). Expression of T. gondii and E. tenella PKG cDNA clones in Toxoplasma yield similarly sized recombinant polypeptides, which co-migrate on SDS-polyacrylamide gels with the corresponding native isoforms. Results of targeted mutagenesis of potential translational initiation sites suggest that parasite isoform-II is a product of alternative translational initiation from an internal initiator methionine codon. Exclusive expression of isoform-II or isoform-I can be achieved by preventing initiation at the respective primary or secondary sites. Immunofluorescence analysis indicates that recombinant isoform-I localizes primarily to the parasite plasma membrane, while isoform-II remains cytosolic. Mutagenesis and metabolic labeling studies reveal that the observed membrane-association of full-length recombinant PKG is mediated by N-terminal myristoylation and palmitoylation at amino acids G2 and C4. We also confirm the functional significance of a putative third PKG allosteric site, common to apicomplexan PKGs but absent from vertebrate or insect PKGs. In assays with transiently transfected parasites, constructs harboring a mutation at this site express markedly lower levels of cGMP-dependent PKG activity, while a triple mutant bearing mutations in all three sites reduces kinase activity to background levels.

Introduction

In a related manuscript, we report the identification of a parasite cGMP-dependent kinase (PKG) from Eimeria tenella as the molecular target of a novel broad-spectrum coccidiostat [1]. The compound (Compound 1), is a trisubstituted pyrrole, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine and is effective both against coccidiosis in poultry and toxoplasmosis in a murine model [1], [2]. Eimeria and Toxoplasma are related coccidian protozoa, a subgroup of the phylum Apicomplexa that includes intestinal parasites of veterinary and clinical significance.

Mammalian cGMP-dependent protein kinases (cGKs) are signal transduction enzymes, encoded by two distinct but related genes, that play different physiological roles in the body [3], [4]. Genetic knock-out studies in mice have implicated mammalian cGK-I as playing a role in smooth muscle physiology and cGK-II in bone growth and fluid homeostasis in the gut [5], [6]. There is no strict physiological counterpart to these processes in protozoan parasites. Although Eimeria and Toxoplasma PKGs are encoded by single-copy genes, two serologically-related parasite PKG isoforms have been detected ([1], Donald et al., submitted). The larger PKG isoform of Eimeria is preferentially associated with parasite membrane fractions and can be extracted with detergents, while the smaller polypeptide is soluble. Native Toxoplasma PKGs fractionate similarly, although the smaller isoform has a faster mobility than the analogous Eimeria polypeptide. A comparison of conceptually translated sequences from cloned PKGs of Toxoplasma gondii (Tg-PKG) and E. tenella (Et-PKG), shows they share 68.5% identity. The parasite enzymes are also functionally related, since the recombinant Eimeria enzyme expressed in Toxoplasma can complement a knock-out in the Toxoplasma PKG gene (Donald et al., submitted).

The identification of an ATP competitive active-site inhibitor selective for the parasite enzyme indicates that therapeutically exploitable structural differences exist between parasite and host enzymes [1]. The apicomplexan parasite enzymes are also 30% larger than their mammalian and insect counterparts. The size increase is due to an expanded N-terminal regulatory domain, which encompasses a putative third cGMP-binding site. This third allosteric site is unique to, and conserved among coccidian parasite PKGs and may be responsible for the enhanced cooperativity and cGMP-dependence of the parasite enzyme compared with the enzymes of the vertebrate host.

Since Eimeria is not amenable to continuous in vitro cultivation, we have performed functional studies of Eimeria and Toxoplasma PKGs in Toxoplasma, where a wide range of molecular tools are available [7], [8], [9]. We show that the two parasite PKG isoforms most likely arise through alternative translation initiation from within the same gene and provide direct evidence that the larger isoform is dually acylated. Finally, a mutational analysis of predicted allosteric sites confirms the functional significance of the putative parasite-specific cGMP binding site.

Section snippets

Host cells and parasite cultures

Cell culture media and reagents were obtained from Life Technologies, Inc. RH strain T. gondii tachyzoites and RHΔHXGPRT knock-out mutants (NIH AIDS Research and Reference Reagent Program, Bethesda, MD; http:www.niaid.nih.gov/regent) were maintained by serial passage in primary human foreskin fibroblast cultures (HFF). Host cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated newborn bovine serum. This culture medium was replaced with modified Eagle's medium

Recombinant expression of Toxoplasma and Eimeria PKGs in toxoplasma

Preparations of native PKG enzyme from Toxoplasma and Eimeria contain two serologically related isoforms of similar size ([1], Donald et al., submitted). Recombinant T. gondii and E. tenella PKG cDNA clones were expressed in Toxoplasma following the addition of FLAG-epitope tags to either the amino terminal (FLAGPKG) or carboxy terminal (PKGFLAG) ends of the clones to facilitate subsequent purification and detection. Parasites stably transformed with FLAGTg-PKG and analyzed by Western blot,

Discussion

Unlike mammalian organisms where two genes code for multiple isoforms of enzyme, PKG in apicomplexan parasites is derived from a single copy gene ([1]; Donald et al., submitted). Using a molecular genetic approach, we have revealed the likely origin of two isoforms of parasite PKG identified in biochemical preparations of native enzyme from T. gondii and E. tenella and confirmed their identity. Enzymatically active recombinant forms of the two polypeptides that co-migrate with native isoforms

Acknowledgements

We thank Tesfaye Biftu for providing Compound 1 for our studies. We also wish to thank the University of Pennsylvania (David Roos) and Stanford University (John Boothroyd) for licenses to use Toxoplasma reagents.

References (26)

Cited by (0)

Note: Nucleotide sequence data reported in this paper are available in the GenBank™ database under accession numbers AF411961 and AF413570.

View full text