Significance and context
Trypanosomatid parasites of the genus Leishmania infect some 12 million people worldwide, with more than 600,000 new clinical cases reported annually. Leishmania species go through a complex life cycle: motile promastigotes survive in the gut of the sandfly vector; mammalian hosts are infected when the vector sucks blood; and, finally, host macrophages internalize Leishmania parasites that need to differentiate into a nonmotile amastigote form to persist in the macrophage lysosomal compartment. Differentiation is characterized by dramatic morphological, genetic and biochemical changes in the parasite. Understanding promastigote-to-amastigote differentiation is crucial for developing methods of blocking Leishmania infection in its mammalian hosts. Duncan et al. have identified genes whose expression changes significantly during promastigote-amastigote differentiation, and which might serve as targets for blocking Leishmania infection.
Two full-length genomic clones of Leishmania donovani, named P9 and A14, were isolated from a genomic library, based on previously identified mRNAs. The P9 gene contains 1,448 base pairs (bp) and encodes a putative protein of 482 amino acids; the A14 gene contains 2,708 bp and encodes a putative protein of 902 amino acids. No homologies with proteins in the database were found. P9 and A14 carry two and four putative membrane-spanning domains, respectively. The expression of P9 and A14, together with that of two known L. donovani genes - c-lpk2, encoding a homolog of the catalytic subunit of cAMP-dependent protein kinase, and mkk, encoding a mitogen-activated protein kinase kinase homolog - was studied during the promastigote- amastigote transition in cells in vitro. The highest expression of P9, c-lpk2 and mkk was found in the promastigote form, whereas A14 was preferentially expressed in amastigotes. These expression patterns were confirmed under in vivo conditions: northern blots using RNA of amastigotes collected from spleens of hamsters infectedwith L. donovani showed that A14 was abundantly expressed in amastigotes but at very low levels in promastigotes, whereas for P9, c-lpk2 and mkk the opposite was observed.
Genomic projects on various Leishmania species are currently underway; for information see the Leishmania genome network.
Duncan et al. report the discovery of two new L. donovani genes that are differentially expressed during promastigote-amastigote differentiation, a process required for infection of mammalian hosts. Thus, these genes are good candidates to be targeted in therapies aimed at blocking parasite development after infection. An expression analysis does not necessarily prove a function for these two genes in differentiation, however, and it would have been more conclusive if the authors had constructed L. donovani knockouts to confirm their involvement. The light-microscopic analysis of morphological changes during promastigote-amastigote differentiation also included in the paper illustrates the complexity of this developmental process, and this complexity implies that many genes and/or gene families are involved. The availability of Leishmania genome sequences, large-scale DNA microarray analyses using RNA prepared from promastigote and amastigote forms, and the construction of a comprehensive series of knockouts should eventually provide information for drug discovery and for developing strategies to arrest Leishmania infection and cure infected humans.