Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17.
Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S(88)VS(90)K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, 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. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.
From the ‡Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei 10617, Taiwan; §Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan;
Juo-Hsin
JH
Lai
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan; ¶Ph.D. Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taiwan;
Jhih-Tian
JT
Yang
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan; ‖Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan; **Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan;
Jeffy
J
Chern
‡‡Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei 11221, Taiwan; §§Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei 11217, Taiwan; ¶¶Department of Medicine, Cheng Hsin General Hospital, Taipei 11220, Taiwan;
Te-Li
TL
Chen
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan;
Wan-Ling
WL
Wu
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan;
Jiahn-Haur
JH
Liao
‖‖Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan; Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli 35053, Taiwan;
Shih-Feng
SF
Tsai
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan; Core Facilities for Protein Structural Analysis, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Suh-Yuen
SY
Liang
§Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan; Core Facilities for Protein Structural Analysis, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Chi-Chi
CC
Chou
From the ‡Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei 10617, Taiwan; §Institute of Biological Chemistry, Academia Sinica. Taipei 11529, Taiwan; ‖Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan; **Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan; shwu@gate.sinica.edu.tw.
Shih-Hsiung
http://orcid.org/0000-0002-1490-1227
SH
Wu
D040981:Acinetobacter baumannii / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D003428:Cross Infection / Q000382:microbiology; D006801:Humans; D015378:Imipenem / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D018440:beta-Lactam Resistance / Q000187:drug effects; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010750:Phosphoproteins; D020543:Proteome; D015378:Imipenem; C048795:AmpC beta-lactamases; D001618:beta-Lactamases