Transcriptome analysis during the process of in vitro differentiation of Leishmania donovani using genomic microarrays

Abstract

Leishmania donovani causes visceral disease (kala-azar), a major health problem throughout the tropics with 500 000 new cases every year. Leishmania differentiates from the promastigote to the amastigote form to establish infection in a mammalian host. To understand the process of differentiation, we assessed the global variation in gene expression in promastigotes, an intermediate stage of differentiation (PA24) and axenic amastigotes in culture using an L. donovani genomic microarray with 4224 clones printed in triplicate. During an intermediate stage of differentiation 24 h after shifting the promastigotes into amastigotes (PA24), there were 41 (approximately 1%) clones with expression > or = 2.0-fold higher than promastigotes, whereas in terminally differentiated amastigotes there were 130 (approximately 3%) such clones. Of particular interest were certain genes that exhibited a transient increase or decrease in expression at the PA24 stage. Kinases showed a transient increase, and surface molecules, PSA and amino acid permease, were prominent clones among those showing a brief decrease at the PA24 stage. The microarray results have been validated using Northern blots or RT-PCR. In summary, our results provide important clues about the genes involved in the differentiation process of L. donovani that may contribute to virulence.