Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.
BACKGROUND
Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced.


RESULTS
We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1.


CONCLUSIONS
The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.
Denise
Hubert
H
Wellcome Centre for Molecular Parasitology and Division of Infection & Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK. Hubert.denise@pfizer.com <Hubert.denise@pfizer.com>
Poot
Jacqueline
J
Jiménez
Maribel
M
Ambit
Audrey
A
Herrmann
Daland C
DC
Vermeulen
Arno N
AN
Coombs
Graham H
GH
Mottram
Jeremy C
JC
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics*; D006801:Humans; D018314:Leishmania infantum / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D007894:Leishmania mexicana / Q000201:enzymology / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000469:parasitology; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D020298:U937 Cells
D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases