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Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections. Author summary: Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, and we still lack a clinically approved vaccine against this infection. The design of efficacious vaccines against Salmonella can benefit from studying overlooked aspects of host immune responses to this pathogen. The cell-to-cell communication facilitated by nano-sized extracellular vesicles (EVs) can affect the immune response. Our earlier work showed that Salmonella infection leads to the generation of immunogenic small EVs by macrophages. Here, we performed a proteomic and metabolomic analysis of the EV cargo, which indicated that EVs generated by Salmonella-infected macrophages stimulate macrophages and promote T-helper 1 (Th1) cell activation. We confirmed in vivo that mucosal administration of these EVs promotes a mucosal immune response against Salmonella infection in BALB/c mice. EVs from infected macrophages increased the CD4+ T cells secreting IL-2, IFN-γ, and TNF-α in response to Salmonella antigens, contributing to Th1 cell bias. These EVs not only stimulated antigen-specific cell-mediated immunity, but also contributed to anti-Salmonella antibody production, which included IgGs recognizing bacterial proteins present in EVs. Our study unraveled a novel role of EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria, thus supporting the future development of preventative approaches to combat Salmonella infection. [ABSTRACT FROM AUTHOR]

The development of new antimicrobial peptides has become an attractive alternative to conventional antibiotics due to the increasing rates of microbial drug resistance. Ib-M corresponds to a family of cationic synthetic peptides, 20 amino acids in length, that have shown inhibitory effect against the non-pathogenic strain Escherichia coli K-12. This work evaluated the antimicrobial potential of Ib-M peptides against the pathogenic E. coli O157: H7 using a reference strain and a clinical isolate. The Ib-M peptides showed antibacterial activity against both strains of E. coli O157: H7; the minimum inhibitory concentration of Ib-M peptides ranged from 1.6 to 12.5 μM and the minimum bactericidal concentration ranged from 3.7 to 22.9 μM, being Ib-M1 and Ib-M2 the peptides that presented the highest inhibitory effect. Time-kill kinetics assay showed a reduction of the bacterial population by more than 95% after 4 hours of exposure to 1xMIC of Ib-M1. Low cytotoxicity was observed in VERO cells with 50% cytotoxic concentration in the range from 197.5 to more than 400 μM. All peptides showed a random structure in hydrophilic environments, except Ib-M1, and all of them transitioned to an α-helical structure when the hydrophobicity of the medium was increased. In conclusion, these findings support the in vitro antimicrobial effect of Ib-M peptides against the pathogenic bacteria E. coli O157: H7 and prove to be promising molecules for the development of new therapeutic alternatives. [ABSTRACT FROM AUTHOR]