PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain Nacylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under ironlimiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DpvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.
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.
The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-L-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-⌬pvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen of vertebrates and a primary pathogen of insects (17). It mainly infects individuals who are immunocompromised, such as human immunodeficiency virus-infected patients, as well as those who have cystic fibrosis. In addition, those having disruptions in normal barriers caused by severe burns or indwelling medical devices are at risk. Hospital-acquired P. aeruginosa pneumonias and septicemias are frequently lethal (2, 3). To facilitate the establishment of infection, P. aeruginosa produces an impressive array of both cell-associated and extracellular virulence factors, such as proteases and phospholipases, and also small molecules, including rhamnolipid, phenazines, and cyanide (17). Expression of many of the extracellular factors is cell density controlled, does not occur until the late logarithmic phase of growth, and is mediated through specific quorum-sensing signal molecules (23). Two of these molecules, N-butanoyl-L-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), have been studied in great detail. In our laboratory, we previously demonstrated that PA2385(pvdQ) from P. aeruginosa PAO1 encodes an acyl-homoserine lactone (AHL) acylase. Analysis of the gene product showed that the posttranslational processing of the acylase as well as the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PvdQ protein is a member of the N-terminal nucleophile hydrolase superfamily. The main AHL signaling molecule of P. aeruginosa PAO1, 3-oxo-C12-HSL, is degraded by PvdQ (16). Addition of the purified protein to PAO1 cu...
Bacteria, although considered for decades to be antisocial organisms whose sole purpose is to find nutrients and multiply are, in fact, highly communicative organisms. Referred to as quorum sensing, cell-to-cell communication mechanisms have been adopted by bacteria in order to co-ordinate their gene expression. By behaving as a community rather than as individuals, bacteria can simultaneously switch on their virulence factor production and establish successful infections in eukaryotes. Understanding pathogen-host interactions requires the use of infection models. As the use of rodents is limited, for ethical considerations and the high costs associated with their use, alternative models based on invertebrates have been developed. Invertebrate models have the benefits of low handling costs, limited space requirements and rapid generation of results. This review presents examples of such models available for studying the pathogenicity of the Gram-negative bacterium Pseudomonas aeruginosa. Quorum sensing interference, known as quorum quenching, suggests a promising disease-control strategy since quorum-quenching mechanisms appear to play important roles in microbe-microbe and host-pathogen interactions. Examples of natural and synthetic quorum sensing inhibitors and their potential as antimicrobials in Pseudomonas-related infections are discussed in the second part of this review.
Pyoverdine biosynthesis in fluorescent Pseudomonas spp. and especially in the opportunistic human pathogen Pseudomonas aeruginosa has been extensively studied. The acylase PvdQ is required for a maturation step in pyoverdine biosynthesis but also has been proven to be effective in degrading long-chain N-acyl homoserine lactones (AHLs). These molecules are used as quorum-sensing molecules by Gram-negative bacteria such as Pseudomonads themselves. Interestingly, the pvdQ gene is part of a pyoverdine cluster in P. aeruginosa and P. syringae but not in other fluorescent Pseudomonas spp. In this study we have compared the activities of PvdQ orthologues from various species and provide evidence for conserved functions in Pseudomonas fluorescens PfO-1, P. putida KT2440 and P. aeruginosa PA14. Despite large differences in genomic organization, expression of each of these pvdQ orthologues is regulated by iron availability. Moreover, PvdQ and its orthologues have conserved substrate specificity for AHLs and play a role in pyoverdine production in all tested Pseudomonas species. These data strongly suggest that the role of PvdQ in pyoverdine biosynthesis is conserved among Pseudomonas spp., while the control that PvdQ exerts in P. aeruginosa over its own quorum-sensing signals seems to be unique to this bacterium.
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