Insights into the regulatory role of bacterial sncRNA and its extracellular delivery via OMVs.

Small noncoding RNAs (sncRNAs) play important regulatory roles in bacterial physiological processes and host-pathogen interactions. Meanwhile, bacterial outer membrane vesicles (OMVs), as naturally secreted outer membrane structures, play a vital role in the interaction between bacteria and their living environment, including the host environment. However, most current studies focus on the biological functions of sncRNAs in bacteria or hosts, while neglecting the roles and regulatory mechanisms of the OMVs that encapsulate these sncRNAs. Therefore, this review aims to summarize the intracellular regulatory roles of bacterial sncRNAs in promoting pathogen survival by regulating virulence, modulating bacterial drug resistance, and regulating iron metabolism, and their extracellular regulatory function for influencing host immunity through host-pathogen interactions. Additionally, we introduce the key role played by OMVs, which serve as important cargoes in bacterial sncRNA-host interactions. We propose emerging pathways of sncRNA action to further discuss the mode of host-pathogen interactions, highlighting that the inhibition of sncRNA delivery by OMVs may prevent the occurrence of infection to some extent. Hence, this review lays the foundation for future prophylactic treatments against bacterial infections and strategies for addressing bacterial drug resistance. KEY POINTS: •sncRNAs have intracellular and extracellular regulatory functions in bacterial physiological processes and host-pathogen interactions. •OMVs are potential mediators between bacterial sncRNAs and host cells. •OMVs encapsulating sncRNAs have more potential biological functions.

Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice.

The widespread prevalence of Helicobacter pylori (H. pylori) infection remains a great challenge to human health. The existing vaccines are not ideal for preventing H. pylori infection; thus, exploring highly effective adjuvants may improve the immunoprotective efficacy of H. pylori vaccines. In a previous study, we found that the outer membrane vesicles (OMVs), a type of nanoscale particle spontaneously produced by Gram-negative bacteria, could act as adjuvants to boost the immune responses to vaccine antigens. In this study, we explored the potential application of OMVs as delivery vectors for adjuvant development. We constructed recombinant OMVs containing eukaryotic expression plasmid of cytokines, including interleukin 17A or interferon-γ, and evaluated their function as adjuvants in combination with inactivated whole-cell vaccine (WCV) or UreB as vaccine antigens. Our results showed that recombinant OMVs as adjuvants could induce stronger humoral and mucosal immune responses in mice than wild-type H. pylori OMVs and the cholera toxin (CT) adjuvant. Additionally, the recombinant OMVs significantly promoted Th1/Th2/Th17-type immune responses. Furthermore, the recombinant OMV adjuvant induced more potent clearance of H. pylori than CT and wild-type OMVs. Our findings suggest that the recombinant OMVs coupled with cytokines may become potent adjuvants for the development of novel and effective vaccines against H. pylori infection.

Disruption of sncRNA Improves the Protective Efficacy of Outer Membrane Vesicles against Helicobacter pylori Infection in a Mouse Model.

The outer membrane vesicles (OMVs) secreted by Helicobacter pylori contain various bacterial components, such as proteins, phospholipids, toxins, and nucleic acids, including small noncoding RNA (sncRNA), which have regulatory functions in cell envelope structure, metabolism, bacterial communication, biofilm formation, and virulence. We previously showed that knocking out sncRNAs sR-989262 and sR-2509025 at the cellular level increased interleukin 8 (IL-8) levels in mice exposed to OMVs. In this study, we show that immunization with ΔsR-989262 and ΔsR-2509025 OMVs intragastrically significantly increased immunoglobulin G (IgG) and secreted IgA levels in mice compared to wild-type OMVs and without weight changes, which indicated that sncRNA-deficient OMVs are relatively safe to immunize mice. The detection of IgG subtypes IgG1 and IgG2c showed that the sncRNA-deficient OMVs primarily stimulate the T helper 2 (Th2)-mediated immune response. Moreover, levels of the cytokines IL-4, IL-13, gamma interferon (IFN-γ), IL-12 (p40), IL-8, and IL-17 indicate that ΔsR-989262 and ΔsR-2509025 OMVs trigger the Th2-type immune response but primarily trigger a Th1-mediated and Th17-mediated immune response. These findings show that OMV-encapsulated sncRNA plays an important role in regulating the immune response in hosts infected by H. pylori at the animal level. Moreover, they show that knocking out of sR-989262 and sR-2509025 improves the immunogenicity and protective efficacy of OMVs, and this may be beneficial to the design of OMV-based H. pylori vaccines.

Outer Membrane Vesicles Derived from Salmonella enterica Serotype Typhimurium Can Deliver Shigella flexneri 2a O-Polysaccharide Antigen To Prevent Shigella flexneri 2a Infection in Mice.

