Signal peptides enhance immunogenicity and protection of recombinant Salmonella enterica serovar Choleraesuis vectors against Streptococcus suis.

Streptococcus suis (S. suis) is prevalent worldwide, threatening the pig farming industry and public health. At present, there is no effective vaccine. Salmonella vectors are highly attractive vaccine platforms that can deliver foreign antigens to induce protective responses. However, the limited secretion and release of foreign antigens limit the immunogenicity. Here, we employed 6-phosphogluconate dehydrogenase (6PGD), a protective antigen of Streptococcus suis serotype 2 (SS2), as a model antigen to evaluate how signal peptide (SP) selection influences antigen expression dynamics and immunogenicity in Salmonella vectors. Three heterologous SP fusion constructs, PelB-6PGD (P-6PGD), DsbA-6PGD (D-6PGD), and Bla/ss-6PGD (B-6PGD), were subjected to comparative analysis of expression efficiency, subcellular localization, and subsequent immune responses. While cytoplasmic expression levels showed no significant differences among constructs, recombinant Salmonella enterica serovar Choleraesuis (S. Choleraesuis) strain rSC0016(pS-P-6PGD) carrying PelB-6PGD exhibited significantly enhanced periplasmic accumulation and superior extracellular secretion efficiency in culture supernatants. The strong ability of PelB SP to secrete proteins leads to better uptake of the foreign antigen by bone marrow-derived dendritic cells (BMDCs). After the strong antigen-specific humoral, cellular, and mucosal immunity induced was characterized, rSC0016(pS-P-6PGD)-immunized mice achieved significantly superior protection than rSC0016(pS-B-6PGD) and rSC0016(pS-D-6PGD) groups. Histopathological assessments further corroborated these findings, revealing minimal SS2-induced lesions in the lungs and brains of rSC0016(pS-P-6PGD)-immunized mice. Collectively, these findings establish PelB as an excellent SP for enhancing extracellular antigen secretion in Salmonella vectors, thereby amplifying both humoral and cellular immunity critical for defense against invasive bacterial pathogens such as SS2. This study provides a strategic framework for optimizing antigen delivery in live-vectored vaccine development.