Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro.

D-eritadenine and (S)-DHPA are aliphatic adenosine analogues known to target S-adenosylhomocysteine hydrolase (SAHH) and potent antiviral compounds. In the present study, we demonstrate that these two compounds also display efficacy against recombinant SAHH enzyme of the protozoan parasite Cryptosporidium parvum, as well as inhibition of parasite growth in vitro. Our data confirm that SAHH could serve as a rational drug target in cryptosporidial infection and antiviral adenosine analogues are potential candidates for drug development against cryptosporidiosis.

Characterization of S-adenosylhomocysteine hydrolase from Cryptosporidium parvum.

The S-adenosylhomocysteine hydrolase from the apicomplexan Cryptosporidium parvum (CpSAHH) has been characterized. CpSAHH is a single-copy, intronless gene of 1479 bp encoding a protein of 493 amino acids with a molecular mass of 55.6 kDa. Reverse transcriptase-polymerase chain reaction analysis confirmed that CpSAHH is expressed both in intracellular stages (in C. parvum-infected HCT-8 cells 24 h after infection) and in sporozoites. CpSAHH was expressed in Escherichia coli TB1 cells as a fusion with maltose-binding protein. The recombinant fusion was cleaved by Factor Xa and the enzymatic activity of both the fusion protein and the purified separated CpSAHH was measured. The enzymatic activity of CpSAHH was inhibited by d-eritadenine, S-DHPA and Ara-A.

Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum.

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.

Direct interaction of Saccharomyces cerevisiae Faa1p with the Omi/HtrA protease orthologue Ynm3p alters lipid homeostasis.

In yeast, long chain acyl-CoA synthetase (ACSL) activity is required for fatty acid uptake, metabolism and fatty acid-dependent transcriptional control. The major ACSL contributing these functions is Faa1p. In a yeast two-hybrid screen, the Omi/HtrA serine protease family orthologue Ynm3p (YNL123w) was identified as a specific interactor with Faa1p. Interaction of Ynm3p and Faa1p was confirmed by co-immunoprecipitation. Disruption of the YNM3 gene encoding Ynm3p resulted in increased fatty acid uptake, triglyceride accumulation and reduced expression of the fatty acid-responsive OLE1 gene encoding the essential Delta(9)-acyl-CoA desaturase. These changes were linked with increased Faa1p and Faa4p ACSL activities. We propose that Ynm3p modulates fatty acid metabolism and gene regulation through negative regulation of ACSL activity. Additional strain-specific phenotypes associated with deletion of YNM3 included inability to grow on non-fermentable carbon sources and altered cellular morphology.

A Narf-like gene from Cryptosporidium parvum resembles homologues observed in aerobic protists and higher eukaryotes.

Here we report a Narf-like gene from the apicomplexan Cryptosporidium parvum (CpNARF). CpNARF is an intronless, single-copy gene of 1680 bp which encodes a putative protein of 560 amino acids with a calculated molecular mass of 63.1 kDa. This gene contains a single highly conserved N-terminal iron-sulfur cluster ([4Fe-4S]) binding site, as well as most of the H-cluster conserved residues. Reverse transcription polymerase chain reaction analysis indicates that CpNARF is expressed by the intracellular stages of C. parvum. Although the function of this gene is as yet unknown, phylogenetic analyses suggest that CpNARF belongs to the group of NARF-like proteins from aerobic protists and higher eukaryotes, which are thought to have had an ancestor in common with [Fe]-hydrogenases.

Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p.

The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.