2-Alkyl-4-quinolone signalling in Pseudomonas and Burkholderia

Pseudomonas and Burkholderia are two related groups of Gram-negative bacteria which are commonly found in water, soil and on the surfaces of plants and animals. Some species are pathogenic for human and plants, some cause food and industrial spoilage problems while others are beneficial and promote plant growth and can be used for biodegradation of hydrocarbons and plastics. This project will focus mainly on Pseudomonas aeruginosa, a human pathogen which is a common cause of infections in hospital patients and a major problem in individuals with cystic fibrosis. P. aeruginosa produces an armoury of virulence factors and is resistant to many conventional antibiotics and so infections with this organism can be difficult to treat and it is almost impossible to eradicate from the lungs of cystic fibrosis patients. Bacteria such as P. aeruginosa use small chemical signal molecules to co-ordinate the activities of the individual cells within the bacterial population. This bacterial cell-to-cell communication device is called quorum sensing (QS). P. aeruginosa uses at least two chemically distinct classes of QS signal molecules, the N-acylhomoserine lactones (AHLs) and the 2-alkyl-4-quinolones (AQs) to control production of virulence factors which enable the organism to combat the immune system and cause tissue damage and disease. Inactivation of QS by mutating the genes responsible for the production and action of the AQ (and AHL) signal molecules renders the organism incapable of causing infections. QS systems are therefore potential targets for new antibacterial agents. Understanding how AQ-signalling works at a molecular level will allow us to develop new strategies for controlling pathogenic bacteria such as P. aeruginosa. In this project we propose to build on our previous work to understand in more detail how P. aeruginosa synthesizes AQ signal molecules, how AQs control virulence factor production, how AQ-signalling is affected by a lack of oxygen and to discover the role of AQs in scavenging for iron (an essential bacterial nutrient) and in stimulating the death of a proportion of the bacterial population under stressful conditions for the benefit of the bacterial population as a whole. We also plan to extend these studies to two Burkholderia species, Burkholderia plantarii and Burkholderia glumae which can cause infections in humans but which are primarily pathogens of rice. From our understanding of the molecular basis of AQ-signalling, we will also explore the potential of AQ-degrading enzymes for controlling Pseudomonas and Burkholderia infections and synthesize a series of analogues of AQs and AQ-precursors which will be screened as possible new antibacterial agents. Thus this research will be of benefit by providing useful scientific tools, by contributing to the understanding of fundamental biological systems and by designing and screening novel antibacterial agents for the prevention and treatment of Pseudomonas and Burkholderia infections

The genera Pseudomonas and Burkholderia contain species pathogenic for animals and plants while others are beneficial for bioremediation and plant growth. In humans, Pseudomonas aeruginosa is a major opportunistic, multi-antibiotic resistant pathogen which produces diverse virulence determinants and secondary metabolites. In P. aeruginosa these are regulated via a sophisticated quorum sensing (QS) regulatory network employing N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. While there has been extensive work on AHL-dependent QS our understanding of AQ-dependent QS in both P. aeruginosa and the Burkholderia is at a much earlier stage. However, P. aeruginosa mutants defective in AQ biosynthesis and signalling are attenuated in plant and animal infection models and consequently AQ signalling is a target for novel anti-infective strategies. We propose to build on our previous work by (i) defining the biochemical roles of PqsABCD, PqsH and PqsL in AQ biosynthesis; (ii) determine how AQ signalling is transduced via PqsE, (iii) elucidate the extent and overlap of the regulons controlled by the AQs, 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-quinolone (HHQ) and 2-heptyl-4-hydroxyquinoline-N-oxide (HHQ-NO) and the regulators PqsE and PqsR, (iv) determine how AQs interact with PqsR to drive gene expression directly, (v) investigate the role of AQs in repressing genes involved in dissimilatory nitrate reduction; (vi) elucidate the function of the AQ-iron complex and its contribution to autolytic cell death, (vii) define the contribution of AQ signaling to the virulence of the rice pathogens Burkholderia plantarii and Burkholderia glumae and (viii) explore AQ-dependent quorum sensing as a target for novel antibacterials via (i) enzymatic degradation of AQs and (ii) the design and synthesis of novel AQ synthesis and signal transduction inhibitors based on analogues of methylanthranilate and PQS.