Karyotype analysis of Leishmania species and its use in classification and clinical diagnosis.

Chromosomes of four species of Leishmania represented by ten different geographic isolates were analyzed by pulsed field gradient gel electrophoresis (PFG) to assess chromosome stability in these parasitic protozoans. Among different geographic isolates of the same subspecies, more than two-thirds of chromosomes had similar sizes, ethidium bromide staining intensities, and locations of alpha,beta-tubulin genes. However, among New World Leishmania, members of different species or subspecies have fewer than one-third of their chromosomes in common. Therefore, PFG karyotypes of Leishmania exhibit intraspecific variability similar to that reported for other parasitic protozoans. The greater similarities of the karyotypes of members of the same Leishmania subspecies may indicate that they represent valid taxa. These similarities also allowed the use of PFG in clinical diagnosis for rapid and accurate typing of patient isolates.

Characterization of a heavy metal ATPase from the apicomplexan Cryptosporidium parvum.

P1-ATPases are transporters which pump heavy metals across membranes, either to provide enzymes with essential cofactors or to remove excess, toxic metal cations from the cytosol. The first protist P1-ATPase (CpATPase2) has been isolated from the apicomplexan Cryptosporidium parvum, an opportunistic pathogen of AIDS patients. This single copy gene encodes 1260 amino acids (aa), predicting a protein of 144.7 kDa. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis confirmed CpATPase2 expression. Immunofluorescence microscopy of C. parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase (GST) fusion protein suggests that CpATPase2 is associated with the plasma- and cytoplasmic membranes. The protein shares greatest overall sequence similarity to previously characterized copper P1-ATPases. Expression and subsequent biochemical analyses of the N-terminal heavy metal binding domain (HMBD, GMxCxxC) of CpATPase2 as a maltose-binding protein (MBP) in Escherichia coli reveals that the protein specifically binds reduced copper, Cu(I), in vitro and in vivo, and that the cysteine residues of HMBD are responsible for heavy metal coordination. Overall, these data show that the apicomplexan C. parvum possesses a heavy metal P-ATPase transporter with a specificity for reduced copper. Since this discovery represents the first time a heavy metal P-ATPase has been identified and characterized from a protist, further molecular and biochemical studies are needed to understand the roles heavy metal P-ATPases play in heavy metal metabolism and potential virulence for this and other apicomplexa.

Molecular analysis of a P-type ATPase from Cryptosporidium parvum.

Eukaryotic P-type ATPases use energy to drive the transport of cations across membranes. A complete P-ATPase gene (CpATPase1) has been isolated from Cryptosporidium parvum, one of the opportunistic pathogens in AIDS patients. The complete gene encodes 1528 amino acids, predicting a protein of 169 kDa. A hydropathy profile of the protein suggested there are eight transmembrane domains (TM). Expression of the gene was confirmed both by Northern blot analysis and RT-PCR. A fragment of the gene has been expressed as a 49 kDa GST-fusion protein. This protein was used to produce rabbit antiserum and fluorescent labeling has localized the protein to the sporozoite apical and perinuclear regions. SDS-PAGE and Western blot analysis show a 160 kDa major protein, close to the predicted size. The protein shares greatest overall identity and similarity to a putative organellar Ca2+ P-ATPase described for Plasmodium falciparum. Unlike P. falciparum, but consistent with all genes so far isolated from C. parvum, the gene contains no introns. The Ca2+ P-ATPases from these two Apicomplexa are large and do not have motifs predicting calmodulin-binding.

Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy.

This study demonstrates that polyamine biosynthesis in Cryptosporidium parvum occurs via a pathway chiefly found in plants and some bacteria. The lead enzyme of this pathway, arginine decarboxylase (ADC) was sensitive to the specific, irreversible inhibitor DL-alpha-difluoromethyl-arginine (IC50 30 microM), and intracellular growth of C. parvum was significantly reduced by inhibitors of ADC. No activity was detected using ornithine as substrate, and the irreversible inhibitor of ornithine decarboxylase, DL-alpha-difluoromethyl-ornithine, had no effect upon ADC activity or upon growth of the parasite. Back-conversion of spermine to spermidine and putrescine via spermidine:spermine-N1-acetyltransferase (SSAT) was also detected. Compounds such as his(ethyl)norspermine, which have been demonstrated to down-regulate SSAT activity in tumor cells, were synergistic in the inhibition of growth when used in combination with inhibitors of the forward pathway. Thus, C. parvum differs fundamentally in its polyamine metabolism from the majority of eukaryotes, including humans. Such differences indicate that polyamine metabolism may serve as a chemotherapeutic target in this organism.

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

We report here the molecular analysis of a Type I fatty acid synthase in the apicomplexan Cryptosporidium parvum (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. parvum, 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. parvum in vitro.

Synthesis, structure, and antiparasitic activity of sulfamoyl derivatives of ribavirin.

The triazole nucleoside derivatives 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-carboxamide (2), 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl) [1,2,4]triazole-3-thiocarboxamide (3), and 1-(5'-O-sulfamoyl-beta-D-ribofuranosyl)-[1,2,4]triazole-3- carbonitrile (4) were synthesized. Suitably protected triazole nucleosides were converted to their corresponding 5'-sulfamoyl derivatives, which on subsequent deprotection gave the desired compounds in good yields. The structures of compounds 2-4 were confirmed by X-ray crystallographic analysis. All three compounds showed significant antiparasitic activity in vitro, while 2 showed significant activity in vivo against Leishmania donovani and Trypanosoma brucei.

Inefficacy of metronidazole in experimental infections of Leishmania donovani, L. mexicana, and Trypanosoma brucei brucei.

Metronidazole has been claimed in several earlier reports to be active in human cases of leishmaniasis and trypanosomiasis. Its efficacy against the protozoa causing these diseases was tested in hamsters infected with Leishmania mexicana or L. donovani, and in mice infected with Trypanosoma brucei brucei. In separate experiments, hamsters were either inoculated intradermally into the nose with 5 million amastigotes of L. mexicana or intracardially with 10-30 million amastigotes of L. donovani, and mice were infected intraperitoneally with 30 million T. b. brucei. Metronidazole was administered in four oral doses on alternate days for a total of 375 mg/kg to hamsters and 500 mg/kg to mice. Sodium stibogluconate (Pentostam) served as a positive control. In hamsters the extent of infection was assessed by the appearance of flagellates in blood agar cultures of nose and spleen, by counting amastigotes in nose and liver impression smears, and by measuring the size of nose lesions. Ultrastructure of nose lesions before and after treatment with metronidazole or Pentostam was also evaluated. Infection in mice was assessed by the extent of parasitemia and/or survival to 30 days. In no case did metronidazole-treated animals differ from untreated controls. Metronidazole shows no activity against experimental infections of leishmaniasis or trypanosomiasis in these animal models.

Cryptosporidium parvum possesses a short-type replication protein A large subunit that differs from its host.

Replication protein A (RPA) consisting of three subunits is a eukaryotic single-stranded DNA (ssDNA)-binding protein involved in DNA replication, repair and recombination. We report here the identification and characterization of a RPA large subunit (CpRPA1) gene from the apicomplexan Cryptosporidium parvum. The CpRPA1 gene encodes a 53.9-kDa peptide that is remarkably smaller than that from other eukaryotes (i.e. approximately 70 kDa) and is actively expressed in both free sporozoites and parasite intracellular stages. This short-type RPA large subunit has also been characterized from one other protist, Crithidia fasciculata. Three distinct domains have been identified in the RPA large subunit of humans and yeasts: an N-terminal protein interaction domain, a central ssDNA-binding area, and a C-terminal subunit-interacting region. Sequence analysis reveals that the short-type RPA large subunit differs from that of other eukaryotes in that only the domains required for ssDNA binding and heterotrimer formation are present. It lacks the N-terminal domain necessary for the binding of proteins mainly involved in DNA repair and recombination. This major structural difference suggests that the mechanism for DNA repair and recombination in some protists differs from that of other eukaryotes. Since replication proteins play an essential role in the cell cycle, the fact that RPA proteins of C. parvum differ from those of its host suggests that RPA be explored as a potential chemotherapeutic target for controlling cryptosporidiosis and/or diseases caused by other apicomplexans.

