N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth ofYersinia pseudotuberculosisandPseudomonas aeruginosa

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Abstract
In gram-negative bacterial pathogens, such asPseudomonas aeruginosaandYersinia pseudotuberculosis, cell-to-cell communication via theN-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures ofY. pseudotuberculosisandP. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the majorP. aeruginosa[N-butanoylhomoserine lactone (C4-HSL) andN-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] andY. pseudotuberculosis[N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL),N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as theN-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, andN-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require anN-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.

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