博士 國立臺灣大學 生化科學研究所 104 Acinetobacter baumannii is an opportunistic bacterial pathogen which is known to infect hospitalized, immunocompromised, or terminal patients. Importantly, this bacterium has become a high-virulent nosocomial pathogen due to the emergence of multidrug-resistant Acinetobacter baumannii (MDRAB) responsible for numerous outbreaks of infections. Global spread of MDRAB has emerged as a serious threat to human health; especially carbapenem resistance has greatly increased over recent decades. Among the many carbapenem derivatives, imipenem has been found to be effective against A. baumannii. It has been reported that imipenem resistance in A. baumannii threatens human health since the early 1990s; however, the underlying molecular mechanisms remain unclear. Accordingly, post-translational modifications (PTMs) of proteins have been found in a wide variety of species from bacteria to human. Among all, phosphorylation is a kind of reversible PTMs, and has been linked to a wide variety of cellular, metabolic, and signaling processes. Measuring the phosphoproteomics of pathogenic bacteria can provide a system view for investigating their roles in virulence regulation networks. In this study, we analyzed the phosphoproteomics of two clinical isolates of A. baumannii: imipenem-sensitive strain SK17-S and -resistant strain SK17-R. Using a shotgun strategy combined with high-accuracy mass spectrometry, we identified 410 phosphosites on 248 unique phosphoproteins from SK17-S and 285 phosphosites on 211 unique phosphoproteins from SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites from SK17-S and SK17-R are 47.0, 27.6, 12.4, 8.0, and 4.9% versus 41.4, 29.5, 17.5, 6.7, and 4.9%, respectively. Among all, the Ser-90 phosphosite, located on the catalytic motif S88VS90K91 of the AmpC β-lactamase, was first identified from SK17-S. We hypothesized that AmpC phosphorylation may control its enzyme activity and the ability of neutralizing imipenem due to the proximity of Ser-90 to the catalytic residue Ser-88. By site-directed mutagenesis, the nonphosphorylatable strain S90A was more resistant to imipenem, by contrast, the phosphorylation-simulated strain S90D was imipenem-sensitive. Also, S90A mutant protein exhibited higher β-lactamase activity and conferred more bacterial protection on SK17-S against imipenem than the wild-type. In summary, our results revealed that in A. baumannii Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem.