Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana.
Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence.
Bart
G
G
Wellcome Unit of Molecular Parasitology, University of Glasgow, The Anderson College, UK.
Frame
M J
MJ
Carter
R
R
Coombs
G H
GH
Mottram
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D017353:Gene Deletion; D015870:Gene Expression; D017125:Genes, Protozoan; D066298:In Vitro Techniques; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D016773:Leishmaniasis, Cutaneous / Q000209:etiology; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D014162:Transfection; D014774:Virulence / Q000235:genetics
D017931:DNA Primers; D010446:Peptide Fragments; D003546:Cysteine Endopeptidases; D002401:Cathepsin B