Participation of the pvd Gene Cluster in the Mechanism of Quorum Sensing and Virulence of Pseudomonas aeruginosa PAO1

Abstract

Objective Determination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes. Methods Supernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains. Abstract Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.