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Functional genomics of the A600 locus in Leishmania mexicana Murray, Angus Stewart

Abstract

Protozoan parasites of the genus Leishmania are the causative agent of a spectrum of important human diseases collectively referred to as leishmaniasis. Leishmania has a digenetic lifecycle alternating between the promastigote and amastigote stages. In the amastigote stage, Leishmania are obligate intracellular parasites that replicate actively in the macrophage phagolysosome. The mechanisms used by amastigotes to survive within the acidic and hydrolytic environment of the phagolysosome and to suppress macrophage activation remain to be determined. The identification and characterization of genes preferentially expressed in the amastigote stage should elucidate novel parasite mechanisms used to establish a persistent infection. The amastigote-specific L. mexicana cDNA, A600, was cloned previously in this laboratory using a suppression-subtraction PCR (SS-PCR) approach. The A600 gene did not share sequence identity with any known genes, although expression of the mRNA transcript was seven-fold higher in amastigotes. Southern blot analysis indicated that multiple A600 coding sequences existed in the L. mexicana genome. In the present study, the multi-gene A600 locus was cloned and restriction mapping identified four open reading frames: A600-1, A600-2.1, A6002.2, and A600-3. The A600-J and A600-3 genes shared 78% DNA sequence identity. A cross-species comparison of the A600 genes using L. mexicana (New World) and L. major (Old World) revealed that divergence of the A600-1 and A600-3 genes occurred in an ancestral Leishmania species. A targeted gene deletion approach was used to determine the cellular function of the A600 genes. An A600-deficient mutant (A600[sup -/-]) of L. mexicana was generated using two rounds of homologous recombination. A600[sup -/-] promastigotes differentiated to amastigotes in response to temperature shift and acidification of culture media, but showed significant growth inhibition. During in vitro infection studies, A600[sup -/-] promastigotes established an early infection, but were deficient in their ability to proliferate as intracellular amastigotes. The ability of A600[sup -/-] amastigotes to proliferate was restored by re-introduction of the A600-1 gene, but not the A600-3 gene. Finally, BALB/c mice infected with L. mexicana A600[sup -/-] cells did not produce lesions, while infection with wildtype cells caused progressive cutaneous lesions. The results from these experiments show that the A600-1 gene was essential for continued proliferation of amastigotes, and potentially for development of chronic leishmaniasis. Leishmania gene expression is regulated post-transcriptionally via mRNA stability or translational control mechanism, usually via regulatory elements in the 3'UTR. This study used a luciferase reporter construct to show that stage-specific expression of the A600-3 mRNA transcript was mediated by the 3'UTR. Progressive deletions generated in the A600-3 3'UTR identified a 250 bp region, RD2, that negatively regulated luciferase protein expression in promastigotes. A 15 nt sequence in the RD2 region was conserved in the divergent A600-1 3'UTR and may constitute a novel regulatory element. It is proposed that trans-acting factors interact with this regulatory element to inhibit translation of the A600 proteins in promastigotes.

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