Project Summary Collectively, the leishmaniases (cutaneous, mucocutaneous, and visceral) afflict approximately 12 million people worldwide, mainly in regions of severe poverty. Current anti-leishmanial therapies can be costly, are often poorly tolerated and the incidence of resistant parasites is increasing. Hence, new and improved drugs are desperately needed. A better understanding of the basic biology of Leishmania would aid in the identification of differences between the parasite and its host that are potentially exploitable for drug development. To this end, we propose to develop and validate a suite of novel tools that will facilitate the analysis of protein function, localization, and regulation in these parasites. The proposed tools are designed to exploit the ability of virally derived 2A peptide sequences to mediate the formation of two polypeptides from a single open reading frame (ORF) via a cotranslational, intra-ribosomal ?cleavage? mechanism. All of these tools will be used to create gene targeting constructs that encode an in-frame genetic fusion between a drug marker, a short 2A peptide sequence linker, and a tagged gene of interest. This will allow integration of the tagged gene into its endogenous chromosomal locus by selecting for drug resistance. Although encoded within the same ORF, cotranslational cleavage mediated by the 2A-peptide sequence will cause the drug marker and protein of interest to be expressed as separate proteins. Tools following this general principle will be created to address two problems. First, traditional systems for expressing tagged proteins in Leishmania can provide unregulated, non-physiological, and often heterogeneous expression. The proposed ?2A-tagging cassettes? will overcome these issues by facilitating integration of a tagged gene at its endogenous locus in a manner that preserves the cognate 5' and 3' flanking sequencing, and potentially maintains normal expression and regulation. We will generate and validate 2A-tagging cassettes encoding 3XHA, GFP, HB tandem affinity tags, DD-FKBP and ltDHFR inducible destabilizing domains, and two different drug markers. Second, commonly used methods for distinguishing between translational and post-translational regulatory mechanisms are cumbersome to apply, particularly to multiple candidates such as those derived from global proteomic studies. We will develop and validate a novel reporter system, employing ?2A-reporter cassettes?, that will allow the contributions of translational and post-translational mechanisms to be readily discerned. Because 2A peptide sequences are functional in every eukaryote yet tested, the 2A-reporter concept will have broad applicability and impact. The modular design of these tools is compatible with our published method for rapidly generating gene targeting vectors, increasing the speed and throughput of the system. Published data support the principles behind both the tagging and reporter systems, making it highly likely that successful completion of the specific aims in this proposal will provide the community with a set of versatile and valuable new tools.