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
88VS
90K 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.Members of the genus
Acinetobacter are nonmotile Gram-negative bacteria, many of which cause severe, life-threatening infections and hospital outbreaks (
1). Although
Acinetobacter baumannii is regarded as an opportunistic pathogen with low virulence, this species infects the soft tissues, bone, bloodstream, and urinary tract and is an important cause of pneumonia and meningitis in immune-compromised patients (
2). Crude mortalities because of nosocomial pneumonia and bloodstream infections caused by
A. baumannii ranged from 30–75% and 25–54%, respectively (
3–
5). In intensive care units (ICU), outbreaks of infection caused by multidrug-resistant
A. baumannii strains exhibit a crude mortality rate as high as 91.7% (
4,
5). The poor outcome in patients with invasive multidrug-resistant
A. baumannii infection highlights the urgent need for new therapeutic agents and vaccines to reduce the associated morbidity and mortality.The survival of
A. baumannii is enhanced by its ability to acquire foreign genes, thus increasing the number of vulnerable hosts, producing biofilms, and displaying an open pan-genome (
6,
7). These abilities enable
A. baumannii to persist in nosocomial environments and to survive even under antibiotic treatment. Numerous studies have reported the emergence of
A. baumannii clinical isolates that are resistant to multiple antimicrobials such as carbapenems, colistin, sulbactam, and tigecycline, thus reducing the number of effective therapeutic options (
8,
9). In epidemiological studies, the incidence rate of carbapenem-resistant
A. baumannii in countries such as Australia, Brazil, Singapore, Canada, South Korea, Taiwan, and Thailand is in the range of 47–80% (
10). A study showed that 11% of nosocomial isolates of
A. baumannii were carbapenem-resistant; resulting in a morbidity and mortality rate of 52% as compared with a rate of 19% of patients infected with carbapenem-sensitive isolates (
4,
11–
13). Among the many carbapenem derivatives, imipenem initially was highly effective in the treatment of patients with
A. baumannii infections; however, imipenem resistance has been confirmed in 53.7% of
Acinetobacter nosocomial infections since the early 1990s (
4,
14,
15). The most common pathways leading to carbapenem resistance are associated with the loss of outer membrane porins, overexpression of efflux pumps, and overproduction of Ambler class B metallo-β-lactamases, class D oxacillinases, and AmpC cephalosporinase (
16–
18). In the case of
Acinetobacter-derived cephalosporinase (ADC)
1, the key upstream insertion sequence (IS) element, IS
Aba1, provides promoter sequences that confer bacterial resistance to broad-spectrum cephalosporins (
3,
19,
20). In a study of
Pseudomonas aeruginosa, the overproduction of AmpC β-lactamase exhibited weak carbapenem-hydrolyzing activity and thus contributed to carbapenem resistance in porin-deficient isolates (
21). Although the study suggested a link between AmpC β-lactamase and carbapenem resistance, the regulatory mechanisms remain unclear.Kinase-induced protein phosphorylation and phosphatase-induced protein dephosphorylation are crucial for signal transduction in both prokaryotic and eukaryotic species (
22–
26). Hence, bacterial phosphoproteomic analysis is a promising and accurate tool to study biological networks, including the mechanisms of antibiotic resistance. In a recent comparative phosphoproteomic study of
A. baumannii ATCC17978 and the multidrug-resistant clinical isolate
A. baumannii Abh12O-A2, the relationship between phosphoproteins and antibiotic resistance remained unclear because of the lack of biological confirmation (
27). In this study, we used two clinical isolates of
A. baumannii to establish comparative phosphoproteomic maps and to conduct biological validation to explore the mechanisms of imipenem resistance (
28). Phosphoproteomic analysis of
A. baumannii SK17 clinical strains was carried out using a shotgun strategy combined with phosphopeptides enrichment techniques and high-performance mass spectrometry, and thus the identified phosphosites were verified by site-directed mutagenesis (
23,
29–
31). Our findings clearly show that AmpC β-lactamase activity is regulated by phosphorylation and is involved in imipenem resistance.
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