Regulation of quorum sensing and virulence in pseudomonas aeruginosa

Abstract

The asocial existence of the bacterial cells has been a major paradigm in microbiology for a long time. However, a huge amount of experimental evidences collected in the last 30 years has revealed that bacteria preferentially live in communities, in which the behavior of individual cells is coordinated by cell-cell communication systems to control phenotypic behaviors at the population level. Bacteria not only form well-organized communities, but they also exploit complex social cooperative and competitive interactions, mimicking multicellular organisms. Bacterial social life mainly relies on their ability to exchange information via chemical communication systems. In some cases, these systems allow a group of bacteria to trigger a unified and coordinated response to metabolic and environmental stimuli, so to accomplish tasks which would be difficult, if not impossible, to achieve for individual bacterial cells. One of the most studied bacterial cell-cell communication systems is quorum sensing (QS), by which a bacterial population coordinately reprograms gene expression in response to cell density. The QS-response is achieved when the concentration of a specific signal molecule, produced and secreted by single bacteria cells, reaches a threshold level, corresponding to a certain cell density, at which it is able to trigger a phenotypic and behavioral change in all the members of the bacterial population. QS in different bacteria is involved in the regulation of a wide variety of physiological processes, including competence, bioluminescence, antibiotic biosynthesis, motility, plasmid conjugal transfer, biofilm formation, and production of bacterial virulence factors in plant, animal and human pathogens. Furthermore, evidence has been accumulated that some bacterial signal molecules are used not only for intra-species communication, but also to exchange information between bacteria of different species or genera occupying the same ecological niche, and also to interact with their eukaryotic hosts. The QS system of the opportunistic human pathogen Pseudomonas aeruginosa is one of the best characterized bacterial communication systems, and it is now considered as a mayor model system for QS studies. In P. aeruginosa QS positively controls the expression of virulence related traits, such as virulence factor production and biofilm formation, and it is consequently involved in both acute and chronic infections. Moreover the QS circuit of P. aeruginosa is one of the most complex communication systems described up to date, since it is made up of four interconnected QS systems interwoven in an intricate regulatory network, the las, rhl, pqs and IQS systems. The four QS systems of P. aeruginosa are hierarchically organized and, in a wide range of cultural conditions, the las system is at the top of this hierarchy, being required for full activation of the other three QS circuits. Overall, the las QS system positively controls the complex regulative cascade involved in the expression of virulence-related phenotypes in P. aeruginosa, and for this reason it is considered one of the most promising target for the development of new anti-virulence drugs. Besides cell density, the las QS system is modulated by many regulators and signalling systems in response to metabolic and environmental cues. In particular, the timing and the extent of the QS response are finely modulated at different levels by a plethora of transcriptional and post-transcriptional regulators. It is believed that this fine-tuning of the las QS system might play a major role during P. aeruginosa infections, even though this hypothesis has been poorly investigated in mammalian models of infection so far. Despite in the last 20 years the huge efforts of the scientific community has lead to a refined knowledge of the molecular mechanisms controlling the expression of the las QS system in P. aeruginosa, our understanding of its regulation and actual role in the infection processes is far from complete. On these bases, the main aim of this PhD project has been to contribute to shed light on some unclear aspects of the las QS system regulation. In particular, this PhD work aimed at (i) identifying novel transcriptional regulators of the las QS system; (ii) evaluating the effect of a dysregulation in the timing and the extent of the las QS response on the ability of P. aeruginosa to establish both acute and chronic infections in a murine model system; (iii) investigating new emerging behavioral properties arising from the peculiar regulatory architecture of the las QS system. Briefly, the search for novel direct regulators of LasR, the QS signal molecule receptor that activates the las QS system, lead to the identification of a new QS transcriptional regulator PA3699. This protein directly represses lasR transcription, and consequent expression of QS-controlled virulence phenotypes in P. aeruginosa. The study of the effect of a dysregulation in the timing and extent of the QS response revealed that an anticipated activation of the las QS system does not significantly affect P. aeruginosa virulence in a mouse model of infection, while an increased activation of the las QS system beyond physiological levels impairs P. aeruginosa ability to establish chronic lung infections in mice. The arrangement of the genes composing the las QS system resembles the architecture of a network motif, the type-1 incoherent feedforward loop (IFFL-1), which is known to confer peculiar regulatory properties to the expression of its output genes. In line with this resemblance, we demonstrated that the las IFFL-1 confers robustness to the expression of its output genes with respect to possible fluctuations in the levels of the LasR activator. The main achievements of this PhD work have been published in two international peerreviewed journals (Longo et al., 2013; Bondí et al., 2014), and have been collected in an additional manuscript almost ready to be submitted (Bondí et al., manuscript in preparation).