RsaL as a negative regulator of quorum sensing in Pseudomonas aeruginosa

Molecular Microbiology

"RsaL provides quorum sensing homeostasis and functions as a global regulator of gene expression in Pseudomonas aeruginosa." By Giordano Rampioni, Martin Schuster, Everett Peter Greenberg, Iris Bertani, Marco Grasso, Vittorio Venturi, Elisabetta Zennaro, Livia Leoni.

Published in Molecular Microbiology. (2007) 66.6: 1557-1565

One of the most virulent and antibiotic resistant pathogens is Pseudomonas aeruginosa. It is commonly found in both soil and water and its effect on humans can be severe. It can cause acute and chronic infections in patients who have compromised immune systems or are suffering from other ailment which may put them at risk for infection. Pseudomonas aeruginosa, "…is the major cause of death in cystic fibrosis patients and the main cause of chronic wounds." (Rampioni, et. al. 1557)

The authors realize the importance of controlling this pathogen and have targeted quorum sensing as a primary first line defense in regulating this bacteria's virulent growth rate and debilitating effect in human beings. Quorum sensing can be compared to a social behavior determinant which helps to regulate the overall population density of the bacterial community of Pseudomonas aeruginosa. In effect it is the controlling mechanism that senses the environment around the bacteria and starts or stops a population explosion of the pathogen. Finding the key chemical in the molecules of this genetic trigger is of primary concern tot he authors. Using Molecular microbiological techniques to control the gene expression may be far more effective that simple antibiotic treatments, which bacterial strains can become immune to over time in just a few generations of the population.

Finding the control mechanism is no small task as quorum sensing in Pseudomonas aeruginosa, as in most organisms, is a rather complex function with more than one causal chemical reactant to trigger the response. One of the key signal compunds is N-O-oxo-dodecanoyl-honioserine lactone (SOC1rHSL). This element is essential is required for completing the entire circuit of reaction. This begins the positive regulation of the genetic expression. However the authors focus is on negative regulators that create homeostasis in the pathogens reproduction. While Pseudomonas aeruginosa quorum sensing is influence by many different chemical genetic controls, the RsaL transcriptional regulator is what the author believed to be the ideal negative regulator for the bacteria.

The RsaL transcriptional regulator, encoded by the rsaL gene, represses LasI expression by binding the promoter of !asl (P/asl) (de Kievit oraL, 1999; Rampioni etal., 2006). Besides LasR, RsaL is the only regulator known to bind Plasl, and among the OS repressors characterized to date it displays the most dramatic effect on 30C12-HSL production. (Rampioni, et. al. 1558)

METHODS RESULTS

Bacterial strains for Pseudorncnas aeruginoss PAOI (wild type) and its reaL derivative strain (rsaL::ISlacZlhah) were obtained from the Washington University's Genome Center's P. aeruglnosa mutant library. The bacterial strains were grown at 37°C in Luria-Bertani broth with aeration and antibiotics to reduce E-coli and other non-specific bacterial growth. The Pseudorncnas aeruginoss double -- mutant strain (PAO-DM1) was generated by using the plasimd pMARLI and then cloning in pMosblue in a polymerase chain reaction. Then a polyclonal antiserum was produced in an emulsification of complete Freund's adjuvant (Sigma) and then used to immunize a rabbit as the subject by intramuscular injections. Three weeks later a second booster in complete Freund's adjuvant was injected and three weeks alter a third booster. The rabbit's blood was taken two weeks later and the serum was stored at 4 degrees C Cells were then cultured with strong aeration in Luria-Bertani medium at 37 degrees C. And pyocyanin levels were measured along with the growth curve of the bacteria.

DISCUSSION

The results of this study seem extremely promising and certainly worth further consideration. They have been cited in numerous other research article on the subject and apperar to be gaining acceptance in the scientific community. The long-range outcome of such forays inot molecular microbiology can certainly be very promising regarding the pathogen Pseudorncnas aeruginos. However even the researcher point out the quorum sensing is a very complex process in any organism and a few chemical transcriptors may not be enough to thoroughly understand and control the process. They believe that is very likely that Rsal does exert a homoestatic effect on the Quorum sensing activity and their results are certainly promising. But even their own results reveal that the difference between the mutated and wild strain showed great variation in results in this experiment and may prove to show even greater results in larger trails of the subject.

While as stated there results are certainly promising, they have opened up the understanding of quorum sensing activity in organism to greater levels and revealed even more complexity than previously thought. This alone is enough to promote much further study into both quorum sensing and the activity of the molecule Rsal on that function.