Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei.
BACKGROUND
Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.


RESULTS
Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the 'greasiness' of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.


CONCLUSIONS
Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.
Biochemistry-Electrochemistry Research Unit, Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Anuwat
A
Aunkham
Biochemistry-Electrochemistry Research Unit, Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Albert
A
Schulte
School of Engineering and Science, Jacobs University, Bremen, Germany.
Mathias
M
Winterhalter
Biochemistry-Electrochemistry Research Unit, Schools of Chemistry and Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Wipa
W
Suginta
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000378:metabolism / Q000494:pharmacology; D016957:Burkholderia pseudomallei / Q000187:drug effects* / Q000235:genetics*; D002511:Cephalosporins / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008554:Melioidosis / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010539:Permeability; D018272:Porins / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013845:Thienamycins / Q000494:pharmacology*; D018440:beta-Lactam Resistance / Q000235:genetics*
D000890:Anti-Infective Agents; D002511:Cephalosporins; D018272:Porins; D011994:Recombinant Proteins; D013845:Thienamycins; C040040:17927 D