Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gramnegative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysinengineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria. Development of novel antibacterials against Gram-negative bacteria is challenging because they possess an outer membrane that prevents many compounds from reaching their intracellular targets 1. Bacteriophages (phages), viruses that infect bacteria, have naturally evolved mechanisms to overcome the outer membrane to infect their bacterial hosts 2,3. In the first step of infection, phages bind to host cells and inject their genetic material across the outer and inner membrane of the bacterial cells into the cytoplasm 4,5. Also, during the final stage of the lytic infection cycle, phages produce proteins within the cell, which destroy the bacterial cell wall, leading to cell lysis 6,7. Thus, the molecular tools developed during phage evolution may be exploited to develop novel phagebased antibacterials that are able to pass the outer membrane and to kill Gram-negative bacteria. Phages recognize their host bacteria by binding to specific surface receptors that may be outer membrane proteins, lipopolysaccharides or components of bacterial capsules, pili and flagella 8-10. The adhesion specificity is mediated by receptor binding proteins (RBPs) that form fibers or spikes at the distal phage tail. A wellcharacterized RBP is the monomeric Pb5, located at the tail tip of the phage T5, which binds irreversibly to the bacterial receptor FhuA during infection of the E. coli host 11,12. FhuA is an outer membrane protein that ...

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections.

Extracellular vesicles large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, evFOUNDRY and VES4US, organized a workshop entitled "massivEVs" to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during "massivEVs", the most important points raised, and the points of consensus reached after active discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing and upscaling challenges and the directions for their resolution highlighted in the workshop was constructive and painted an optimistic future for the dynamically expanding EV field.

Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a major cause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, we describe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independent A. pleuropneumoniae immunogens. Potential immunogens were identified in the complete genomes of three A. pleuropneumoniae strains belonging to different serovars using the following parameters: predicted outer-membrane subcellular localization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in all known A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regarding immunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressed and further characterized, both in silico and in vitro. Additionally, we analysed outer membrane vesicles (OMVs) of A. pleuropneumoniae MIDG2331 as potential immunogens, and compared deletions in degS and nlpI for increasing yields of OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturally expressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ were shown to possess a high immunogenic potential in vitro. These findings favour the immunogen selection protocol used, and suggest that OMVs, along with ApfA and VacJ, could represent effective immunogens for the prevention of A. pleuropneumoniae infections in a serovar-independent manner. This hypothesis is nonetheless predictive in nature, and in vivo testing in a relevant animal model will be necessary to verify its validity.Electronic supplementary materialThe online version of this article (10.1186/s13567-017-0479-5) contains supplementary material, which is available to authorized users.

Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns.