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Pseudomonas aeruginosa ( P.a ) is one of the most critical antibiotic resistant bacteria in the world and is the most prevalent pathogen in cystic fibrosis (CF), causing chronic lung infections that are considered one of the major causes of mortality in CF patients. Although several studies have contributed to understanding P.a within-host adaptive evolution at a genomic level, it is still difficult to establish direct relationships between the observed mutations, expression of clinically relevant phenotypes, and clinical outcomes. Here, we performed a comparative untargeted LC/HRMS-based metabolomics analysis of sequential isolates from chronically infected CF patients to obtain a functional view of P.a adaptation. Metabolic profiles were integrated with expression of bacterial phenotypes and clinical measurements following multiscale analysis methods. Our results highlighted significant associations between P.a "metabotypes", expression of antibiotic resistance and virulence phenotypes, and frequency of clinical exacerbations, thus identifying promising biomarkers and therapeutic targets for difficult-to-treat P.a infections.

Pseudomonas aeruginosa (Pa) is a significant cause of morbidity and mortality, especially in cystic fibrosis patients. Its eradication is difficult due to a wide phenotypic adaptability and an increase of its resistance to antibiotics. After the failure of several recombinant vaccines which mainly triggered humoral response, live-attenuated vaccines received attention thanks to their ability to elicit a broad immunity with both humoral- and cell-mediated responses, essential to fight this pathogen. In this study, we developed an innovative and safer live-attenuated Pa vaccine based on a Killed But Metabolically Active (KBMA) attenuation method. KBMA Pa has been further rationally designed to overexpress beneficial effectors like the type 3 secretion system apparatus. We demonstrated that KBMA Pa elicits a high and broad humoral response in mice against several antigens of particular interest such as OprF and PcrV proteins. Moreover, we assessed cytokines in the serum of immunized mice and showed that KBMA Pa elicits Th1, Th2 and especially Th17 pathways of cell-mediated immune responses. Th17 pathway involvement was also confirmed after specific stimulation of helper T cells in immunized mice. Finally, we showed that this vaccine is safe and has a protective effect in a murine acute pulmonary infectious challenge. In conclusion, KBMA Pa is a new platform with high potential for the development of a vaccine against Pa., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E. coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8 + T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

The use of bacterial vectors, which offer ease of production and efficiency, has become an important mechanism for the delivery of protein antigens to antigen-presenting cells (APCs) in vivo. Proof of concept studies has been carried out utilizing different bacteria in various cancer models with some in clinical trials. Here we described the way to prepare Pseudomonas aeruginosa (P. aeruginosa) vaccines based on a virulence-attenuated strain to test the efficacy of different fragments of a well-known tumor antigen. This protocol could be applied to efficacy studies in murine models of human cancers.

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.

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the "killed but metabolically active" (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.

Active immunotherapy based on live attenuated bacterial vectors has matured in terms of industrial development and develops through a combination of three phenomena. First, active immunotherapy that stimulates an antigen-specific cytotoxic T-cell immune response has become a reality after several years of work. Second, there is still a need to identify vectors that can deliver antigens to the cytosol of antigen-presenting cells in vivo. Third, the recent progress in the understanding of bacterial lifestyle and in developing genetic engineering tools has enabled the design of bioengineered bugs that are capable of delivering antigens. Here, we review the mechanisms by which clinical bacterial vectors deliver antigens into the cytosol of antigen-presenting cells and summarize the development strategy of the three identified firms in this field.

DivIB(FtsQ), FtsL, and DivIC(FtsB) are enigmatic membrane proteins that are central to the process of bacterial cell division. DivIB(FtsQ) is dispensable in specific conditions in some species, and appears to be absent in other bacterial species. The presence of FtsL and DivIC(FtsB) appears to be conserved despite very low sequence conservation. The three proteins form a complex at the division site, FtsL and DivIC(FtsB) being associated through their extracellular coiled-coil region. We report here structural investigations by NMR, small-angle neutron and x-ray scattering, and interaction studies by surface plasmon resonance, of the complex of DivIB, FtsL, and DivIC from Streptococcus pneumoniae, using soluble truncated forms of the proteins. We found that one side of the "bean"-shaped central beta-domain of DivIB interacts with the C-terminal regions of the dimer of FtsL and DivIC. This finding is corroborated by sequence comparisons across bacterial genomes. Indeed, DivIB is absent from species with shorter FtsL and DivIC proteins that have an extracellular domain consisting only of the coiled-coil segment without C-terminal conserved regions (Campylobacterales). We propose that the main role of the interaction of DivIB with FtsL and DivIC is to help the formation, or to stabilize, the coiled-coil of the latter proteins. The coiled-coil of FtsL and DivIC, itself or with transmembrane regions, could be free to interact with other partners.

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of a divIB deletion mutant of Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the Delta divIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the Delta divIB mutant in defined medium. Functional mapping of the three distinct alpha, beta, and gamma domains of the extracellular region of DivIB revealed that a complete beta domain is required to fully rescue the deletion mutant. DivIB with a truncated beta domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.

DivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer.