Charles A. Yowell

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula.

Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.

Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum

A clone encoding the aspartic proteinase (PFAPD) from Plasmodium falciparum strain HB3 was obtained during the course of a project designed to sequence and identify the protein coding regions of the parasites genome. The protein encoded by the clone contains a sequence identical to the N-terminal sequence determined for an aspartic proteinase isolated from the digestive vacuole of P. falciparum and demonstrated to participate in the hemoglobin digestive pathway (D. Goldberg, personal communication). The translated polypeptide sequence encompasses a number of features characteristic of aspartic proteinases, having > 30% identity and > 50% similarity overall to human cathepsin D, cathepsin E and renin. A model of the three-dimensional structure of PFAPD was constructed using rule-based procedures. This confirms that the primary sequence may be folded as a single chain into a three dimensional structure closely resembling those of other known aspartic proteinases. It includes a lengthy prosegment, two typical-hydrophobic-hydrophobic-Asp-Thr/Ser-Gly motifs and a tyrosine residue positioned in a beta-hairpin loop. The distribution of hydrophobic residues throughout the active site cleft is indicative of a likely preference for hydrophobic polypeptide substrates. The recombinant form of this enzyme expressed using the pGEX2T vector in Escherichia coli is active in digesting hemoglobin at acidic pH and in hydrolyzing a synthetic peptide corresponding to the putative initial cleavage site in hemoglobin. Activity is inhibited completely by pepstatin, confirming the identity of PFAPD as a member of the aspartic proteinase family. Specific mRNA for PFAPD is expressed in the erythrocytic stages of the life cycle.

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function

Knockout mutants of Plasmodium falciparum lacking pfpm1, pfpm2 and pfhap (triple‐PM KO), and mutants lacking all four digestive vacuole (DV) plasmepsins (pfpm4, pfpm1, pfpm2 and pfhap; quadruple‐PM KO), were prepared by double cross‐over integration effecting chromosomal deletions of up to 14.6 kb. The triple‐PM KO was similar to the parental line (3D7) in growth rate, morphology and sensitivity to proteinase inhibitors. The quadruple‐PM KO showed a significantly slower rate of growth in standard medium, which manifested as delayed schizont maturation accompanied by reduced formation of haemozoin. In amino acid‐limited medium, the reduction in growth rate of the quadruple‐PM KO was pronounced. The sensitivity of both the triple‐ and quadruple‐PM KOs to six different HIV aspartic proteinase inhibitors was comparable to that of 3D7, thus establishing that the DV plasmepsins were not the primary targets of the antimalarial activity of these clinically important compounds. Electron microscopic analysis revealed the presence of multilamellar bodies resembling ceroid in the DV of the quadruple‐PM KO, and intermediates of the autophagic pathway accumulated as determined by Western blot analysis. Thus, the DV plasmepsins, although not essential, contribute significantly to the fitness of the parasite and are required for efficient degradation of endosomal vesicles delivered to the DV.

Sarcocystis falcatula from Passerine and Psittacine Birds: Synonymy with Sarcocystis neurona, Agent of Equine Protozoal Myeloencephalitis

Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis neurona. The horse is a dead-end host for S. neurona and the definitive and intermediate hosts have not previously been identified. We hypothesized that S. neurona is actually Sarcocystis falcatula, a parasite that cycles in nature between Virginia opossums (Didelphis virginiana) and any of a variety of avian intermediate hosts. We extracted DNA from S. falcatula sarcocysts in the muscle of a brown-headed cowbird (Molothrus ater) and from schizonts in a fixed specimen of lung from a Moluccan cockatoo (Cacatua moluccensis). Three segments of the small subunit ribosomal RNA (SSURNA) gene, containing a total of 742 nucleotides, were amplified by the polymerase chain reaction, sequenced, and compared with the SSURNA sequence from two isolates of S. neurona. The S. falcatula sequence was identical to the sequence of the S. neurona isolate UCD-1 and differed in only 3 positions from isolate SN5. Recent evidence, also based on SSURNA sequences, implicates the opossum as the definitive host of S. neurona. Based on the SSURNA gene sequences S. falcatula and S. neurona are synonymous, thus the parasite cycles between opossums and birds maintaining a reservoir of the organism from which horses can be infected.

Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.

Plasmepsins are aspartic proteinases of the malaria parasite, and seven groups of plasmepsins have been identified by comparing genomic sequence data available for the genes encoding these enzymes from Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium berghei, and Plasmodium yoelii. The food vacuole plasmepsins typified by plasmepsin 4 from P. falciparum (PfPM4) constitute one of these groups. Genes encoding the ortholog of PfPM4 have been cloned from Plasmodium ovale, Plasmodium malariae, and P. vivax. In addition, P. falciparum contains three paralagous food vacuole plasmepsins or plasmepsin-like enzymes that appear to have arisen by gene duplication, plasmepsins 1 (PfPM1), 2 (PfPM2) and HAP, and all four were localized to purified food vacuole preparations by two-dimensional gel electrophoresis and mass spectroscopic analysis. The three paralogs of PfPM4 do not have counterparts in the six other Plasmodium spp. examined by genomic DNA blot analysis and by review of available genomic sequence data. The presence of these paralogs among the food vacuole plasmepsins in P. falciparum as compared with the other three species causing malaria in man will impact efforts to rationally design antimalarials targeting the food vacuole plasmepsins.

Haemonchus placei and Haemonchus contortus are distinct species based on mtDNA evidence

Debates continue over the extent to which the parasitic trichostrongylids Haemonchus placei and Haemonchus contortus hybridise in nature, and whether they deserve species status. Mitochondrial ND4 gene sequences from individuals of each putative species collected from populations around the United States indicate that the two species are highly differentiated at the mtDNA level. Furthermore, there was no evidence of introgressive hybridisation occurring in wild populations.

Phylogenetic Relationship of Cowdria ruminantium, Agent of Heartwater, to Anaplasma marginale and Other Members of the Order Rickettsiales Determined on the Basis of 16s rRNA Sequence?

The phylogenetic relationship between Cowdria ruminantium and representative members of the orders Rickettsiales and Chlamydiales has been examined on the basis of the sequence of the 16S rRNA. Phylogeny reconstruction by using both parsimony and distance methods supports the conclusion that C. ruminantium is closely related to the Rickettsiales and in particular to the family Anaplasmataceae. A signature of nine base substitutions delineated the linkage of Anaplasma marginale with C. ruminantium and differentiated these two species from the 45 other members of the alpha group of Proteobacteria examined, and five of these base substitutions were unique among all members of the class Proteobacteria examined to date.

Structural insights into the activation of P. vivax plasmepsin.

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.

Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus

The ribosomal RNA gene unit of the protozoan parasite Tritrichomonas foetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb.