Membrane-bound PenA β-lactamase of Burkholderia pseudomallei

Linnell B. Randall, Karen Dobos, Krisztina M. Papp-Wallace, Robert A. Bonomo, Herbert P. Schweizer

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1509-1514
Number of pages6
JournalAntimicrobial Agents and Chemotherapy
Volume60
Issue number3
DOIs
StatePublished - Mar 2016
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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