ABSTRACT
The antibacterial drug azithromycin has clinically beneficial effects at sub-inhibitory concentrations for the treatment of conditions characterized by chronic Pseudomonas aeruginosa infection, such as cystic fibrosis. These effects are, in part, the result of inhibition of bacterial biofilm formation. Herein, the efficient synthesis of azithromycin in 4 steps from erythromycin and validation of the drug's ability to inhibit biofilm formation at sub-MIC (minimum inhibitory concentration) values are reported. Furthermore, the synthesis of immobilized and biotin-tagged azithromycin analogues is described. These chemical probes were used in pull-down assays in an effort to identify azithromycin's binding partners in vivo. Results from these assays revealed, as expected, mainly ribosomal-related protein binding partners, suggesting that this is the primary target of the drug. This was further confirmed by studies using a P. aeruginosa strain containing plasmid-encoded ermC, which expresses a protein that modifies 23S rRNA and so blocks macrolide entry to the ribosome. In this strain, no biofilm inhibition was observed. This work supports the hypothesis that the sub-inhibitory effects of azithromycin are mediated through the ribosome. Moreover, the synthesis of these chemical probes, and proof of their utility, is of value in global target identification in P. aeruginosa and other species.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/metabolism , Azithromycin/chemical synthesis , Azithromycin/metabolism , Biofilms/drug effects , Biotin/metabolism , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/physiologyABSTRACT
Production of virulence factors and secondary metabolites is regulated in the phytopathogen Erwinia carotovora by quorum sensing involving N-acylated homoserine lactone (AHL) signaling molecules. Non-hydrolyzable AHL analogues were synthesized and screened in vivo. The biological activity of each compound was correlated with its ability to bind Erwinia AHL receptor proteins (LuxR homologues) in vitro. There is an excellent correlation between carbapenem production in vivo and in vitro binding to CarR. However, no such correlation could be found between exoprotease production and analogue binding to EccR. Our data are consistent with the involvement of a third, as yet uncharacterized LuxR homologue.
Subject(s)
4-Butyrolactone/analogs & derivatives , Pectobacterium carotovorum/chemistry , Repressor Proteins/metabolism , Signal Transduction , Trans-Activators/metabolism , 4-Butyrolactone/chemical synthesis , 4-Butyrolactone/pharmacology , Bacterial Proteins/biosynthesis , Binding Sites , Carbapenems/biosynthesis , Colony Count, Microbial , Exopeptidases/biosynthesis , Gene Expression Regulation/drug effects , Pectobacterium carotovorum/cytology , Pectobacterium carotovorum/metabolism , Structure-Activity RelationshipABSTRACT
Over the last decade or so, a wealth of research has established that bacteria communicate with one another using small molecules. These signals enable the individuals in a population to coordinate their behaviour. In the case of pathogens, this behaviour may include decisions such as when to attack a host organism or form a biofilm. Consequently, such signalling systems are excellent targets for the development of new antibacterial therapies. In this review, we assess how Gram-negative bacteria use small molecules for cell-cell communication, and discuss the main approaches that have been developed to interfere with it.
Subject(s)
Cell Communication/physiology , Gram-Negative Bacteria/physiology , Gram-Negative Bacteria/pathogenicity , VirulenceABSTRACT
The human pathogen Pseudomonas aeruginosa uses N-butyryl-L-homoserine lactone (BHL) and N-(3-oxododecanyl)-L-homoserine lactone (OdDHL) as small molecule intercellular signals in a phenomenon known as quorum sensing (QS). QS modulators are effective at attenuating P. aeruginosa virulence; therefore, they are a potential new class of antibacterial agent. The lactone in BHL and OdDHL is hydrolysed under physiological conditions. The hydrolysis proceeds at a rate faster than racemisation of the alpha-chiral centre. Non-hydrolysable, non-racemic analogues (small molecule probes) were designed and synthesised, replacing the lactone with a ketone. OdDHL analogues were found to be relatively unstable to decomposition unless they were difluorinated between the beta-keto amide. Stability studies on a non-hydrolysable, cyclohexanone analogue indicated that racemisation of the alpha-chiral centre was relatively slow. This analogue was assayed to show that the L-isomer is likely to be responsible for the QS autoinducing activity in P. aeruginosa and Serratia strain ATCC39006.
Subject(s)
Biochemistry/methods , Molecular Probes/chemistry , Pseudomonas aeruginosa/physiology , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/chemistry , 4-Butyrolactone/metabolism , Cyclohexanones/chemistry , Drug Evaluation, Preclinical/methods , Fluorine/chemistry , Homoserine/analogs & derivatives , Homoserine/chemistry , Hydrolysis , Isomerism , Ketones/chemistry , Molecular Probes/chemical synthesis , Molecular Probes/metabolism , Pseudomonas aeruginosa/pathogenicity , Serratia/physiology , Structure-Activity RelationshipABSTRACT
A number of bacteria, including some significant pathogens, utilize N-acylhomoserine lactones (AHLs) as quorum sensing signals. There is considerable interest in the therapeutic potential of disrupting quorum sensing. Recently, a number of bacteria have been identified which are capable of enzymic inactivation of AHLs. These enzymes show considerable promise as 'quenchers' of quorum sensing. However, the assumption that the natural function of these enzymes is to disrupt or modulate quorum sensing has yet to be established. This review surveys the progress made to date in this field and examines what implications these findings have for our understanding of the role played by these enzymes in vivo.
