MOLECULAR GENETICS OF LEISHMANIA

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Parasites of the trypanosomatid protozoan genus Leishmania are responsible for a spectrum of tropical diseases that affect more than 10 million people worldwide, and depending on the specific species and immune status of the infected person, can be severe or fatal. In Brasil, Africa and the Mediterranean basin, leishmaniasis is a common opportunistic infection in AIDS patients. The goal of this proposal is to develop methods for the genetic identification of genes and proteins likely to play important roles in the infectious cycle of the protozoan parasite Leishmania. The premise is that the availability of powerful molecular genetic tools, and identification of genes important to parasite virulence, will radically advance our ability to develop improved control strategies. In previous studies methods were developed for manipulating the parasite genome in a variety of ways, and performing functional genetic rescue. These led to the identification of several candidate virulence genes. The role of genes such as UBF], implicated in parasite DNA repair pathways essential for survival, will be pursued. With rapid progress in the Leishmania genome project, and recent breakthroughs in the development of new methods suitable for genome-wide surveys of parasite gene expression, the time is appropriate to initiate a series of 'functional genomic' studies oriented towards characterizing expression from the complete parasite genome. The goal is to identify genes showing patterns of expression suggestive of functional roles in the infectious cycle, for example stage-specific expression or alterations in response to relevant stimuli. We will use mariner-based transpositional systems to systematically generate GFP gene fusions in defined segments of the parasite genome in vitro or the entire genome in vivo. These methods have the advantage of studying expression at the protein level, allowing the cumulative effect of regulation at both nucleic acid and protein levels to be evaluated. Promising preliminary data with DNA microarrays have been gathered, and a series of investigations of Leishmania gene expression under a variety of relevant developmental stages and environmental treatments will be carried out.