The genome of the simian and human malaria parasite Plasmodium knowlesi.

Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

Antibodies raised against Bcvir15, an extrachromosomal double-stranded RNA-encoded protein from Babesia canis, inhibit the in vitro growth of the parasite.

As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis, we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M(1) to I(141)), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S-transferase-Bcvir15 (at a concentration of 160 micro g/ml) induced 50% inhibition of the in vitro growth of B. canis, and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein.

Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes.

The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi.

In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA was cloned and sequenced. Protein sequence database searches suggested that the 41-kDa protein belongs to the phosphofructokinase B type family (PFK-B). However, because of the low level sequence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding site. However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds rather than sugar ones. Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nM, whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of the PFK-B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.

Exchange of domain I from Bacillus thuringiensis Cry1 Toxins Influences protoxin stability and crystal formation.

Influence of domain I exchange on the stability and production of Bacillus thuringiensis Cry1 protoxins as well as on the shape of inclusion and toxicity to Spodoptera exigua and Plutella xylostella larvae was investigated. Chimeric genes were prepared by exchanging the regions coding for domain I between Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The AcCC chimera accumulated into bipyramidal inclusion bodies, whereas CEE produced round-shaped inclusion bodies, and ECC and AaEE protoxins produced small granules. AbEE and EAaAa did not produce any inclusion body and were visualized by immunodetection only. AcCC, CEE, ECC, and AaEE were stable to trypsin, whereas AbEE and EAaAa were not. Bioassays showed that the chimeras were not toxic in vivo. However, S. exigua larvae fed with the activated AcCC toxin displayed a lower growth rate.