Molecular Microbiology Rsal Provides Quorum

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.

RESULTS

The first noticeable result was at first disappointing but turned around later in the trail. At first the rsaL mutation did not have any effect on the production of 30C12-HSL nor on lasl transcription, but it began to appear later in the logarithmic phase of propagation at which time the 30C12-HSL production along with Plasl activity reached homoeostasis in the wild strain while continuing to increase rsaL in the mutant strain of the pathogen. However the sturdy found that:

The increase in rsaL level coincides with the point at which the production of 30C12-HSL diverges in the parent and mutant strains. This is consistent with the conclusion that Rsal induction by 30C12-HSL results in sufficient Rsal to keep 30C12-HSL production at a steady level, balancing the positive feedback action on lasl expression (Rampioni, et. al. 1558)

From this the authors concluded that Rsal is the key molecule that effects the homeostasis of 30C12-HSL production. This could provide the first step in creating the quorum sensing trigger thereby effecting, halting and possibly even reversing the virulent reproduction of the pathogen's population.

The latter part of the previous conclusion is pointed out by the study as well. They believe that further findings suggest that RsaL is a negative autoregulator for the quorum sensing process. Their research has found that the binding of Rsal upon a unique site in the lasl-rsal inergenic region does trigger the divergent transcription on both of the genes sequencing. They refer to this area as Plasl or Prsal dependent on the consideration of the pasl or rsal being considered. There fore their final conclusion of these results is, "that the major funciont of Rsal in P. aeruginossp physiology is to govern the homeostasis of 30C12-HSL by controlling, in concert with Lasl, the expression of rsal and lasl. Besides this Rsal is an integral part of the QS [quorum sensing] signaling network that controls gene expression though different and not mutually exclusive mechanisms… (Rampioni, et. al. 1562)