SMALL RNAS AND REGULATION OF VIRULENCE TRAITS IN PSEUDOMONAS AERUGINOSA

This PhD project aimed at the functional characterization of novel Pseudomonas aeruginosa small RNAs (sRNAs) involved in virulence-associated regulatory networks. Following an initial phase of screenings for sRNA candidates involved in the regulation of virulence traits, the work focused on the sRNAs SPA0122 and SPA0084. SPA0122, renamed ErsA, is transcribed from the same genomic context of the well-known Escherichia coli Spot 42. We show that, different from Spot 42, ErsA is under the transcriptional control of the envelope stress response, which is known to impact the pathogenesis of P. aeruginosa through the activity of the alternative sigma factor σ22. The transcriptional responsiveness of ErsA RNA also spans infection-relevant cues that P. aeruginosa can experience in mammalian hosts, such as limited iron availability, temperature shifts from environmental to body temperature and reduced oxygen conditions. Another difference between Spot 42 and ErsA is that ErsA does not seem to be involved in the regulation of carbon source catabolism. Instead, our results suggest that ErsA is linked to anabolic functions for the synthesis of exoproducts from sugar precursors. We show that ErsA directly operates in the negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several P. aeruginosa polysaccharides. Like ErsA, the activation of algC expression is also dependent on σ22. Altogether, our results suggest that ErsA and σ22 combine in an incoherent feed-forward loop to fine-tune AlgC enzyme expression. As regards SPA0084, our results indicate that is embedded in the P. aeruginosa quorum sensing (QS) with the role of wiring las to pqs systems. In fact, we show that SPA0084 responds to the las regulator LasR and impacts positively the synthesis of the pqs quinolone signal PQS. Our results suggest that the stimulation of PQS synthesis is mediated by a positive post-transcriptional effect of SPA0084 on the pqsC gene belonging to the pqsABCD cluster involved in the biosynthesis of the PQS-precursor HHQ. We suggest that a fine balancing between the different already known regulatory effects of LasR on PQS synthesis and this one mediated by SPA0084 can influence timing and magnitude of expression of QS-regulated virulence factors. This view is consistent with the evidence that perturbations of SPA0084 levels affect pyocyanin synthesis, biofilm formation and swarming motility, processes that are known to be influenced by PQS synthesis. Besides being regulated by LasR, SPA0084 responds to infection relevant cues that P. aeruginosa can experience in mammalian hosts such as temperature and oxygen availability. Furthermore, SPA0084 shows a growth phasedependent pattern of expression, being up-regulated in stationary phase. Sequence analysis of the spa0084 promoter region strongly suggests that the growth phase-dependent pattern of SPA0084 expression is due to the activity of the alternative σ factor RpoS whose significance as global factor controlling quorum sensing gene expression was shown previously. Together, these SPA0084 regulations are expected to contribute to the fine co-modulation of PQS synthesis.