Development of a dry, stable and inhalable acyl-homoserine-lactone-acylase powder formulation for the treatment of pulmonary Pseudomonas aeruginosa infections.

In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl-homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20°C and 55°C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55°C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 µm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation.

Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions.

PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.