Characterization of the AmpC β-Lactamase from Burkholderia multivorans

Abstract

Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due to Bmultivorans, which possesses two β-lactamase genes, bla_penA and bla_AmpC PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae carbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams in Bmultivorans Here, we characterize AmpC from Bmultivorans ATCC 17616. AmpC possesses only 38 to 46% protein identity with non-Burkholderia AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of Bmultivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (K_iapp > 600 μM), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in Bmultivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in Bmultivorans compared to PenA, which is the dominant β-lactamase in Bmultivorans ATCC 17616.

Keywords: AmpC; Burkholderia; β-lactam; β-lactamases.

FIG 1

Comparison of AmpC1 from B. multivorans (AmpC1_Bm) with AmpC from B. cenocepacia J2315 (AmpC_Bc) and other prevalent class C β-lactamases (PDC_1 and E. coli AmpC [EcAmpC]). (A) Amino acid sequence alignments using Clustal Ω. Black shading indicates identity, dark-gray shading is similarity, and light-gray and white shading are differences. AmpC motifs (SXXK, YSN, and KTG) are in red. (B) Percent identity and homology between prevalent AmpC proteins and AmpC1.

FIG 2

(A) Homology models of AmpC1 combined with a loop refinement protocol suggest high flexibility of the L117-to-H126 loop (model 1, purple; model 2, cyan; multiple possible conformations from loop refinement, yellow; best energetically favorable conformation, magenta). The Q120 region has the largest movement (up to 10 Å), possibly changing the shape of the active-site entrance. (B) The amino acids that are replaced in the 27 AmpC variants (Table 2) are highlighted in green. Natural variants of AmpC have amino acid substitutions in two active-site motifs, the Ω loop (blue) (residues D193, R196, and T205) and the R2 loop (orange) (position K290); other active-site regions are the SXXK (with the nucleophilic S64), YSN, and KTG motifs (pink).

FIG 3

(A) Mass spectrometry of AmpC1 apoenzyme after 15 min (shaded) and 24 h (nonshaded) of incubation. Shown is mass spectrometry of the AmpC1-avibactam acyl-enzyme complex after 15 min and 24 h of incubation at 1:1 and 1:10 enzyme/avibactam ratios. (B) Summary of mass spectrometry data, including description of peaks formed.

FIG 4

Michaelis-Menten complex of AmpC1 (gray) and avibactam (cyan) revealing nonproductive interactions of avibactam with active-site residues. Potential hydrogen bonding interactions are indicated by dashed green lines.

FIG 5

Immunoblotting reveals that protein expression of AmpC1 and PenA is inducible in B. multivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem for different amounts of time. The anti-RecA antibody was used as a loading control.