SagE induces highly effective protective immunity against Streptococcus iniae mainly through an immunogenic domain in the extracellular region

Abstract

Background: Streptococcus iniae is a Gram-positive bacterium and a severe pathogen of a wide range of farmed fish. S. iniae possesses a virulence-associated streptolysin S cluster composed of several components, one of which is SagE. SagE a transmembrane protein with one major extracellular region named ECR. This study aimed to develop a SagE-based DNA candidate vaccine against streptococcosis and examine the immunoprotective mechanism of the vaccine.

Results: We constructed a DNA vaccine, pSagE, based on the sagE gene and examined its immunological property in a Japanese flounder (Paralichthys olivaceus) model. The results showed that at 7 days post-vaccination, expression of SagE at transcription and translation levels was detected in the tissues of the vaccinated fish. After challenge with S. iniae at one and two months post-vaccination, pSagE-vaccinated fish exhibited relative percent survival (RPS) of 95% and 88% respectively. Immunological analysis showed that (i) pSagE significantly upregulated the expression of a wide range of immune genes, (ii) pSagE induced the production of specific serum antibodies that bound whole-cell S. iniae, and (iii) treatment of S. iniae with pSagE-induced antibodies blocked bacterial invasion of host cells. To localize the immunoprotective domain of SagE, the ECR-expressing DNA vaccine pSagEECR was constructed. Immunization analysis showed that flounder vaccinated with pSagEECR exhibited a RPS of 68%, and that pSagEECR induced serum antibody production and immune gene expression in a manner similar to, though to lower magnitudes than, those induced by pSagE.

Conclusions: We in this study developed a DNA vaccine, pSagE, which induces highly protective immunity against S. iniae. The protective effect of pSagE is probably due to its ability to elicit systemic immune response, in particular that of the humoral branch, which leads to production of specific serum antibodies that impair bacterial infection. These results add insights to the immunoprotective mechanism of fish DNA vaccine.

Figure 1

Detection of vaccine plasmids (A) and expression of the vaccine-encoding genes (B and C) in fish tissues. (A) Flounder were vaccinated with pSagE (lanes 3, 5, 7, and 9) or pCN3 (lanes 2, 4, 6, and 8). At 7 days post-vaccination, DNA and RNA were extracted from muscle (lanes 2 and 3), spleen (lanes 4 and 5), liver (lanes 6 and 7), and kidney (lanes 8 and 9) and used for PCR (A) and RT-PCR (B and C) analysis. PCR was performed using primers specific to pSagE or pCN3. RT-PCR was performed using primers specific to sagE(B) or α-tubulin (internal reference) (C). Lane 1, DNA markers.

Figure 2

Detection of SagE in vaccinated fish. Muscle tissues were taken from flounder vaccinated with pSagE (A) and pCN3 (B) at 7 days post-vaccination and used for immunocolloidal gold electron microscopy with gold-labeled antibody. Arrows indicate gold particles. Bar = 100 nm.

Figure 3

Expression of immune genes in vaccinated fish. Flounder were vaccinated with pSagE, pSagEECR, or pCN3 (control). Spleen was taken from the fish at 24 h post-challenge. Total RNA was extracted from the spleen and used for quantitative real time RT-PCR. For each gene, the mRNA level of the control fish was set as 1. Data are presented as means ± SE (N = 3). ^*P < 0.05; ^**P < 0.01.

Figure 4

Serum antibody production in vaccinated fish. Sera were taken from flounder vaccinated with pSagE, pSagECR, and PBS (control). Serum antibodies against ECR were determined by ELISA. Values are shown as means ± SE (N = 5). ^*P < 0.05; ^**P < 0.01.

Figure 5

Interaction between vaccine-induced antibodies and bacterial cells.Streptococcus iniae was incubated with serum from pSagE-vaccinated fish (A and C) or with serum from control fish (B and D). Cell-bound antibodies were detected with FITC-labeled antibody. The cells were observed under a microscope with (A and B) or without (C and D) fluorescence.