Identification and characterization of regulatory networks controlling the expression and activity of the alternative sigma factor pvds in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is one of the most dreaded opportunistic pathogens in the hospital setting and represents the first cause of morbidity and mortality in cystic fibrosis patients. P. aeruginosa is able to produce several virulence factors; among them, the siderophore pyoverdine plays a critical role in P. aeruginosa pathogenicity. This siderophore is not only the main iron uptake system of P. aeruginosa but also a signaling molecule which promotes the expression of other virulence factors (i.e. exotoxin A, extracellular protease PrpL). The key role of pyoverdine in P. aeruginosa infections makes this siderophore a promising target for the development of anti-virulence drugs. The antimicrobial agents currently used in clinical practice induce a strong selective pressure which causes the development and spreading of resistant bacteria. An alternative antibacterial strategy, which should reduce the emergence of resistance, is the development of anti-virulence drugs able to disarm pathogens without inhibiting their growth. In the first part of this PhD thesis the use of a drug repurposing approach to search for anti-pyoverdine compounds is described. This approach is based on the search for side activities in old drugs already used in humans. A library of 1,120 marketed drugs was screened with a purpose-generated reporter strain, leading to the identification of a promising hit compound, the antimycotic drug flucytosine, which strongly reduced pyoverdine production in vitro without affecting bacterial growth. The anti-virulence activity of flucytosine was also confirmed in vivo in a mouse model of lung infection. This work provided the first evidence that pyoverdine inhibition is a suitable strategy for anti-virulence therapy against P. aeruginosa, and that drug repurposing is a cheap and rapid approach to search for novel anti-virulence compounds. The prominent role of pyoverdine in P. aeruginosa virulence emphasized the importance of knowledge of pyoverdine regulation full understand P. aeruginosa pathogenicity. Previous transcriptomic studies suggested that the Gac system, which regulates the switch between the planktonic and biofilm lifestyles, could also influence pyoverdine gene expression in P. aeruginosa, although different studies obtained opposite results. In the second part of this PhD thesis the role of Gac system in the regulation of pyoverdine production has been investigated. It has been demonstrated that the Gac system and high intracellular levels of the intracellular signaling molecule bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) coordinately promote pyoverdine production. In more detail, these two systems indirectly control pyoverdine genes by modulating the expression of the operons encoding the enzymes involved in the synthesis of the two aggregative exopolysaccharides Pel and Psl. The deletion of both the pel and psl operons caused a strong reduction in the production of pyoverdine and pyoverdine-dependent virulence factors, irrespective of the Gac activation state or the intracellular c-di-GMP levels, indicating that these exopolysaccharides plays an important role in the regulation of this siderophore. We also found that the effect of Pel and Psl on pyoverdine production depends on their ability to support the formation of planktonic aggregates, rather than on the exopolysaccharides per se. Indeed, we observed that the simulation of aggregation in a Pel- and Psl-independent manner is able to restore pyoverdine production in the exopolysaccharide-null mutant. These results indicate that pyoverdine, and consequently, pyoverdine-dependent virulence factors are also controlled by a new regulatory system activated by the cellular aggregation. In conclusion, this thesis shows that cell aggregation is not only important in the first stages of biofilm formation, but also in the regulation of virulence in P. aeruginosa. The formation of cellular aggregates depends on the production of the Pel and Psl exopolysaccharides, which are regulated by the Gac system. Therefore, given the role of Gac also in the activation of pyoverdine-dependent virulence, this system could represent a potential target to inhibit simultaneously biofilm formation and the production of several virulence factors. The drug repurposing approach was thus used to search for Gac inhibitors. Although the initial screening led to identify three promising anti-Gac compounds, subsequent experiments revealed that these molecules promote, rather than inhibit, biofilm formation in P. aeruginosa, in a Gac-independent manner. Consequently, they were not further investigated as potential anti-virulence drugs.