Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA⁸⁷⁷ and SadA²⁶⁹) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158–172aa and UreB349–363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA⁸⁷⁷ or SadA¹²⁹². Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA⁸⁷⁷ or SadA¹²⁹² as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA¹²⁹². The oral administrated pSadBA¹²⁹²-FUM/StmΔygeAΔmurI and pSadBA⁸⁷⁷-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Background: Outer Membrane Vesicles (OMVs), nanosized particles derived from Gram-negative bacteria, are promising vaccine carriers due to innate immunogenicity and self-adjuvant properties. Yet the systematic evaluations of OMV-associated toxicity remain limited. Methods: We developed a CRISPR/Cas9-engineered Salmonella enterica serovar Typhimurium ΔmsbB mutant (Mut4_STM) to produce detoxified OMVs (Mut4_OMVs) with enhanced yield. Subcutaneous immunization of BALB/c mice with Mut4_OMVs to evaluate safety, and the adjuvant efficacy was also determined by injecting Mut4_OMVs with Yersinia pestis F1Vmut or Bacillus anthracis PA_D4 antigens, mixing formulation, respectively. Results: Mut4_OMVs showed a 9-fold protein yield increase over wild-type OMVs. While all mice injected with wild-type OMVs died, 100% survival was observed in those receiving Mut4_OMVs. However, dose-dependent pathological alterations became evident in specific organs as the administration dose escalated, such as induced splenic extramedullary hematopoiesis and renal edema. Despite residual toxicity, 2–3 doses of 10 μg Mut4_OMVs elicited antigen-specific antibody titers comparable to aluminum adjuvant controls and superior T-cell immune responses. Conclusion: While Mut4_OMVs retain potent adjuvant activity, their residual toxicity necessitates further biocompatibility optimization for safe vaccine applications.

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae.