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Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana
1Wellcome Unit of Molecular Parasitology, University of Glasgow, The Anderson College, UK.
Abstract:
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.
Insights
Leishmania mexicana cysteine proteinase (cpc) gene disruption reduced parasite infectivity to macrophages. Re-expressing cpc restored infectivity, suggesting cpc is important for host cell interaction but not essential for virulence.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Leishmania mexicana is an important human pathogen.
- Cysteine proteinases play roles in parasite survival and virulence.
- The specific role of cathepsin B-like cysteine proteinase (cpc) in Leishmania mexicana infectivity is not fully understood.
Purpose of the Study:
- To investigate the role of the cathepsin B-like cysteine proteinase (cpc) gene in Leishmania mexicana.
- To determine the impact of cpc gene deletion on parasite infectivity and virulence.
Main Methods:
- Targeted gene disruption was used to create cpc null mutants.
- Polymerase chain reaction (PCR) confirmed gene deletion.
- Re-expression of cpc was achieved using an episomal vector.
- Western blotting detected enzyme re-expression.
- In vitro infectivity assays with macrophages were performed.
- Lesion development in mice was monitored.
Main Results:
- cpc null mutants exhibited normal growth in axenic cultures.
- Mutants showed significantly reduced infectivity to macrophages in vitro.
- Re-expression of cpc restored in vitro infectivity.
- Null mutants formed lesions in mice at rates similar to wild-type, but lesions were smaller.
Conclusions:
- The cysteine proteinase encoded by cpc plays a role in Leishmania mexicana's interaction with macrophages.
- cpc is important for parasite infectivity in vitro.
- cpc alone is not essential for overall infectivity or virulence in vivo.
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