PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily

Abstract

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like b-lactam acylases, PA0305 undergoes post-translational processing resulting in a-and b-subunits, with the catalytic serine as the first amino acid of the b-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C 8 -HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K cat /K m values of 0.14¾10 4 M "1 s "1 towards C 8 -HSL and 7.8¾10 4 M "1 s "1 towards 3-oxo-C 12 -HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C 12 -HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Dpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Dpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards longchain homoserine lactones, including 3-oxo-C 12 -HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…PvdQ (PA2385) is an AHL aclyase with specificity for long-chain AHLs (42,44), and crystallization studies revealed that AHL hydrolysis proceeds by a mechanism typical of the Ntn hydrolase superfamily (41). The observation that a pvdQ mutant of PAO1 retained its ability to grow on longchain AHLs suggested that other AHL-degrading enzymes were also employed by P. aeruginosa (42), and additional studies confirmed that two other Ntn hydrolase homologues, QuiP (PA1032) and HacB (PA0305), also function as AHL acylases with substrate specificity similar to that of PvdQ (45,52). Importantly, although PvdQ, QuiP, and HacB were all capable of modulating the accumulation of the endogenous P. aeruginosa signal 3OC12HSL (44,45,52), their abilities to modulate P. aeruginosa behavior and virulence in vivo were distinct.…”

“…The observation that a pvdQ mutant of PAO1 retained its ability to grow on longchain AHLs suggested that other AHL-degrading enzymes were also employed by P. aeruginosa (42), and additional studies confirmed that two other Ntn hydrolase homologues, QuiP (PA1032) and HacB (PA0305), also function as AHL acylases with substrate specificity similar to that of PvdQ (45,52). Importantly, although PvdQ, QuiP, and HacB were all capable of modulating the accumulation of the endogenous P. aeruginosa signal 3OC12HSL (44,45,52), their abilities to modulate P. aeruginosa behavior and virulence in vivo were distinct. Whereas overproduction of PvdQ in P. aeruginosa or exogenous addition of purified PvdQ protein attenuated Pseudomonas virulence in both fast and slow killing assays in the nematode Caenorhabditis elegans (106), overproduction of HacB showed only limited effects on pathogenesis in nematodes, with differences in virulence being detectable only after 5 days of infection (52).…”

“…Interestingly, homologues of AiiD were also found in various other Ralstonia and Pseudomonas spp. (46), and these bacteria were later confirmed to also possess AHL acylase activity (42,44,45,47,51,52). Biochemical analysis of the fate of 3OC10HSL incubated with purified AiiD protein confirmed that AiiD functions as an AHL acylase, releasing HSL and 3-oxodecanoic acid as the major degradation products (46).…”

“…To date, most bacteria known to be capable of AHL inactivation have been found to contain only one or two enzymes responsible for this activity. However, there are exceptions, including P. aeruginosa, which employs multiple AHL acylases (PvdQ, QuiP, HacB, and PA3922) (42,44,45,52). One unique exception is the bacterium Rhizobium sp.…”

“…Importantly, although PvdQ, QuiP, and HacB were all capable of modulating the accumulation of the endogenous P. aeruginosa signal 3OC12HSL (44,45,52), their abilities to modulate P. aeruginosa behavior and virulence in vivo were distinct. Whereas overproduction of PvdQ in P. aeruginosa or exogenous addition of purified PvdQ protein attenuated Pseudomonas virulence in both fast and slow killing assays in the nematode Caenorhabditis elegans (106), overproduction of HacB showed only limited effects on pathogenesis in nematodes, with differences in virulence being detectable only after 5 days of infection (52). These disparate effects help to underscore the importance of in vivo studies of all AHL-targeting enzymes even when their substrate specificities are comparable.…”