Actividad nº 5 : “Descubrimos los nombres” (ANEXO V)

AHLs are composed of a homoserine lactone ring and a fatty acid side chain and are synthesised primarily through the actions of a LuxI synthase or homologue [73, 75, 93, 94, 98, 99, 109, 127, 146]. Acyl-acyl carrier proteins, such as acyl-CoA provide the fatty acid hydrocarbon tail, while S- adenosylmethionine (SAM) from the activated methyl cycle (AMC) is the source of the homoserine lactone ring [94, 109, 146]. LuxI acts as the catalyst for the formation of an amide bond between these two molecules [94, 110, 146]. Once the amide bond has been formed, lactonisation of the reaction intermediate and the release of methylthioadenosine lead to the formation of the AHL signal molecule [75].

All AHLs have the same homoserine lactone ring structure, but can vary in the length and structure of the fatty acid chain [73, 91, 143, 155, 175]. The side chain, or R-group, can be between 4 and 18 carbons in length, and can be unmodified, or may have a hydroxyl, oxygen or a fully reduced alkyl group on the third carbon (Figure 7) [91, 93, 94, 109, 127, 143, 155, 175].

In addition there are two stereoisomers of AHL, differing in orientation at the α-centre of the lactone ring; the L-isomer and the D-isomer. The L-isomer is able to activate the ligand- binding domain of the LuxR protein, while the D-isomers are not [93].

Using radiolabelled N-(3-oxohexanoyl)-L-HSL both Kaplan and Greenberg and Pearson et al showed the AHL readily diffused across the membranes of V. fischeri and E. coli as both influx and efflux [176, 177].

Pearson et al compared the internalisation of N-butanoyl-L-HSL and N-(3- oxododecanoyl)-L-HSL in Ps. aeruginosa [178]. Both AHLs were found to diffuse into and out of the bacteria during the study. Using changes in concentration of the AHL, internal concentrations of N-butanoyl-L-HSL were observed to reach a steady level quickly which nearly equalled the external concentration of the AHL. This result suggested passive diffusion of the signal across the cell membrane. In contrast, N-(3-oxododecanoyl)-L-HSL took longer to reach a steady

Non-modified AHL

N-(3-oxo)-DL-HSL

N-(3-hydroxy)-DL- HSL

Figure 7 - Chemical structures of non-modified and modified AHLs

concentration, which was higher inside the cell compared to the external milieu. Further investigation suggested active efflux of the long chain AHL out of Ps. aeruginosa required the MexAB-OprM efflux pump [178]. These observations in Ps. aeruginosa suggest mechanistic differences in AHL uptake depending on chain length, which is likely to be conserved across bacterial species [98, 109, 178].

The length of the fatty acid chain affects the physical characteristics of the signals, such as solubility, mobility and hydrophobicity [175]. An AHL with a side chain of between 4 and 8 carbons in length, classed as short chain AHLs, are highly soluble in water and readily diffuse across the bacterial cell membrane [175]. The AHLs with the longer R-groups become progressively less soluble as the number of carbons increases, and the ability to diffuse across the cell membrane decreases in relation to the size of the molecule [98, 175]. When modified, for example addition of an oxo-group to the third carbon, the molecule becomes more hydrophilic and solubility in water increases, however the diffusion across the membrane is unchanged from the non-modified form [143, 175].

The structure and thereby activity of AHLs can also be affected by pH and enzyme activity [103]. In order to maintain the structure of the homoserine lactone ring, a more acidic pH is required [73, 93]. Exposure to a pH of more than 6.5 for an extended period resulted in the opening of the lactone ring, thereby rendering the AHL inactive [93]. By decreasing the pH to between pH1 and 2 for up to 24 hours the lactone ring can be reformed, activating the AHL molecule [93, 94] (Personal Communication, Williams. P, February 2010). The long chain AHLs are more resistant to alkaline hydrolysis compared to the short chain and modified AHLs, but prolonged exposure also caused adverse changes in structure of these longer signal molecules [103, 175].

Enzymes produced by some gram positive and gram negative bacteria can inactivate the AHLs. Lactonases open the lactone ring by hydrolysing the ester bond, whilst acylases cleave

the side chain by hydrolysis of the amide bond between the fatty acid and the lactone ring [73, 100, 110]. These enzymes decrease the amount of gram-negative specific signal in the external milieu, thereby reducing the potential for interspecies competition within a given niche [110].

1.3.2.1 Phenotypic switching

AHL synthesis occurs constitutively at a low level, maintaining a background concentration of the signal in the external milieu [139, 179]. The concentration of signal has been shown to increase relative to cell density [75]. Upon achieving a critical signal concentration threshold which reflects the size of the population, synthesis of specific AHL signals would rapidly increase as a result of a positive transcription feedback loop. The signals bind the cognate LuxR protein resulting in a phenotypic change in the bacterial population (Figure 8) [61, 75, 98, 155, 165]. As the signal is internalised or the bacterial population is reduced through translocation from the defined QS area, the concentration of AHL would decrease giving rise to an oscillating pattern of AHL production [61, 75, 98, 155, 165].

The phenotypic switch in the bacterial population is a result of up- or down-regulation of transcription [75, 94, 110]. These changes alter the regulation of pathogenic traits, such as production of toxins, or of secondary metabolites such as antimicrobials, proteases or siderophores (Table 2) [143, 155]. An important target for regulator binding is the luxI promoter as this increases signal production further [155].