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Sub-cellular localization of the PenA β-lactamase in Burkholderia pseudomallei




Randall, Linnell B., author
Schweizer, Herbert P., advisor
Borlee, Brad R., committee member
Argueso, Juan Lucas, committee member

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Burkholderia pseudomallei, a Gram-negative soil bacterium found in tropical regions, is the etiologic agent of melioidosis. B. pseudomallei is intrinsically resistant to many antibiotics, and melioidosis treatment involves prolonged antibiotic therapy. PenA, a chromosomal beta-lactamase in B. pseudomallei, confers resistance to many beta-lactams. Point mutations in penA leading to PenA amino acid changes can cause resistance to ceftazidime and amoxicillin-clavulanate, which can result in treatment failure. Typically beta-lactamase enzymes are found in the periplasm of Gram-negative bacteria. Previous studies have shown that PenA is secreted via the twin arginine translocase (Tat) system, but failed to demonstrate periplasmic localization. The purpose of this study was to determine the sub-cellular localization of PenA in B. pseudomallei. Using alkaline phosphatase as a periplasmic marker, we optimized a method for extracting periplasmic proteins from B. pseudomallei. Through subcellular fractionations, immunoblotting, and colorimetric enzyme assays, we have shown that PenA does not localize to the periplasm. Rather, it is present in a detergent-soluble fraction of the cellular membranes. Further experiments including site-directed mutagenesis, metabolic labeling with 14C-palmitate, globomycin treatment, and mass spectrometry indicate that PenA is likely a lipoprotein with post-translational lipid modification of the cysteine residue at position 23. This work implicates PenA as the first example of a beta-lactamase that is a Tat-secreted lipoprotein, and provides a better physiological understanding of an important antibiotic resistance mechanism in B. pseudomallei.


2013 Summer.
Includes bibliographical references.

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