Engineered Streptococcus suis Bacterial Ghosts via Lytic Plasmid and Cell‐Penetrating Peptides for Enhanced Protective Immunity

ABSTRACT Bacterial ghosts (BGs) prepared by biological methods effectively preserve their morphological and antigenic characteristics, making them excellent vaccine carriers for robust immune response. Streptococcus suis is a zoonotic pathogen that causes severe human infections such as meningitis and bacteremia, while also imposing significant economic losses in livestock, posing dual threats to public health and food safety. This study identified that CrfP, a murein hydrolase that induces BG production in S. suis (SSBGs), functions by forming pores during natural transformation. A shuttle plasmid pKSET CrfP is then constructed to achieve anhydrotetracycline‐inducible SSBGs generation. The screened WR 12 , a cell‐penetrating peptide (CPP), significantly enhanced the production of SSBGs and achieved a 100% inactivation for vaccine preparation. Immunization with SSBGs elicited a robust immune response, characterized by the production of IgG1 and IgG2a antibodies and the activation of CD4 + T lymphocytes, crucial for humoral immunity. The SSBGs vaccine exhibited excellent protective efficacy against S. suis challenge in a mouse model. These findings establish a scalable method for Gram‐positive BG vaccines and introduce CrfP as a template for lytic enzyme discovery in other pathogens. The work advances a versatile platform for next‐generation vaccines against emerging infections.