In vivo genetic analysis of Pseudomonas aeruginosa carbon catabolic repression through the study of CrcZ pseudo-revertants shows that Crc-mediated metabolic robustness is needed for proficient bacterial virulence and antibiotic resistance

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Abstract
Pseudomonas aeruginosa is an opportunistic bacterial pathogen able to colonize a variety of habitats. Its success in colonizing these habitats relies on its metabolic robustness and its capability of efficiently using available carbon sources in a hierarchical way. P. aeruginosa carbon catabolic repression is post-transcriptionally regulated by Hfq and Crc, which form a complex that binds and impedes the translation of their target mRNAs. Under no catabolic repression conditions, the complex is sequestered by the small RNA CrcZ, allowing the translation of the involved mRNAs. In addition to regulating carbon sources use, Crc and Hfq modulate P. aeruginosa virulence and antibiotic resistance. In the absence of CrcZ, catabolic repression should be constitutive, severely impairing P. aeruginosa fitness. A Δ crcZ mutant was generated. As predicted, it presents severe fitness defects and alterations in virulence and antibiotic resistance. Pseudo-revertants that restore P. aeruginosa fitness, antibiotic resistance, and virulence were selected. Notably, most pseudo-revertants presented mutations in crc , despite Hfq, not Crc, being the RNA-binding protein of the complex. The analysis of several traits, including antibiotic resistance and bacterial virulence of these mutants, indicates that they can be grouped into two categories, those in which Crc is fully inactivated and those presenting smaller structural changes. The phenotypes of the latter resembling those of the wild-type strain. Notably, even when Hfq is not sequestered by CrcZ, in the Δ crcZ mutant, the lack of Crc impedes a proficient catabolic repression, indicating that Crc is strictly required for keeping P. aeruginosa metabolic robustness, virulence, and antibiotic resistance. Iimportance: Hfq and Crc regulate P. aeruginosa carbon catabolic repression at the post-transcriptional level. In vitro work has shown that Hfq binds the target RNAs and Crc stabilizes the complex. A third element in the regulation is the small RNA CrcZ, which sequesters the Crc-Hfq complex under no catabolic repression conditions, allowing the translation of the target mRNAs. A Δ crcZ mutant was generated and presented fitness defects and alterations in its virulence potential and antibiotic resistance. Eight pseudo-revertants that present different degrees of fitness compensation were selected. Notably, although Hfq is the RNA binding protein, most mutations occurred in Crc. This indicates that Crc is strictly needed for P. aeruginosa efficient carbon catabolic repression in vivo . The compensatory mutations restore in a different degree the alterations in antibiotic susceptibility and virulence of the Δ crcZ mutant, supporting that Crc plays a fundamental role linking P. aeruginosa metabolic robustness, virulence, and antibiotic resistance.

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