Leishmania mexicana: identification of genes that are preferentially expressed in amastigotes

The protozoan parasite Leishmania has a digenetic life cycle, alternating between the promastigote and the amastigote stages. Amastigotes infect macrophage cells and reside in the hydrolytic environment of the phagolysosome. Leishmania show distinct morphological and biochemical changes during differentiation into amastigotes. These alterations are believed to be regulated by stage-specific expression of a discrete number of genes. Selective-suppression PCR, a PCR-based subtractive hybridization technique, identified two genes preferentially expressed in L. mexicana lesion amastigotes: a novel gene family, A600, and a differentially expressed beta-tubulin gene. Northern blot analysis confirmed amastigote-specific expression of these genes and quantitation showed a sixfold higher abundance of A600 and beta-tubulin transcripts in lesion amastigotes. The A600 gene was predicted to contain a 293-bp open reading frame (ORF) that was tandemly repeated in the L. mexicana genome. Sequence analysis predicted that the A600 ORF encodes either a membrane-bound or a secreted protein that may have a functional role in amastigote differentiation or intraphagolysosomal parasite survival.