Shigellosis has become a serious threat to health in many developing countries due to the severe diarrhea it causes. Shigella flexneri 2a is the principal species responsible for this endemic disease. Despite multiple attempts to design a vaccine against shigellosis, no effective vaccine has been developed yet. Lipopolysaccharide (LPS) is both an essential virulence factor and an antigen protective against Shigella, due to its outer domain, termed O-polysaccharide antigen. In the present study, S. flexneri 2a O-polysaccharide antigen was innovatively biosynthesized in Salmonella and attached to core-lipid A via the ligase WaaL, with purified outer membrane vesicles (OMVs) utilized as vaccine vectors. Here, we identified the expression of the heterologous O-antigen and have described the isolation, characterization, and immune protection efficiency of the OMV vaccine. Furthermore, the results of animal experiments indicated that immunization of mice with the OMV vaccine induced significant specific anti-Shigella LPS antibodies in the serum, with similar trends in IgA levels from vaginal secretions and fluid from bronchopulmonary lavage, both intranasally and intraperitoneally. The OMV vaccine derived from both routes of administration provided significant protection against virulent S. flexneri 2a infection, as judged by a serum bactericidal assay, opsonization assay, and challenge test. This vaccination strategy represents a novel and improved approach to control shigellosis by the combination of Salmonella glycosyl carrier lipid bioconjugation with OMVs. IMPORTANCEShigella, the cause of shigellosis or bacillary dysentery, is a major public health concern, especially for children in developing countries. An effective vaccine would control the spread of the disease to some extent. However, no licensed vaccine against Shigella infection in humans has so far been developed. The Shigella O-antigen polysaccharide is effective in stimulating the production of protective antibodies and so could represent a vaccine antigen candidate. In addition, bacterial outer membrane vesicles (OMVs) have been used as antigen delivery platforms due to their nanoscale properties and ease of antigen delivery to trigger an immune response. Therefore, the present study provides a new strategy for vaccine design, combining a glycoconjugated vaccine with OMVs. The design concept of this strategy is the expression of Shigella O-antigen via the LPS synthesis pathway in recombinant Salmonella, from which the OMV vaccine is then isolated. Based on these findings, we believe that the novel vaccine design strategy in which polysaccharide antigens are delivered via bacterial OMVs will be effective for the development and clinical application of an effective Shigella vaccine.

Immunization With Outer Membrane Vesicles Derived From Major Outer Membrane Protein-Deficient Salmonella Typhimurium Mutants for Cross Protection Against Salmonella Enteritidis and Avian Pathogenic Escherichia coli O78 Infection in Chickens.

Colibacillosis is an economically important infectious disease in poultry, caused by avian pathogenic Escherichia coli (APEC). Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne diseases in human circulated through poultry-derived products, including meat and chicken eggs. Vaccine control is the mainstream approach for combating these infections, but it is difficult to create a vaccine for the broad-spectrum protection of poultry due to multiple serotypes of these pathogens. Our previous studies have shown that outer membrane vesicles (OMVs) derived from S. enterica serovar Typhimurium mutants with a remodeled outer membrane could induce cross-protection against heteroserotypic Salmonella infection. Therefore, in this study, we further evaluated the potential of broad-spectrum vaccines based on major outer membrane protein (OMP)-deficient OMVs, including ΔompA, ΔompC, and ΔompD, and determined the protection effectiveness of these candidate vaccines in murine and chicken infection models. The results showed that ΔompA led to an increase in the production of OMVs. Notably, ΔompAΔompCΔompD OMVs showed significantly better cross-protection against S. enterica serovar Choleraesuis, S. Enteritidis, APEC O78, and Shigella flexneri 2a than did other omp-deficient OMVs, with the exception of ΔompA OMVs. Subsequently, we verified the results in the chicken model, in which ΔompAΔompCΔompD OMVs elicited significant cross-protection against S. Enteritidis and APEC O78 infections. These findings further confirmed the feasibility of improving the immunogenicity of OMVs by remodeling the outer membrane and provide a new perspective for the development of broad-spectrum vaccines based on OMVs.

Outer Membrane Vesicles of Helicobacter pylori 7.13 as Adjuvants Promote Protective Efficacy Against Helicobacter pylori Infection.

Helicobacter pylori(H. pylori), a gram-negative bacterium in the human stomach with global prevalence, is relevant to chronic gastrointestinal diseases. Due to its increasing drug resistance and the low protective efficacy of some anti-H. pylori vaccines, it is necessary to find a suitable adjuvant to improve antigen efficiency. In our previous study, we determined that outer membrane vesicles (OMVs), a multicomponent secretion generated by gram-negative bacteria, of H. pylori were safe and could induce long-term and robust immune responses against H. pylori in mice. In this study, we employed two common vaccines, outer membrane proteins (OMPs) and whole cell vaccine (WCV) to assess the adjuvanticity of OMVs in mice. A standard adjuvant, cholera toxin (CT), was used as a control. Purified H. pylori OMVs used as adjuvants generated lasting anti-H. pylori resistance for 12 weeks. Additionally, both systematic and gastric mucosal immunity, as well as humoral immunity, of mice immunized with vaccine and OMVs combinations were significantly enhanced. Moreover, OMVs efficiently promoted Th1 immune response, but the response was skewed toward Th2 and Th17 immunity when compared with that induced by the CT adjuvant. Most importantly, OMVs as adjuvants enhanced the eradication of H. pylori. Thus, OMVs have potential applications as adjuvants in the development of a new generation of vaccines to treat H. pylori infection.