Abstract:Streptococcus agalactiae (S. agalactiae) is a major pathogen in tilapia aquaculture, causing severe economic losses worldwide. Infected fish often develop systemic infection accompanied by neurological symptoms such as meningitis, reflecting successful invasion of the central nervous system. Among the different stages of infection, traversal of the blood-brain barrier (BBB) represents a critical step in disease progression. However, the mechanisms underlying its pathogenicity, particularly those related to host interaction and BBB crossing, remain poorly understood. Successful infection involves multiple steps, including host colonization, immune evasion, and dissemination to the central nervous system, highlighting the complexity of this process. The ability to cross the BBB is closely associated with the development of neurological symptoms and disease severity in infected fish. Disruption of BBB integrity facilitates bacterial entry into the central nervous system and is a key factor contributing to disease progression. In this study, we investigated the role of the transcriptional regulator Spx in the virulence of a tilapia-derived S. agalactiae HN016 strain. A spx deletion mutant (Δspx) was constructed in the wild-type strain HN016, and a complemented strain (CΔspx) was generated to restore gene function. The role of spx was evaluated by using a combination of in vitro and in vivo approaches, including analysis of cell wall-related gene expression, antibiotic susceptibility, biofilm formation, host-pathogen interaction, and blood-brain barrier (BBB) crossing. A tilapia infection model was further employed to assess bacterial pathogenicity, BBB permeability, and host responses under physiological conditions. Together, these experiments provided a comprehensive assessment of spx-associated phenotypes across multiple experimental systems. This experimental design enabled the characterization of spx-associated phenotypes across key aspects of bacterial physiology and host–pathogen interactions. Gene expression analysis showed that the deletion of spx significantly downregulated several genes of the mur family genes involved in peptidoglycan biosynthesis, including murA, mur1, and mur2. Notably, murA is involved in the initial step of peptidoglycan precursor synthesis, and Mur proteins are associated with cell wall biosynthetic processes. Consistent with these transcriptional changes, the Δspx mutant exhibited increased susceptibility to the cell wall-targeting antibiotic vancomycin, indicating altered cell wall-associated properties. In addition, the Δspx mutant displayed a markedly reduced capacity for biofilm formation (P < 0.05), which may affect its ability to persist under environmental conditions. Loss of spx further affected bacterial interaction with host cells. Compared with the WT strain, the Δspx mutant showed significantly reduced adhesion to (P < 0.01) and invasion of (P < 0.0001) tilapia brain microvascular endothelial cells (TiB). In macrophage infection assays, the Δspx mutant was more readily phagocytosed (P < 0.001) and induced lower cytotoxicity, as reflected by decreased lactate dehydrogenase (LDH) release (P < 0.0001). These observations are consistent with a reduced capacity of the mutant strain to resist host immune clearance. To assess the role of spx in BBB traversal, an in vitro BBB model based on human brain microvascular endothelial cells (hBMEC) was established. The Δspx mutant exhibited significantly reduced translocation across the BBB model compared with the WT strain (P < 0.01). In vivo experiments using a tilapia infection model further supported these observations. Evans blue (EB) permeability assays showed reduced dye extravasation in the brains of fish infected with the Δspx mutant, indicating decreased BBB permeability. This observation is in line with the reduced translocation observed in the in vitro BBB model, suggesting a consistent effect of spx deletion on BBB-associated phenotypes under both experimental conditions. These results were further supported by histopathological analysis. The WT-infected group exhibited typical acute meningitis, characterized by pronounced meningeal thickening and inflammatory cell infiltration extending into the brain parenchyma. In contrast, no obvious pathological damage was observed in the Δspx-infected group, whereas the complemented strain (CΔspx) restored these pathological features to levels comparable to the WT strain. These findings are consistent with a reduced capacity of the Δspx mutant to induce brain tissue damage. To further evaluate host responses, the expression of inflammatory cytokines was analyzed in infected tilapia. Infection with the Δspx mutant resulted in reduced expression of pro-inflammatory cytokines (IL-6 and TNF-α, P < 0.05), accompanied by a significant increase in the anti-inflammatory cytokine IL-10 (P < 0.01), indicating an attenuated inflammatory response during infection. In conclusion, deletion of spx affects multiple phenotypes associated with virulence in S. agalactiae, including cell wall-related characteristics, biofilm formation, host cell interaction, and BBB crossing, ultimately leading to reduced pathogenicity in vivo. These phenotypic differences were consistently observed across multiple experimental models. Collectively, the results highlight the contribution of spx to several aspects of bacterial infection. These findings were consistent across both in vitro and in vivo experimental systems, supporting the robustness of the observed phenotypic differences. These findings contribute to the current understanding of streptococcosis in tilapia and may inform disease control strategies in aquaculture, as well as provide a potential target for the development of novel vaccines or antimicrobial agents.
