Benoit Polack

Activation of the Pseudomonas aeruginosa Type III Secretion System Requires an Intact Pyruvate Dehydrogenase aceAB Operon

ABSTRACT Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.

Alveolar Response to Pseudomonas aeruginosa: Role of the Type III Secretion System

ABSTRACT The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1β, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.

PsrA Is a Positive Transcriptional Regulator of the Type III Secretion System in Pseudomonas aeruginosa

ABSTRACT The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced in vivo upon contact with eukaryotic cells and in vitro by calcium depletion in culture medium. We have observed a previously identified protein, PsrA, necessary for full activation of TTSS gene expression in P. aeruginosa. Electrophoretic mobility shift assays showed that recombinant PsrA could bind to the exsCEBA promoter region. A mutant with a deletion in the psrA gene was constructed. Using transcriptional fusions, we demonstrated that PsrA is required for the full activation of transcription of the TTSS regulatory operon exsCEBA and effector exoS, although the deletion mutant still responded to calcium depletion, to serum, and to host cell contact. The psrA mutant showed a marked decrease in the secretion of the type III effectors and weak resistance to phagocyte-like PLB-985 cells. The defect in TTSS transcription and secretion in the psrA mutant could be complemented by expression in trans of psrA. PsrA was previously identified as a transcriptional activator of RpoS, a central regulator during stationary phase. We confirmed with our strain that RpoS has a negative effect on TTSS gene expression. Taken altogether, these results suggest that PsrA is a newly identified activator that is involved in the expression of the TTSS by enhancing the exsCEBA transcriptional level.

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.

Live-attenuated bacteria as a cancer vaccine vector

In the emerging field of active and specific cancer immunotherapy, strategies using live-attenuated bacterial vectors have matured in terms of academic and industrial development. Different bacterial species can be genetically engineered to deliver antigen to APCs with strong adjuvant effects due to their microbial origin. Proteic or DNA-encoding antigen delivery routes and natural bacterial tropisms might differ among species, permitting different applications. After many academic efforts to resolve safety and efficacy issues, some firms have recently engaged clinical trials with live Listeria or Salmonella spp. We describe here the main technological advances that allowed bacteria to become one of the most promising vectors in cancer immunotherapy.

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.

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

ABSTRACT During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8+ T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA-deficient mutant was the most efficient at a low dose (105), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (106), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (107), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.

High-Yield Production of Secreted Active Proteins by the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.

Antithrombin-Independent Effects of Heparins on Fibrin Clot Nanostructure

Objective—Because of the widespread clinical use of heparins, their effects on the enzymatic cascade are very well known. In contrast, little is known about the direct effect of heparins on the nanostructure of fibrin fibers, even though this nanostructure plays a major role in the mechanical strength and lysis of clots. This lack of reliable data can be correlated with the lack of a nonintrusive, quantitative method to determine this structure. We recently developed such a method that allows the simultaneous determination of the average fiber radius and the protein content using spectrometric data. In this study, we assessed the nanostructure of fibrin in a system composed of human thrombin and fibrinogen. Methods and Results—We provide quantitative evidence showing that both unfractionated heparin and low molecular weight heparin directly alter the nanostructure of fibrin fibers independent of their other actions on the coagulation cascade; as expected, the pentasaccharide fondaparinux has no effect. Conclusion—Our results show that in addition to the effect of heparin on the coagulation cascade, modifications of the fibrin nanostructure may also contribute to improved fibrinolysis.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRP-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2125-243, TRP2L125-376 and TRP2S291-376. However, only immunization with TRP2L125-376 induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8+ epitopes, and strong CD4+ Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapide screening and evaluation of antigens of varying length and epitope composition.