Impacto del regulador global Crc en la fisiología de P. aeruginosa

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen which causes serious infections in immunocompromised patients or in those with previous pathologies, such as cystic fibrosis. It is also an ubiquitous and versatile microorganism that lives in different environments such as the sea water or the rhizosphere. One of the regulatory mechanism that allows the environmental adaptation of P. aeruginosa is the catabolite repression, which is the regulatory mechanism that allows a hierarchical assimilation of carbon sources in complex media. Crc is the main regulator of catabolite repression in P. aeruginosa, and its inactivation causes pleiotropic changes in phenotypes related not just with metabolism, but with virulence and antibiotic resistance also. Crc acts with Hfq being both post‐transcriptional repressors of the target genes. When there is a preferential carbon source in the media, Crc represses the translation of the target genes that encode enzymes, transporters and regulators involved in the assimilation of secondary carbon sources. When the preferential carbon source is consumed, a small RNA, CrcZ is expressed, sequestering the Crc/Hfq complex. The mRNA is freed and the protein which participates in the assimilation of the secondary carbon source is translated. However, the mechanism of molecular interactions among Crc, Hfq and CrcZ remains to be elucidated in full. In this PhD Thesis, we go in depth inside the knowledge in the impact of Crc in bacterial physiology, and we characterized in greater detail the regulation through this system. To distinguish the direct and indirect regulation by Crc, a transcriptome and a proteome of a Δcrc mutant were performed. Post‐transcriptional regulation of Crc over 2000 genes was characterized in detail, leading to a precise map of the post‐transcriptional action of Crc protein, including its role in the central metabolism. The regulation of Crc over the enzyme Zwf and the EDEMP cycle is an important element that maintains the homeostasis of the cellular redox state and an adequate response to oxidative stress. Moreover, the targets of Crc include several transporters and enzymes of secondary carbon sources and the proteins that belong to the systems of iron uptake. The specific impact of Crc in the secretion of proteinaceous virulence factors was analyzed by the analysis of the secretome of a Δcrc mutant in two fractions corresponding to the proteome of the outer membrane vesicles secretome and the vesicles‐free secretome. The Δcrc mutant presents more outer membrane vesicles and those are bigger. The virulence factors, ToxA, CbpD, PiV and Hcp1 are present in less amount in the Δcrc exoproteome than in the wildtype one. Another secreted virulence factor is the type three secretion system. The Δcrc mutant presents less type three secretion proteins in the secretome, but does not show a defect on their production, indicating that the Δcrc mutant presents a defect in the export of type three secretion proteins. As a result, the Δcrc mutant is less virulent and cytotoxic than the wild type strain. A strategy based on suppressor mutations to genetically define the regulatory network of Crc was used to track more precisely the Crc regulation network. Pseudorevertants of the Δcrc and the ΔcrcZ mutants were selected with different degrees of catabolite repression. All the mutations were in Hfq and in Crc, which demonstrates that Hfq, Crc and CrcZ are functionally linked. The different variants selected in our screening might be used to characterize the interaction between these elements.