Minimizing DNA recombination during long RT-PCR.

Recent developments have made it possible to reverse transcribe RNA and amplify cDNA molecules of > 10 kb in length, including the HIV-1 genome. To use long reverse transcription combined with polymerase chain reaction (RT-PCR) to best advantage, it is necessary to determine the frequency of recombination during the combined procedure and then take steps to reduce it. We investigated the requirements for minimizing DNA recombination during long RT-PCR of HIV-1 by experimenting with three different aspects of the procedure: conditions for RT, conditions for PCR, and the molar ratios of different templates. We used two distinct HIV-1 strains as templates and strain-specific probes to detect recombination. The data showed that strategies aimed at completing DNA strand synthesis and the addition of proofreading function to the PCR were most effective in reducing recombination during the combined procedure. This study demonstrated that by adjusting reaction conditions, the recombination frequency during RT-PCR can be controlled and greatly reduced.

Ornithine decarboxylase and trypanothione reductase genes in Leishmania braziliensis guyanensis.

Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC 1. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania.

Infectivity of Leishmania donovani amastigotes and promastigotes for golden hamsters.

Intracardial injection of hamsters with from 5 to 114 million amastigotes or promastigotes of Leishmania donovani and screening of the 8th-day liver impression smears, provides a rapid and reproducible method for assaying infectivity. Amastigotes are at least 10X more infective than promastigotes, and log-phase promastigotes act as a single infective population for hamsters.

What is the phylogenetic position of Cryptosporidium?

The phylogenetic position of Cryptosporidium is elusive. Although previous studies based solely upon small-subunit (SSU) rRNA sequences suggested that the genus was an early emerging lineage among the Apicomplexa, bootstrap support for this placement was low. Here, the phylogenetic position of Cryptosporidium has been re-evaluated for SSU rRNA, fused SSU/large-subunit (LSU) rRNA and six protein sequences using traditional distance-based neighbour-joining, maximum-parsimony and maximum-likelihood methods of phylogenetic reconstruction as well as the new Slow-Fast analysis, which focuses upon the slowly evolving positions within sequences and is especially useful if a long-branch attraction (LBA) artefact is suspected. All the methods of reconstruction indicated a trend for the early emergence of Cryptosporidium at the base of the Apicomplexa and showed that an LBA artefact plays no role in this placement. Although the inclusion of additional numbers of neither species nor genes has significantly enhanced the bootstrap support for this phylogenetic position, recent biochemical, molecular and ultrastructural data are congruent with it. Therefore, we favour a working hypothesis that this genus constitutes an early emerging branch of the Apicomplexa.

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.

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

Cryptosporidium parvum: functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast.

We report here the identification of a novel multiprotein bridging factor type 1 from the apicomplexan Cryptosporidium parvum (CpMBF1), one of the opportunistic pathogens in AIDS patients. In slime molds, insects, and humans, MBF1-regulated systems have been associated with cell differentiation, which indicates that CpMBF1 could be responsible for the activation of similar systems in C. parvum during its complex life cycle. Because of the difficulties and high cost in obtaining sufficient and purified C. parvum material for molecular and biochemical analyses, well-characterized yeast genetic systems may be useful for investigating the functions of C. parvum genes. In this study, the function of CpMBF1 as an interconnecting element between a DNA-binding regulator and TATA-box-binding protein (TBP) was confirmed using a yeast complementation assay. Under conditions of histidine starvation, an MBF1-deficient strain of Saccharomyces cerevisiae was unable to activate the HIS3 gene, which encodes imidazoleglycerol-phosphate dehydratase (IGPDH), and thus became sensitive to 3-amino triazole, an inhibitor of this enzyme. Upon introduction of parasite CpMBF1 into S. cerevisiae, 3-amino triazole resistance of the MBF1-deficient strain was restored to wild-type levels, and Northern blot analysis revealed that CpMBF1 was able to activate HIS3 transcription in response to histidine starvation.

