A Putative ABC Transporter Permease is Necessary For Resistance to Acidified Nitrite and EDTA in Pseudomonas aeruginosa Under Aerobic, Anaerobic, Planktonic or Biofilm Conditions
Cameron McDaniel, Warunya Panmanee, Shengchang Su, Renuka Kapoor, Kevin Cox, Andrew Paul, Gee Lau, Seung-Hyun Ko, Joel Mortensen, Joseph S. Lam, Daniel Muruve and Daniel Hassett
Frontiers in Microbiology, Front. Microbiol. | doi: 10.3389/fmicb.2016.00291
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 25 mM (aerobic), 15 mM (anaerobic) A-NO2- and EDTA (1 mM, aerobic, anaerobic), respectively. 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.
Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions
Sang Sun Yoon, Ray Coakley, Gee W. Lau, Sergei V. Lymar, Benjamin Gaston, Ahmet C. Karabulut, Robert F. Hennigan, Sung-Hei Hwang, Garry Buettner, Michael J. Schurr, Joel E. Mortensen, Jane L. Burns, David Speert, Richard C. Boucher, and Daniel J. Hassett
The Journal of Clinical Investigation 2006 Feb;116(2):436-46. Epub 2006 Jan 26, PMID: 16440061, PMCID: PMC1350997
Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2–) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2– also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.