P. aeruginosa is an opportunistic pathogen capable of infecting a wide host range. It possesses a large arsenal of virulence factors. The type III secretion system (TTSS) is a major virulence factor whose regulation is complex to allow the most accurate adaptation of the bacteria during infection. We were interested to determine the potential role of new actors in the adaptation of P. aeruginosa during infection. OprF represents the most abundant protein of the outer membrane of P. aeruginosa. This protein allows bacteria to assess the activation status of the host's immune system to adapt its virulence. In P. aeruginosa, tryptophan is the precursor of kynurenines that are also produced by the host from tryptophan and in the latter context, are immunomodulators. Little or no studies have been done to determine a possible role of bacterial kynurenines in immune modulation or virulence. Initially, we were interested in a signal previously discovered in the laboratory and which suppresses the expression of TTSS at high bacterial density. We have shown that this signal exerts a post-transcriptional regulation in addition to inhibition of TTSS genes transcription. The metabolism of tryptophan and anthranilate appears to be at the heart of this regulatory process. By inactivating pathways of tryptophan catabolism, we showed that production of this signal depends partly on the kynurenines pathway but does not depend neither classical ways of quorum sensing or phnAB operon involved in the synthesis of anthranilate. However, the phenazines pathway could be involved in the production of this signal. By HPLC coupled with mass spectrometry, we were able to separate molecular species suppressing the TTSS and which are contained in this signal, but accurate identification requires further investigation. In a second time, we were interested to kynurenines produced by the bacterium. We confirmed that P. aeruginosa produces kynurenines and KynA is the key gene in the synthesis of these metabolites. We showed that tryptophan and kynurenine upregulate the production of kynurenines by acting on the expression of key genes. Other shares, we found that the bacterium modulates the activity of the kynurenines pathway depending on its state of growth. We showed that during the dialogue bacteria / host, the pathway of kynurenines in P. aeruginosa is stimulated by certain immune system components. With an acute lung infection model, we proved that kynurenines produced by the bacterium are important to its virulence. We hypothesized that the kynurenines could have an effect on the immune response, but this remains to be determined. In a third time, we focused on the protein OprF. We showed that mutation ΔoprF is causing an alteration in production but probably not the secretion of TTSS exotoxins. One known ligand of OprF is the gamma interferon. It modulates the pathway of kynurenines. OprF could therefore have a central role in various aspects of the regulation of virulence. So, we produced monoclonal anti-OprF which recognizes specifically the protein OprF. To verify the effectiveness of these antibodies, neutralization experiments of the bacteria in vitro and in vivo will be realized.