Direct isolation of DNA from patient stools for polymerase chain reaction detection of Cryptosporidium parvum.

Although polymerase chain reaction (PCR) can sensitively detect parasitic or other infections, its use with fecal samples is extremely limited, primarily because of the presence of substances that inhibit DNA extension. Here an improved protocol is reported for directly isolating DNA from aged or fresh formalin-fixed stools, which can then be used to detect Cryptosporidium parvum by nested PCR. This method is highly reproducible, sensitive, and specific. It detects <1 pg of C. parvum DNA in human stool, and there are no cross-reactions with other parasites commonly found there.

Evaluation of in vitro activity of aurones and related compounds against Cryptosporidium parvum.

The efficacy of a series of aurones, auronols and 4-methoxy-alpha-pyrones has been screened for the ability to inhibit the intracellular growth of the parasitic protist Cryptosporidium parvum using an in vitro enzyme linked immunosorbent assay (ELISA). All aurones of this series were active at 25 to 100 microM. 10 of 19 aurones inhibited the intracellular growth of C. parvum by > 90 % with moderate to no toxicity. The most active of these was 3',4',6-trihydroxy-2-[phenylmethylene]-3(2H)-benzofuranone.

Response of babesiosis to pentamidine therapy.

Three nonsplenectomized patients were infected with Babesia microti. One had fever, abdominal pain suggesting gallbladder disease, and evidence of disseminated intravascular coagulation; another was considered to have lymphoma, partly because two smears for Babesia before admission were negative. All three patients were treated with pentamidine isethionate and improved clinically. Parasites were no longer seen on smears after 5 days of therapy, but Babesia could still be recovered by hamster inoculation 5 weeks after therapy in one of the patients tested, underscoring the need for this test to properly evaluate eradication of the organism. In one patient, pentamidine was stopped after 7 days because of increased creatinine concentration, and this amount of drug appeared adequate to control the parasitemia. Pain at drug injection sites was a major side effect in all three patients. Pentamidine appears to be useful in controlling clinical manifestations of babesiosis and decreasing parasitemia, but it does not eradicate the organism.

Haemonchus contortus: cloning and functional expression of a cDNA encoding ornithine decarboxylase and development of a screen for inhibitors.

Polyamines (PA) are essential for viability and replication of all cells; organisms either synthesize PA or acquire them from the environment. How nematodes that parasitize the gut satisfy their PA requirement has not been resolved. The primary regulatory enzyme in PA biosynthesis in most animals is ornithine decarboxylase (ODC). This enzyme has recently been characterized in free-living nematodes and in the parasitic species. Haemonchus contortus. Nematode and mammalian ODC are reported to differ in subcellular localization, kinetics, and sensitivity to inhibitors. We cloned an H. contortus cDNA that encodes a full-length ODC (sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF016538 and AF016891). This cDNA was functionally expressed in strains of Escherichia coli and Saccharomyces cerevisiae that lack ODC and are dependent upon exogenous PA for survival. Expression of nematode ODC reversed the PA-dependence phenotype of both microorganisms. The complemented yeast strain was used to develop a nutrient-dependent viability screen for selective inhibitors of nematode ODC. The antiprotozoal drug stilbamidine isethionate was identified as active in this screen, but biochemical characterization revealed that this compound did not inhibit ODC. Instead, like other cationic diamidines, stilbamidine probably inhibits yeast S-adenosylmethionine decarboxylase. Nonetheless, the activity in the screen of the known ODC inhibitor difluoromethylornithine (DFMO) validates the concept that specific recombinant microorganisms can serve as the basis for extremely selective and facile screens.