5. Quorum sensing interference: novel biocontrol strategies for pathogenic bacteria

Objective: To isolate and identify Streptomyces spp. with AHLs degrading activity and to explore their                  antagonistic activity against the quorum-sensing dependent bacteria.

Background information

                 Quorum sensing (QS) is a population-density dependent gene regulation.  It appears to be one of the most important regulatory systems activating virulence gene expression in pathogens. Cells perceive their population density by sensing the concentration of small signal molecules called autoinducers. The most common autoinducer found in pathogenic bacteria are N-acyl homoserine lactones (AHLs). Since QS regulatory systems are often required for pathogenesis, interference of QS signaling or quorum quenching may offer a promising strategy in controlling virulence of pathogenic bacteria. N-Acyl homoserine lactone acylase (AHL-acylase) is a quorum quenching enzyme which is capable of cleaving acyl side chain and blocking quorum sensing in bacteria. To date only three AHL-acylase genes have been identified from Rastonia isolate, Pseudomonas aeruginosa PAO1 and Streptomyces M664. As a source of AHL-degrading enzymes, micro-organisms are increasingly being investigated, and we also are interested in isolating AHL-degrading bacteria for the purpose of disrupting and manipulating QS signaling in phytopathogenic bacteria. Since QS regulatory systems are often required for pathogenesis, interference with QS signaling may offer a means of controlling bacterial diseases of plants. Consideration of the possibility to use as the biocontrol agent, we focus on isolation of AHL-degrading enzyme from Streptomyces, since this bacterium possesses the ability to synthesize and secrete a variety of secondary metabolites, as well as various extracellular hydrolytic enzymes. Two phytopathogenic bacteria (Pectobacterium carotovorum and Xanthomonas campestris) and a both plant and human pathogenic bacterium (P. aeruginosa) will be selected as the target for quorum quenching activity since both P. carotovorum and X. campestris were important plant pathogens and course disease in many crop plants. Whereas P. aeruginosa was selected since it was an important human pathogen which also reported to cause plant disease.

Hypothesis: Interfering of QS signaling may offer a promising strategy in controlling virulence of pathogenic bacteria.
Framwork:
1. Screening and isolation of Streptomyces producing AHL-degrading enzyme from soil sample
2. Isolation of AHL-degrading Streptomyces

        - Detection system  kindly provided by Prof. Dr. Stephen K Farrand
         - Screening of AHL-degrading Streptomyces
3. Cloning and expression of AHL-acylase from Streptomyces
4. Kinetic study of AHL-acylase enzyme
 5. Study effect of AHL-acylase on QS-bacteria