Quorum sensing and pyoverdine signaling :possible targets for the development of inhibitors of Pseudomonas aeruginosa virulence

Abstract

Pseudomonas aeruginosa is a major opportunistic pathogen in the hospital setting, and P. aeruginosa chronic lung infection is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa infections are difficult to eradicate by conventional antibiotic therapy due to the inherent antimicrobial resistance of the bacterium and its propensity to acquire new resistances. The search for new drugs capable of inhibiting specific virulence-related traits of P. aeruginosa is a promising strategy to face with infections caused by multidrug-resistant P. aeruginosa. This approach offers the advantage of reducing the bacterial adaptability to the host environment, thus providing the host immune system with a better chance of clearing the infection without creating the selective pressure generally caused by conventional antibiotics. In P. aeruginosa, quorum sensing (QS) and pyoverdine signaling systems regulate a number of virulence phenotypes which are critical for P. aeruginosa pathogenicity, and therefore represent ideal targets for the development of novel anti-Pseudomonas drugs. QS is a cell-density dependent global regulatory system which relies in the production of a signal molecule that accumulates in the growth medium in a way that is proportional to the cell density. P. aeruginosa has three interconnected QS systems, namely las, rhl and pqs, based on the production of distinct signal molecules and organized in a hierarchical fashion with the las QS system on top. In the whole, these QS systems regulate ca. 10% of P. aeruginosa genes, including several virulence genes. Pyoverdine is a high-affinity iron chelator (siderophore) which binds Fe(III) in the extracellular milieu and delivers it to the cell through receptor-mediated active transport. Pyoverdine also acts as signal molecule, enhancing the expression of a number of P. aeruginosa virulence factors. The aims of my PhD work were the development of novel high-throughput screening systems to be used for the detection of compounds able to inhibit QS and pyoverdine signaling and, subsequently, the characterization of the effects on P. aeruginosa virulence of the molecules identified by the screening experiments. With regard to the screening system for QS inhibitors, a reporter system (PA14-R3) was developed, consisting in a P. aeruginosa lasI defective mutant, unable to produce the N-3-oxo-dodecanoyl-homoserin lactone (3OC12-HSL) signal molecule and carrying the lux bioluminescence genes under the control of the rsaL promoter, inserted as a single copy in a neutral site of the P. aeruginosa chromosome. PA14-R3 was able to detect 3OC12-HSL concentrations in a 150 pM - 3 μM range, and showed high specificity towards 3OC12-HSL, while not being significantly influenced by other acyl-HSLs produced by P. aeruginosa or by other bacterial pathogens that can coexist with P. aeruginosa in the lung of CF patients. The wide range of response and the high sensitivity and specificity of PA14-R3 made this biosensor a convenient tool for the direct micro-volumetric determination of 3OC12-HSL levels in P. aeruginosa laboratory cultures as well as in clinical samples, by non-linear interpolation from a standard curve obtained with purified 3OC12-HSL. The PA14-R3 biosensor was efficiently employed for direct measurements of the levels of 3OC12-HSL in culture supernatants of P. aeruginosa strains that differ in 3OC12-HSL production and for the quantification of 3OC12-HSL in sputa from CF patients with known history of colonization by P. aeruginosa. A novel screening system for QS inhibitors (QSI) was developed, based on the co-cultivation of PA14-R3 with the P. aeruginosa wild-type strain PA14. In this system, the wild-type strain provides the PA14-R3 biosensor with the 3OC12-HSL required for luminescence emission. The addition of a molecule with inhibitory activity towards any process related to QS would reduce the emission of luminescence by the biosensor with respect to a control co-culture without any inhibitor added. This system would allow the detection of any inhibitory compound targeting one or more of the following steps: i) expression/activity of signal molecule biosynthesis enzymes; ii) expression/activity of signal receptors; iii) import/export of signal molecules. The PA14-R3/PA14 co-culture was validated as screening system for QSI compounds by testing the response of the system to furanone C30, a well-known QSI, and subsequently employed in a pilot screening for potential QSI in a commercial chemical library consisting of 1,120 FDA-approved drugs, selected for their high chemical and pharmacological diversity as well as for their known bioavailability and safety in humans. The screening led to the identification of a number of QS-inhibiting compounds, among which a teniacide drug (QSI-1), able to cause a decrease in the 3OC12-HSL production of about 50% and active at physiologically meaningful concentrations. The ability of QSI-1 to equally reduce the bioluminescence emission in both a PA14-R3/PA14 co-culture and a culture of PA14-R3 alone in presence of exogenously added 3OC12-HSL suggested that QSI-1 was likely to inhibit the sensing, rather than the synthesis or import/export of the signal molecule. In addition to 3OC12-HSL, QSI-1 also showed a relevant inhibitory effect on the production of the rhl QS signal molecule, namely C4-HSL. The effect of QSI-1 on the production of several QS-dependent virulence determinants in P. aeruginosa was thus investigated. The presence of a 10 μM QSI-1 in the culture medium determined a strong reduction of pyocyanine, rhamnolipids, and elastase production. Finally, the QSI-1 was tested for its ability to inhibit P. aeruginosa virulence in the Galleria mellonella (major wax moth) model of infection. A concentration of 15 μM QSI-1 almost completely abrogated the pathogenicity of P. aeruginosa in larvae inoculated with a lethal dose of P. aeruginosa PA14. A screening system for pyoverdine signaling inhibitors (PSI) was developed, consisting in a P. aeruginosa PAO1 strain carrying the lux genes under the control of the signaling-responding pvdE promoter. In this system, the addition of a molecule with inhibitory activity towards pyoverdine signalling and/or pyoverdine synthesis would determine a decrease in the emission of luminescence by the biosensor and in pyoverdine-specific fluorescence. This system was used to screen a commercial chemical library for putative PSI, leading to the identification of an antimycotic drug (PSI-1) capable of reducing pyoverdine signaling and pyoverdine production in P. aeruginosa PAO1 by > 50%. In addition to the type I pyoverdine produced by P. aeruginosa PAO1, PSI-1 was also shown to reduce pvdE transcription and pyoverdine synthesis in type II and III pyoverdine-producing P. aeruginosa strains. A preliminary investigation was conducted to tentatively determine the molecular target(s) of PSI-1. PSI-1 was active against selected mutant strains impaired in different steps of the pyoverdine signaling pathway, suggesting that the inhibitory activity of PSI-1 does not depend on inhibition of pyoverdine signaling. Further experiments showed that PSI-1 was capable of reducing the transcription of the pvdS gene, encoding the alternative sigma factor PvdS, which is a central regulator essential not only for pyoverdine production but also for the expression of a number of P. aeruginosa virulence genes. PSI-1 at 10 μM concentration determined a relevant decrease in both transcription and expression of (i) pyoverdine synthesis genes, (ii) the toxA gene, which encodes the exotoxin A, a major determinant of P. aeruginosa virulence, and (iii) the prpL gene, encoding the extracellular protease PrpL. Further experiments are in progress to assess the effect of QSI-1 and PSI-1 in a mouse model of infection. The evidence that QSI-1 and PSI-1 are effective in inhibiting crucial signaling processes for P. aeruginosa virulence and consequently reduce the expression of many virulence factors of P. aeruginosa sets the basis for further investigation focused on the characterization of the mode of action these drugs with the ultimate aim of using these compounds for the development of an anti-virulence strategy against P. aeruginosa infection.