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CARDOSO, A. R. Construction of a global map of protein-protein interactions in c-diGMP signalling pathways of Pseudomonas aeruginosa. 2016. 107 p. Dissertacao (Mestrado em Ciencias) Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Sao Carlos, 2016. Persister bacteria are correlated to biofilm formation and have been a source of great medical concern due to its close association with the impairment of traditional treatment in combating chronic infections. On the other hand, using bacterial biofilms to create original biotechnological applications or even as a means of therapeutic treatment in medical settings constitutes a promising prospect. There is, therefore, a great interest in understanding the mechanisms that allow bacteria to leave the free-living planktonic lifestyle and associate in these highly complex cellular aggregates. Over the last decades, the second messenger c-diGMP – and also the molecules involved in its synthesis (diguanylate ciclases) and degradation (phosphodiesterases) along with its receptors – has been established as a key element implicated in regulation of a series of cellular responses that determine biofilm formation or dispersion. Curiously, the proteins that play a part in the metabolism of this second messenger are frequently coded multiple times in single bacterial genomes. Taking this into account, recent studies indicate that, in order to control such a wide range of phenotypes, this system operates via high specificity of signaling – which means that the signal metabolized by a certain set of diguanylate ciclases and phosphodiesterases has specific cellular targets. Robust but yet isolated evidence indicate that a means by which a signal is segregated with its correlated phenotypic response is through direct protein-protein interaction involving the components of these signaling pathways. Even more, there has been strikingly evidence that, in some of these pathways, signal transduction occurs exclusively through protein-protein interaction, entirely dismissing any mediation by the signal molecule. In order to validate and evaluate the global relevance of this type of mechanism, this study proposed the investigation of the entire network of interactions between proteins typically associated with c-di-GMP signaling pathways of Pseudomonas aeruginosa by employing bacterial two-hybrid system assays. To make that possible, two DNA libraries were constructed and interaction essays were performed in a strategic way so that all possibilities of interaction between target proteins were explored. The results obtained from these experiments allowed the construction of a broad map of interactions that, although still primitive, indicates that, chances are, the mechanisms previously described are both recurrent and relevant to signaling regulation in this organism. Some of the interaction partners found are particularly interesting and will be further investigated in future studies.

Mapeamento global de interações proteicas nas vias de sinalização mediadas por c-di-GMP de <i>Pseudomonas aeruginosa</i>