TY - JOUR
T1 - The Leishmania mexicana A600 genes are functionally required for amastigote replication
AU - Murray, Angus S.
AU - Lynn, Miriam A.
AU - McMaster, W. Robert
N1 - Funding Information:
We thank Dr. David Lynn and Dr. Corinna Warburton for assistance with manuscript preparation and Dr. Alice Mui for technical advice. This study was supported by the Canadian Institutes for Health Research grant number MOP 92194 .
PY - 2010/8
Y1 - 2010/8
N2 - Leishmania parasites, the causative agent of leishmaniasis, have a digenetic lifecycle consisting of the morphologically distinct insect vector stage (promastigote) and the mammalian infective amastigote stage. Differentiation of promastigotes to the amastigote stage involves significant morphological and biochemical changes, however, very few genes have been characterised as being differentially expressed in the two stages. The Leishmania A600 genes are one of the few gene families that exhibit stage-specific expression and, as such, they are of interest as potential virulent factors. In this study, we characterize the A600 family in several Leishmania species and investigate their role in amastigote differentiation and proliferation. Four open reading frames, A600-1, A600-2, A600-3, and A600-4, were identified at the multi-gene L. mexicana A600 locus via cloning and restriction mapping. Homology searching identified A600 homologues in other Leishmania species, L. major, L. braziliensis and L. infantum but not in the closely related Trypanosoma family. A targeted gene deletion approach was utilized to determine the cellular function of the L. mexicana A600 genes. A600-/- promastigotes differentiated to axenic amastigotes in response to temperature shift and acidification of culture media, but showed significant growth arrest. Similarly, during in vitro macrophage infection studies, A600-/- promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600-/- amastigotes to proliferate in mouse peritoneal macrophages was restored by re-introduction of the A600-1 gene, but not the A600-4 gene. The results from these experiments show that the A600-1 gene is essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Furthermore, these results suggest a potential role for the L. mexicana A600-deficient mutant as a vaccine candidate.
AB - Leishmania parasites, the causative agent of leishmaniasis, have a digenetic lifecycle consisting of the morphologically distinct insect vector stage (promastigote) and the mammalian infective amastigote stage. Differentiation of promastigotes to the amastigote stage involves significant morphological and biochemical changes, however, very few genes have been characterised as being differentially expressed in the two stages. The Leishmania A600 genes are one of the few gene families that exhibit stage-specific expression and, as such, they are of interest as potential virulent factors. In this study, we characterize the A600 family in several Leishmania species and investigate their role in amastigote differentiation and proliferation. Four open reading frames, A600-1, A600-2, A600-3, and A600-4, were identified at the multi-gene L. mexicana A600 locus via cloning and restriction mapping. Homology searching identified A600 homologues in other Leishmania species, L. major, L. braziliensis and L. infantum but not in the closely related Trypanosoma family. A targeted gene deletion approach was utilized to determine the cellular function of the L. mexicana A600 genes. A600-/- promastigotes differentiated to axenic amastigotes in response to temperature shift and acidification of culture media, but showed significant growth arrest. Similarly, during in vitro macrophage infection studies, A600-/- promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600-/- amastigotes to proliferate in mouse peritoneal macrophages was restored by re-introduction of the A600-1 gene, but not the A600-4 gene. The results from these experiments show that the A600-1 gene is essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Furthermore, these results suggest a potential role for the L. mexicana A600-deficient mutant as a vaccine candidate.
KW - A600 genes
KW - Amastigote
KW - Leishmania
KW - Targeted gene deletion
UR - https://www.scopus.com/pages/publications/77953028152
U2 - 10.1016/j.molbiopara.2010.03.008
DO - 10.1016/j.molbiopara.2010.03.008
M3 - Article
C2 - 20307588
AN - SCOPUS:77953028152
SN - 0166-6851
VL - 172
SP - 80
EP - 89
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -