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Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

ABSTRACT: Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.

SUBMITTER: Randall LB

PROVIDER: S-EPMC4775993 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium's intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation ?-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted ?-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine ?-lactams tested and ?16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible P(tac) promoter, increased resistance levels for all ?-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ?85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ?tatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression.

Project description:The unwelcome evolution of resistance to the advanced generation cephalosporin antibiotic, ceftazidime is hindering the effective therapy of Burkholderia cepacia complex (BCC) infections. Regrettably, BCC organisms are highly resistant to most antibiotics, including polymyxins; ceftazidime and trimethoprim-sulfamethoxazole are the most effective treatment options. Unfortunately, resistance to ceftazidime is increasing and posing a health threat to populations susceptible to BCC infection. We found that up to 36% of 146 tested BCC clinical isolates were nonsusceptible to ceftazidime (MICs ≥ 8 μg/ml). To date, the biochemical basis for ceftazidime resistance in BCC is largely undefined. In this study, we investigated the role of the Ω-loop in mediating ceftazidime resistance in the PenA β-lactamase from Burkholderia multivorans, a species within the BCC. Single amino acid substitutions were engineered at selected positions (R164, T167, L169, and D179) in the PenA β-lactamase. Cell-based susceptibility testing revealed that 21 of 75 PenA variants engineered in this study were resistant to ceftazidime, with MICs of >8 μg/ml. Under steady-state conditions, each of the selected variants (R164S, T167G, L169A, and D179N) demonstrated a substrate preference for ceftazidime compared to wild-type PenA (32- to 320-fold difference). Notably, the L169A variant hydrolyzed ceftazidime significantly faster than PenA and possessed an ∼65-fold-lower apparent Ki (Kiapp) than that of PenA. To understand why these amino acid substitutions result in enhanced ceftazidime binding and/or turnover, we employed molecular dynamics simulation (MDS). The MDS suggested that the L169A variant starts with the most energetically favorable conformation (-28.1 kcal/mol), whereas PenA possessed the most unfavorable initial conformation (136.07 kcal/mol). In addition, we observed that the spatial arrangement of E166, N170, and the hydrolytic water molecules may be critical for enhanced ceftazidime hydrolysis by the L169A variant. Importantly, we found that two clinical isolates of B. multivorans possessed L169 amino acid substitutions (L169F and L169P) in PenA and were highly resistant to ceftazidime (MICs ≥ 512 μg/ml). In conclusion, substitutions in the Ω-loop alter the positioning of the hydrolytic machinery as well as allow for a larger opening of the active site to accommodate the bulky R1 and R2 side chains of ceftazidime, resulting in resistance. This analysis provides insights into the emerging phenotype of ceftazidime-resistant BCC and explains the evolution of amino acid substitutions in the Ω-loop of PenA of this significant clinical pathogen.

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Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

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