Protein prenylation is an important post-translational modification that enhances membrane localization and protein-protein interactions. Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase) catalyze the addition of 15-and 20-carbon isoprenoid groups, respectively. Further modifications of prenylated proteins include, but are not limited to, proteolysis of the last three amino acids, methylation, and palmitoylation. FTase and GGTase require a cysteine 4 amino acids from the C-terminus as a minimal substrate. Canonical substrates are described by the Ca1a2X paradigm where C is a cysteine, followed by two aliphatic amino acids, and an X group, which is proposed to determine specificity for FTase or GGTase. While these consensus sequences identify many prenylated proteins, including G-proteins Ras, Rab, and Rho, the entire complement of prenylated proteins in vivo has not yet been determined. Recent experiments have demonstrated that the infectious bacterium Legionella pneumophila (and possibly other bacteria) requires host cell farnesylation machinery to induce disease. Inhalation of Legionella- containing water aerosol can cause Legionnaire's disease, a pneumonia-like illness. Ankyrin B (ankB), an essential Legionella effector protein, is farnesylated by the host FTase. This modification is important for both evasion of the endocytic pathway and for the function of ankB. Furthermore, prenylation of effector proteins may be a general method used by pathogenic bacteria to anchor proteins to bacterial vacuolar membranes in host cells. To investigate the prevalence of prenylation of bacterial proteins, I propose to measure the reactivity of mammalian FTase and GGTase both in vivo and in vitro with peptide sequences derived from proteins expressed in pathogenic bacteria, including: (1) measuring the kinetic parameters for prenylation of a library of TKCaaX peptides catalyzed by FTase and GGTase;(2) determining the cellular localization of eGFP-CaaX fusion proteins in mammalian cells using fluorescence microscopyl and (3) analyzing the C-terminal structure of the modified eGFP-CaaX proteins using mass spectrometry (in collaboration with Prof. K. Hakansson). These experiments will facilitate identification of modified proteins from a variety of bacteria. Knowledge of how effecto proteins are modified by eukaryotic host cells may lead to the identification of novel pathways that could be exploited for the development of antibacterials.
With the continuing development of multi-drug resistant bacteria, such as Staphylococcus aureus, it is clear that the current repertoire of antibiotics is rapidly becoming outdated;novel treatments are needed to fight bacterial infections, but few new targets are available for drug development. The recent discovery that lipidation of bacterial effectors is important for pathogenesis suggests that this step might be a target for antibacterials since inhibitors of these enzymes are well-tolerated by humans. The proposed study will identify the essential bacterial proteins that are modified and probe the function of ths modification.