Structure-Function Analysis of the Transmembrane Protein AmpG from Pseudomonas aeruginosa

Abstract

AmpG is a transmembrane protein with permease activity that transports meuropeptide from the periplasm to the cytoplasm, which is essential for the induction of the ampC encoding β-lactamase. To obtain new insights into the relationship between AmpG structure and function, comparative genomics analysis, secondary and tertiary structure modeling, site-directed mutational analyses and genetic complementation experiments were performed in this study. AmpGs from different genera of bacteria (Escherichia coli, Vibrio cholerae and Acinetobacter baumannii) could complement AmpG function in Pseudomonas aeruginosa. The minimal inhibitory concentration (MIC) to ampicillin is 512 μg/ml for wild type strain PAO1, while it is 32 μg/ml for an ampG deletion mutant strain (PAO1ΔampG) with a corresponding decrease in the activity of the ampC-encoded β-lactamase. Site-directed mutagenesis of conserved AmpG residues (G29, A129, Q131 and A197) resulted in a loss of function, resulting in a loss of resistance to ampicillin in PAO1ΔampG. The G29A, G29V, A129T, A129V, A129D, A197S and A197D mutants had lower resistance to ampicillin and significantly decreased activity of the AmpC β-lactamase. The G29A, G29V, A129V, A197S and A197D mutants had decreased ampG mRNA transcript levels. The A129T and A129D mutants had normal ampG mRNA transcript levels, but the function of the protein was drastically reduced. Our experimental results demonstrate that the conserved amino acids played essential roles in maintaining the function of AmpG. Combined with the AmpG structural information, these critical amino acids can be targeted for the development of new anti-bacterial agents.

Peptidoglycan Muropeptides: Release, Perception, and Functions as Signaling Molecules

“…The amount of PG turnover products released to the environment is dependent on the PG recycling pathway, and so, it is expected that bacteria regulate this process. Information regarding the regulation of AmpG or other transporters with similar activity is therefore crucial for a complete understanding of the function(s) of released anhydromuropeptides and other PG fragments; however, few studies have focused on this kind of protein (Cheng and Park, 2002; Chahboune et al., 2005; Zhang et al., 2010; Chan and Dillard, 2016; Li et al., 2016). A recently described assay to quantify AmpG-mediated transport (Perley-Robertson et al., 2016) may help to understand important aspects regarding the regulation of this permease in years to come.…”

Class C β-Lactamases: Molecular Characteristics

“…freundii, E. cloacae, and P. aeruginosa (243,244). AmpR loss leads to susceptibility to βlactams, whereas constitutively high levels of ampC expression were reported for clinical isolates and in vitro strains with mutations resulting in AmpR amino-acid substitutions: C. AmpG, an intrinsic membrane protein, displays permease activity mediating the transport of muropeptides from the periplasm to the cytoplasm, such transport being essential for the induction of class C β-lactamases (252)(253)(254). Deletion of the gene encoding this protein in P. aeruginosa results in a lower level of bacterial resistance to ampicillin.…”

Role of AmpG in the resistance to β-lactam agents, including cephalosporins and carbapenems: candidate for a novel antimicrobial target

“…Based on the homology modeling structure of AmpG KE-Y1, the activation motif residues are located inside the transmembrane. Therefore, it is expected to be similar to the previously known AmpG modeling structure [ 22 ].

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