Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

Watt, Samantha C.

doi:10.1016/0378-1097(94)90341-7

Cited by 11 publications

(16 citation statements)

References 20 publications

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“…AERUGINOSA g-MVs PREY ON OTHER BACTERIA 2771 on May 13, 2018 by guest http://jb.asm.org/ certain surface-active agents (e.g., EDTA, cationic peptides, lysozyme, and cell wall-active antibiotics, including aminoglycosides) seem to be able to deregulate autolysins associated with the peptidoglycan layer so that they are artificially activated and lyse the bacteria (2,8,13,17,24,36,48,52,55). The addition of lysozyme increases the lytic rate, which suggests that the surface-active agents have perturbed the outer membrane so as to allow entry of the enzyme (2,24).…”

Section: Vol 178 1996mentioning

confidence: 99%

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics

1996

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Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.

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Section: Vol 178 1996mentioning

confidence: 99%

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics

1996

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“…2B). This possibly is due to the fact that EDTA can cause the release of protein-lipopolysaccharide complexes of P. aeruginosa (16). The pvdA mutant is more sensitive to the iron chelator acetohydroxamic acid and EDTA than wild-type PAO1 (Fig.…”

mentioning

confidence: 99%

Synergistic Activities of an Efflux Pump Inhibitor and Iron Chelators against Pseudomonas aeruginosa Growth and Biofilm Formation

Liu

Yang

Molin

2010

Antimicrob Agents Chemother

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The efflux pump inhibitor phenyl-arginine-␤-naphthylamide (PA␤N) was paired with iron chelators 2,2-dipyridyl, acetohydroxamic acid, and EDTA to assess synergistic activities against Pseudomonas aeruginosa growth and biofilm formation. All of the tested iron chelators synergistically inhibited P. aeruginosa growth and biofilm formation with PA␤N. PA␤N-EDTA showed the most promising activity against P. aeruginosa growth and biofilm formation.Pseudomonas aeruginosa is an important opportunistic pathogen that can cause a wide range of human infections (4). P. aeruginosa is notorious for its tolerance to antimicrobial agents and continues to cause a serious public health problem worldwide (1). The intrinsic multidrug resistance of P. aeruginosa is due, to a large extent, to the expression of efflux pump systems, which include MexAB-oprM, MexXY-OprM, . These efflux systems are reported to promote the export of antibiotics, organic solvents, biocides, and dyes (14). Recently, efflux pump inhibitors (EPIs), which specifically target the efflux activity and pump components, have been identified and proposed as novel agents to combat drug efflux mechanisms of pathogen (10). For example, phenyl-arginine-␤-naphthylamide (PA␤N) has been reported to be an efficient EPI for P. aeruginosa, as it can potentiate fluoroquinolone activity in resistant P. aeruginosa strains (9).Among all of the reported P. aeruginosa Mex efflux systems, MexAB-OprM is the only system that is expressed constitutively in cells grown in standard laboratory media (15). This suggests that the export of antimicrobial agents is not the primary function of the MexAB-OprM efflux system. The MexAB-OprM system was identified initially by growing P. aeruginosa in iron-depleted minimal medium containing 2,2Ј-dipyridyl (Dipy) (13). In that study, Poole and colleagues reported that MexAB-OprM is overexpressed under severe iron limitation conditions, suggesting that the MexAB-OprM system plays an essential role for P. aeruginosa survival under iron limitation conditions (13). The study thus suggests that the combination of efflux inhibitors and iron chelators synergistically inhibits P. aeruginosa growth. In the present study, we evaluated the synergistic activities of EPI with iron chelators against P. aeruginosa growth and biofilm formation.The wild-type P. aeruginosa strain PAO1 (7) and two isogenic mutants, pvdA (deficient in the synthesis of the iron siderophore pyoverdine [18]) and mexAB-oprM (deficient in the synthesis of the entire MexAB-OprM efflux pump [11]) were used in this study. Bacterial strains were cultivated in AB minimal medium supplemented with 30 mg/liter glucose (5). The AB minimal medium is an iron-restricted medium that promotes the production of the iron siderophore pyoverdine in P. aeruginosa (17). Stock solutions of 100 mg/ml Dipy (SigmaAldrich) in ethanol, 100 mg/ml acetohydroxamic acid (SigmaAldrich) in water, and 100 mg/ml EDTA (Sigma-Aldrich) in water were kept at 4°C until use. A stock solution of 10 mg/ml PA␤N (Sigma-Aldrich) in wat...

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“…After 30 min, the gels were transferred to fresh renaturation buffer and further incubated for 18 h at 37°C. Lytic bands were visualized as clear bands against a blue background after the gels were stained with 0.01% methylene blue in 0.1% KOH for 3 h, followed by destaining with deionized water (3,4). For quantification and presentation purposes, the gels were scanned using a BioRad Gel Doc 1000 equipped with a Mitsubishi video copy processor.…”

Section: Zymogram Analysismentioning

confidence: 99%

“…The renatured autolysins then catalyze the solubilization of the peptidoglycan in situ and detection of the clearing zones is facilitated by staining with methylene blue. This method has led to the detection of multiple autolysins in a variety of gram-negative pathogenic bacteria and also permitted preliminary characterization of their biochemical properties (3,4).…”

mentioning

confidence: 99%

“…This overlay system is still widely used but recent developments involved the incorporation of substrates directly into to polyacrylamide separating gel. We recently applied this technique for the detection of peptidoglycan hydrolases (autolysins) from gram-negative bacteria (3,4). Insoluble peptidoglycan is suspended in sodium dodecyl sulfate (SDS) PAGE gels and after electrophoresis, the slab gels are transferred to a renaturation solution which removes the bulk of the SDS and permits refolding of the separated proteins.…”

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confidence: 99%

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