A putative ABC transporter permease is necessary for resistance to acidified nitrite and EDTA in Pseudomonas aeruginosa under aerobic and anaerobic planktonic and biofilm conditions

Cameron McDaniel, Shengchang Su, Warunya Panmanee, Gee Lau, Tristan Browne, Kevin Cox, Andrew T. Paul, Seung Hyun B. Ko, Joel E. Mortensen, Joseph S. Lam, Daniel A. Muruve, Daniel J. Hassett

Research output: Contribution to journalArticle

Abstract

Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite (A-NO2-, pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to A-NO2-. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to A-NO2-, but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with A-NO2- plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM A-NO2-, and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to A-NO2- in biofilms. A-NO2- sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, A-NO2- as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains.

Original languageEnglish (US)
Article number291
JournalFrontiers in Microbiology
Volume7
Issue numberAPR
DOIs
StatePublished - Apr 1 2016

Fingerprint

ATP-Binding Cassette Transporters
Membrane Transport Proteins
Biofilms
Nitrites
Edetic Acid
Pseudomonas aeruginosa
Anti-Bacterial Agents
Bacteria
Colistin
Membranes
Doxycycline
Chloramphenicol
Cystic Fibrosis
Mutagenesis
Chronic Disease
Cell Membrane
Therapeutics
Growth
Pharmaceutical Preparations
Genes

Keywords

  • ABC transporter permease
  • Acidified nitrite
  • Biofilms
  • EDTA
  • Pseudomonas aeruginosa

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

A putative ABC transporter permease is necessary for resistance to acidified nitrite and EDTA in Pseudomonas aeruginosa under aerobic and anaerobic planktonic and biofilm conditions. / McDaniel, Cameron; Su, Shengchang; Panmanee, Warunya; Lau, Gee; Browne, Tristan; Cox, Kevin; Paul, Andrew T.; Ko, Seung Hyun B.; Mortensen, Joel E.; Lam, Joseph S.; Muruve, Daniel A.; Hassett, Daniel J.

In: Frontiers in Microbiology, Vol. 7, No. APR, 291, 01.04.2016.

Research output: Contribution to journalArticle

McDaniel, Cameron ; Su, Shengchang ; Panmanee, Warunya ; Lau, Gee ; Browne, Tristan ; Cox, Kevin ; Paul, Andrew T. ; Ko, Seung Hyun B. ; Mortensen, Joel E. ; Lam, Joseph S. ; Muruve, Daniel A. ; Hassett, Daniel J. / A putative ABC transporter permease is necessary for resistance to acidified nitrite and EDTA in Pseudomonas aeruginosa under aerobic and anaerobic planktonic and biofilm conditions. In: Frontiers in Microbiology. 2016 ; Vol. 7, No. APR.
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AU - McDaniel, Cameron

AU - Su, Shengchang

AU - Panmanee, Warunya

AU - Lau, Gee

AU - Browne, Tristan

AU - Cox, Kevin

AU - Paul, Andrew T.

AU - Ko, Seung Hyun B.

AU - Mortensen, Joel E.

AU - Lam, Joseph S.

AU - Muruve, Daniel A.

AU - Hassett, Daniel J.

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AB - Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite (A-NO2-, pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to A-NO2-. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to A-NO2-, but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with A-NO2- plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM A-NO2-, and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to A-NO2- in biofilms. A-NO2- sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, A-NO2- as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains.

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