Dynamic Proteome Response of Pseudomonas aeruginosa to Tobramycin Antibiotic Treatment

Genetically susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by subinhibitory concentrations of tobramycin in Pseudomonas aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 μg/ml), while less dramatic at 0.1 μg/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 μg/ml) but not at 1 μg/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 h (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collectively, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments. Genetically susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by subinhibitory concentrations of tobramycin in Pseudomonas aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 μg/ml), while less dramatic at 0.1 μg/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 μg/ml) but not at 1 μg/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 h (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collectively, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments. The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the natural environment and causes human infections (1.Trautmann M. Lepper P.M. Haller M. Ecology of Pseudomonas aeruginosa in the intensive care unit and the evolving role of water outlets as a reservoir of the organism.Am. J. Infection Control. 2005; 33: S41-49Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). P. aeruginosa can metabolize various carbon and nitrogen compounds and persists under nutrient-poor and hostile growth environments (2.Williams H.D. Zlosnik J.E. Ryall B. Oxygen, cyanide and energy generation in the cystic fibrosis pathogen Pseudomonas aeruginosa.Adv. Microbial Physiol. 2007; 52: 1-71Crossref PubMed Scopus (135) Google Scholar, 3.Frimmersdorf E. Horatzek S. Pelnikevich A. Wiehlmann L. Schomburg D. How Pseudomonas aeruginosa adapts to various environments: A metabolomic approach.Env. Microbiol. 2010; 12: 1734-1747Crossref PubMed Scopus (104) Google Scholar). One example is P. aeruginosa pulmonary infection of cystic fibrosis (CF) patients. Despite stress induced by host defenses and high concentrations of antibiotics, P. aeruginosa cells are able to persistently colonize CF airways (4.Goss C.H. Burns J.L. Exacerbations in cystic fibrosis. 1: Epidemiology and pathogenesis.Thorax. 2007; 62: 360-367Crossref PubMed Scopus (334) Google Scholar). The aminoglycoside tobramycin is a front-line drug currently used in the treatment of P. aeruginosa in CF and other diseases. It is supplied in the forms of inhaled solution (TOBI) and intravenous injection. The tobramycin concentrations in airways after 300-mg dosage TOBI inhalation can reach 1,000 μg per g of sputum (5.Geller D.E. Pitlick W.H. Nardella P.A. Tracewell W.G. Ramsey B.W. Pharmacokinetics and bioavailability of aerosolized tobramycin in cystic fibrosis.Chest. 2002; 122: 219-226Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 6.Cheer S.M. Waugh J. Noble S. Inhaled tobramycin (TOBI): a review of its use in the management of Pseudomonas aeruginosa infections in patients with cystic fibrosis.Drugs. 2003; 63: 2501-2520Crossref PubMed Scopus (101) Google Scholar). This concentration is in the range of 10 to 1,000 times of the minimal inhibitory concentration (MIC) for P. aeruginosa clinical isolates tested ex vivo (6.Cheer S.M. Waugh J. Noble S. Inhaled tobramycin (TOBI): a review of its use in the management of Pseudomonas aeruginosa infections in patients with cystic fibrosis.Drugs. 2003; 63: 2501-2520Crossref PubMed Scopus (101) Google Scholar). However, even with such high tobramycin concentrations, chronic P. aeruginosa infections are rarely eradicated (6.Cheer S.M. Waugh J. Noble S. Inhaled tobramycin (TOBI): a review of its use in the management of Pseudomonas aeruginosa infections in patients with cystic fibrosis.Drugs. 2003; 63: 2501-2520Crossref PubMed Scopus (101) Google Scholar). This is true even when the infecting bacteria are antibiotic sensitive, as is the case early in disease (7.Mayer-Hamblett N. Kronmal R.A. Gibson R.L. Rosenfeld M. Retsch-Bogart G. Treggiari M.M. Burns J.L. Khan U. Ramsey B.W. Initial Pseudomonas aeruginosa treatment failure is associated with exacerbations in cystic fibrosis.Ped. Pulmonol. 2012; 47: 125-134Crossref PubMed Scopus (67) Google Scholar). One possible reason for P. aeruginosa persistence in vivo could relate to the time dependence of local concentrations of tobramycin experienced by P. aeruginosa in CF patient airways. Many factors, including inflammatory responses, blood and lymphatic circulations, and air flow distribution (for inhaled antibiotics), can alter the local antibiotic concentrations. In addition, P. aeruginosa cells can form biofilms in CF lungs and other infection sites (8.Singh P.K. Schaefer A.L. Parsek M.R. Moninger T.O. Welsh M.J. Greenberg E.P. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.Nature. 2000; 407: 762-764Crossref PubMed Scopus (1202) Google Scholar), and biofilm exopolysaccharide layers may slow the diffusion of tobramycin (9.Slack M.P. Nichols W.W. The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudomonas aeruginosa.Lancet. 1981; 2: 502-503Abstract PubMed Scopus (60) Google Scholar, 10.Tseng B.S. Zhang W. Harrison J.J. Quach T.P. Song J.L. Penterman J. Singh P.K. Chopp D.L. Packman A.I. Parsek M.R. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.Env. Microbiol. 2013; 15: 2865-2878PubMed Google Scholar). P. aeruginosa cells in the inner layers of biofilms may experience lower concentrations and more gradual increase of tobramycin levels than those in outer layers (10.Tseng B.S. Zhang W. Harrison J.J. Quach T.P. Song J.L. Penterman J. Singh P.K. Chopp D.L. Packman A.I. Parsek M.R. The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin.Env. Microbiol. 2013; 15: 2865-2878PubMed Google Scholar, 11.Davison W.M. Pitts B. Stewart P.S. Spatial and temporal patterns of biocide action against Staphylococcus epidermidis biofilms.Antimicrob. Agents Chemother. 2010; 54: 2920-2927Crossref PubMed Scopus (98) Google Scholar). Furthermore, even if final tobramycin concentration levels inside the biofilm eventually grow to match the highest levels experienced elsewhere, bacteria in these inner regions have experienced a slower increase, during which time proteome levels could be altered to promote the “adapted resistant state” (12.Szomolay B. Klapper I. Dockery J. Stewart P.S. Adaptive to in Microbiol. 2005; PubMed Scopus Google Scholar). Adaptive can be induced in planktonic P. aeruginosa Adaptive to aminoglycoside antibiotics from PubMed Scopus Google Scholar, Adaptive of in a in vitro Agents Chemother. PubMed Scopus (98) Google Scholar), and are not to the bacteria higher to antibiotic treatments Adaptive to aminoglycoside antibiotics from PubMed Scopus Google Scholar, Adaptive of in a in vitro Agents Chemother. 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