Scavenging of reactive oxygen species by tryptophan metabolites helps Pseudomonas aeruginosa escape neutrophil killing.

Pseudomonas aeruginosa is responsible for persistent infections in cystic fibrosis patients, suggesting an ability to circumvent innate immune defenses. This bacterium uses the kynurenine pathway to catabolize tryptophan. Interestingly, many host cells also produce kynurenine, which is known to control immune system homeostasis. We showed that most strains of P. aeruginosa isolated from cystic fibrosis patients produce a high level of kynurenine. Moreover, a strong transcriptional activation of kynA (the first gene involved in the kynurenine pathway) was observed upon contact with immune cells and particularly with neutrophils. In addition, using coculture of human neutrophils with various strains of P. aeruginosa producing no (ΔkynA) or a high level of kynurenine (ΔkynU or ΔkynA pkynA), we demonstrated that kynurenine promotes bacterial survival. In addition, increasing the amount kynurenine inhibits reactive oxygen species production by activated neutrophils, as evaluated by chemiluminescence with luminol or isoluminol or SOD-sensitive cytochrome c reduction assay. This inhibition is due neither to a phagocytosis defect nor to direct NADPH oxidase inhibition. Indeed, kynurenine has no effect on oxygen consumption by neutrophils activated by PMA or opsonized zymosan. Using in vitro reactive oxygen species-producing systems, we showed that kynurenine scavenges hydrogen peroxide and, to a lesser extent, superoxide. Kynurenine׳s scavenging effect occurs mainly intracellularly after bacterial stimulation, probably in the phagosome. In conclusion, the kynurenine pathway allows P. aeruginosa to circumvent the innate immune response by scavenging neutrophil reactive oxygen species production.

Optimization of antitumor immunotherapy mediated by type III secretion system-based live attenuated bacterial vectors.

Recently, due to their effective ability to deliver antigen to antigen-presenting cells in vivo, type III secretion system-based attenuated bacterial vectors have increasingly attracted attention for their potential interest in cancer vaccine development. We have previously developed live attenuated Pseudomonas aeruginosa type III secretion system-based vectors to deliver in vivo tumor antigens. In this work, we improved the performance of these bacterial vectors through several approaches in different murine cancer models involving non-self-antigens or self-antigens. First, by modulating injection frequency and interval, bacterial vaccination-activated immune response could be enhanced and the in vivo therapeutic efficacy of bacterial vaccines could be improved. The optimized vaccination scheme induced long-lasting CD8+ T cells' response. Second, a dual antigen delivery pattern was successfully applied in our bacterial vectors. Compared with a single antigen delivery vector, biantigen delivery vectors demonstrated several advantages including better tumor rejection efficiency, simplicity of use, and safety. Third, 1 more attenuated mutant-CHA-OAL strain that is totally avirulent in mice was further adapted to grow in a chemically defined medium to comply with current good manufacturing processes. The poor infectivity of this new strain could be overcome by vaccinations at multiple loci, yielding an efficiently improved vaccination performance. Taken together, our results highlight the potential of our live attenuated P. aeruginosa vectors for applications in relevant clinical trials.

Full virulence of Pseudomonas aeruginosa requires OprF.

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.

High-cell-density regulation of the Pseudomonas aeruginosa type III secretion system: implications for tryptophan catabolites.

The Pseudomonas aeruginosa type III secretion system (T3SS) is known to be a very important virulence factor in acute human infections, but it is less important in maintaining chronic infections in which T3SS genes are downregulated. In vitro, the activation of T3SS expression involves a positive activating loop that acts on the transcriptional regulator ExsA. We have observed that in vivo T3SS expression is cell density-dependent in a manner that does not need known quorum-sensing (QS) signals. In addition, stationary-phase culture supernatants added to exponential-phase growing strains can inhibit T3SS expression. The analysis of transposon insertion mutants showed that the production of such T3SS-inhibiting signals might depend on tryptophan synthase and hence tryptophan, which is the precursor of signalling molecules such as indole-3-acetic acid (IAA), kynurenine and Pseudomonas quinolone signal (PQS). Commercially available tryptophan-derived molecules were tested for their role in the regulation of T3SS expression. At millimolar concentrations, IAA, 1-naphthalacetic acid (NAA) and 3-hydroxykynurenine inhibited T3SS expression. Inactivation of the tryptophan dioxygenase-encoding kynA gene resulted in a decrease in the T3SS-inhibiting activity of supernatants. These observations suggest that tryptophan catabolites are involved in the downregulation of T3SS expression in the transition from a low- to a high-cell-density state.