Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C 12 -HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest that Pseudomonas 3-oxo-C 12 -HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C 12 -HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C 12 -HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.
Quorum-sensing systems have been reported to play a critical role in the pathogenesis of several bacterial infections. Recent data have demonstrated that Pseudomonas N-3-oxododecanoyl-l-homoserine lactone (3-oxo-C12-homoserine lactone, 3-oxo-C12-HSL), but not N-butanoyl-l-homoserine lactone (C4-HSL), induces apoptosis in macrophages and neutrophils. In the present study, the effects of active immunization with 3-oxo-C12-HSL–carrier protein conjugate on acute P. aeruginosa lung infection in mice were investigated. Immunization with 3-oxo-C12-HSL–BSA conjugate (subcutaneous, four times, at 2-week intervals) elaborated significant amounts of specific antibody in serum. Control and immunized mice were intranasally challenged with approximately 3×106 c.f.u. P. aeruginosa PAO1, and survival was then compared. All control mice died by day 2 post bacterial challenge, while 36 % of immunized mice survived to day 4 (P<0.05). Interestingly, bacterial numbers in the lungs did not differ between control and immunized groups, whereas the levels of pulmonary tumour necrosis factor (TNF)-α in the immunized mice were significantly lower than those of control mice (P<0.05). Furthermore, the extractable 3-oxo-C12-HSL levels in serum and lung homogenate were also significantly diminished in the immunized mice. Immune serum completely rescued reduction of cell viability by 3-oxo-C12-HSL-mediated apoptosis in macrophages in vitro. These results demonstrated that specific antibody to 3-oxo-C12-HSL plays a protective role in acute P. aeruginosa infection, probably through blocking of host inflammatory responses, without altering lung bacterial burden. The present data identify a promising potential vaccine strategy targeting bacterial quorum-sensing molecules, including autoinducers.
BackgroundBacteria release a wide variety of small molecules including cell-to-cell signaling compounds. Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones (acyl-HSLs) as signal compounds for quorum sensing (QS) within and between bacterial species. QS plays a significant role in the pathogenesis of infectious diseases and in beneficial symbiosis by responding to acyl-HSLs in Pseudomonas aeruginosa. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to the regulation of virulence and provides a growth advantage in several environments. In this study, we hypothesized that RND-type efflux pump system MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis.ResultsLoss of MexAB-OprM due to deletion of mexB caused increases in QS responses, as shown by the expression of gfp located downstream of the lasB promoter and LasB elastase activity, which is regulated by a LasR-3-oxo-C12-HSL complex. Either complementation with a plasmid containing wild-type mexB or the addition of a LasR-specific inhibitor, patulin, repressed these high responses to 3-oxo-acyl-HSLs. Furthermore, it was shown that the acyl-HSLs-dependent response of P. aeruginosa was affected by the inhibition of MexB transport activity and the mexB mutant. The P. aeruginosa MexAB-OprM deletion mutant showed a strong QS response to 3-oxo-C10-HSL produced by Vibrio anguillarum in a bacterial cross-talk experiment.ConclusionThis work demonstrated that MexAB-OprM does not control the binding of LasR to 3-oxo-Cn-HSLs but rather accessibility of non-cognate acyl-HSLs to LasR in P. aeruginosa. MexAB-OprM not only influences multidrug resistance, but also selects acyl-HSLs and regulates QS in P. aeruginosa. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P. aeruginosa.
Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C 12 -HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C 12 -HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C 12 -HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 mM 3-oxo-C 12 -HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C 12 -HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C 12 -HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone. INTRODUCTIONBacteria respond and adapt to a constantly changing environment because they have to compete with other micro-organisms for nutrients. To sense their own environmental conditions, bacteria utilize quorum sensing (QS) systems that operate via small molecules known as autoinducers (Miller & Bassler, 2001). These systems regulate the expression of a number of genes synchronously across the bacterial population. Gram-negative bacteria utilize lipid-based molecules, termed acylhomoserine lactones (AHLs) to communicate with each other, whereas Gram-positive bacteria employ peptide-based molecules, termed autoinducing peptides (Federle & Bassler, 2003;Fuqua & Greenberg, 2002). Autoinducer-2 has been found in a variety of bacteria and is considered to be a universal QS molecule (Xavier & Bassler, 2003). When an autoinducer accumulates to the threshold concentration in a populationdensity manner, the expression triggers induction or repression of certain sets of genes that co-ordinate the behaviour of the bacterial population, including the expression of virulence factors. The mechanisms of QS have been extensively characterized in the pathogenic bacterium Pseudomonas aeruginosa, an important causative agent of nosocomial respiratory and urinary tract infections (Willcox et al., 2008). Ps. aeruginosa produces several virulence factors, including LasB elastase, rhamnolipids, pyocyanin, lipase and hydrogen cyanide that contribute to its pathogenesis (Winstanley & Fothergill, 2009). The las-and rhl-QS systems regulate the production of these virulence factors via N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) and N-butanoyl-L-homoserine lactone (C 4 -HSL), respectively. METHODSBacterial strains and culture media. Bacterial strains used ...
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