摘要：

Pseudomonas aeruginosa and Burkholderia cenocepacia, beside their relevance as a nosocomial pathogens, cause lethal chronic lung infections in the vast majority of cystic fibrosis (CF) patients. Once established, the CF lung infection is impossible to eradicate with traditional antimicrobial therapies.
The main body of this project has been focused on P. aeruginosa. In this bacterium the Quorum Sensing (QS) system coordinates the production of virulence factors in a cell density-dependent manner via the secretion of specific signal molecules. During growth the bacteria secrete the signal molecules, which accumulate in the surrounding environment as the population density increases. When a threshold concentration is reached, the receptor is activated by the binding with the cognate signal molecule and triggers the expression of several genes, including virulence genes.
P. aeruginosa has two QS systems relying on the production of acylated- homoserine lactone (acyl-HSL) as signal molecules: the las and rhl systems. The lasI gene encodes the LasI synthase producing the N-3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) signal molecule, that binds to the LasR receptor encoded by lasR. The rsaL and qscR genes encode repressors of lasI and constitute an integral part of the las QS system, since they respond to 3-oxo-C12-HSL. The rhl system is organized similarly to the las one: the rhlI and rhlR genes encode, respectively, for the RhlI synthase and the RhlR receptor of the N-butyryl-homoserin lactone (C4-HSL) signal molecule. The signal-activated LasR and RhlR receptors regulate transcription of hundreds of genes, including the genes encoding a third QS system, based on the production of a different signal molecule, and the vast majority of virulence genes.
In P. aeruginosa many regulators have been found to affect the expression of the las and rhl genes, although their mechanism of action remains largely unknown. In particular, only the Vfr protein has been shown to directly activate lasR transcription. Besides las and rhl mutants, also a vfr mutant shows reduced virulence in murine models, suggesting that also the QS regulators could be feasible drug-targets.
The main objective of this project has been the identification and functional characterization of transcriptional factors that directly regulate the las and rhl genes. To achieve this objective, six DNA fragments corresponding to the promoters of lasR, lasI, rsaL, qscR, rhlR and rhlI were independently conjugated to a chromatography resin and incubated with protein crude extracts prepared from P. aeruginosa cultures. The proteins
able to specifically bind each DNA bait were recovered, separated by SDSPAGE
and identified by MALDI-TOF mass spectrometry.
Overall, 25 proteins bound on the promoter regions of the QS genes were
identified. Out of these, fifteen factors were selected for further analysis as
possible QS regulators. Noteworthy, the activator Vfr, already known to
directly bind the lasR promoter, was recovered using this promoter as a bait.
A set of fifteen P. aeruginosa mutants, each one deleted in one of the
genes encoding the selected proteins, was generated. In each mutant the
activity of correspondent target promoter was compared with that of the
wild type, by means of transcriptional fusions between the promoter region
of the target genes and the reporter system lux. In this system the production
of bioluminescence is proportional to the promoter activity. Among the
newly identified factors, we showed for the first time that the histon-like
proteins MvaT and MvaU directly control the transcription of the las genes.
In particular, MvaU is a repressor of both rsaL and lasI transcription, while
MvaT represses lasI and qscR transcription and activates lasR transcription.
MvaT and MvaU are global regulators of gene expression and on some
promoters can also interact

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