Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection - PubMed (original) (raw)

. 2020 Feb;104(4):1633-1646.

doi: 10.1007/s00253-019-10316-0. Epub 2020 Jan 7.

Hengchang Sun 1 2 3 4, Mei Shang 1 2 3 4, Hongye Jiang 2 3 4, Huimin Dong 1, Xinyi Zhou 2 3 4, Zhipeng Lin 2 3 4, Cunbin Shi 6, Pengli Ren 2 3 4, Lu Zhao 2 3 4, Mengchen Shi 2 3 4, Lina Zhou 2 3 4, Houjun Pan 6, Ouqin Chang 6, Xuerong Li 2 3 4, Yan Huang 7 8 9, Xinbing Yu 10 11 12

Affiliations

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection

Hengchang Sun et al. Appl Microbiol Biotechnol. 2020 Feb.

Abstract

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

Keywords: Bacillus subtilis spore; Clonorchis sinensis; Grass carp; Intestinal microbiota; Oral vaccine; Paramyosin.

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Conflict of interest statement

The authors declares that they have no conflict of interest.

Figures

Fig. 1

Fig. 1

Analysis of CsPmy expression on spore surface. A 12% SDS-PAGE analysis of CsPmy expression on B. subtilis spore at the different time point. The dark arrows indicated the band of CsPmy. M, protein marker; CotC, B.s-CotC spores; 6 h, 12 h, 18 h, 24 h, sporulation induction time of B.s-CotC-CsPmy. B Western blotting of CsPmy expression in coat proteins of B.s-CotC-CsPmy spores. Lane 1, recombinant protein of CsPmy; lane 2, coat protein of B.s-CotC spore; lane 3, propagules of B.s-CotC-CsPmy; lane 4–6, coat protein of B.s-CotC-CsPmy spore-induced for 6 h, 12 h, and 24 h, respectively. C Flow cytometry; Control, B. subtilis spores containing no plasmid; B.s-CotC, B.subtilis spore containing PEB03-CotC plasmid; B.s-CotC-CsPmy, B. subtilis spore harboring PEB03-CotC-CsPmy plasmid. D LC-MS/MS analysis of coat proteins of B.s-CotC-CsPmy. The paramyosin from C. sinensis was identified in coat proteins

Fig. 2

Fig. 2

Specific anti-CsPmy IgM levels in oral immunized grass carp Grass carp was orally administrated with different dosage of recombinant spores. Specific anti-CsPmy antibodies were determined by ELISA in week 2, 4, and 6 after the beginning of the oral immunization. (A) sera, (B) skin mucus, (C) intestinal mucus, (D) bile. Naïve, basal diet group; B.s-CotC-108: basal diet plus 1 × 108 CFU/g B.s-CotC spores. B.s-CotC-CsPmy-105, 108, 1011, basal diet plus 105, 108, 1011 CFU/g B.s-CotC-CsPmy spores, respectively. Data were represented as mean ± SD. Statistical significance was analyzed by the Dunnett’s test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Fig. 3

The relative mRNA levels of IgM and IgZ in the head kidney and spleen. The mRNA levels of IgM and IgZ were analyzed by qRT-PCR at 6 weeks after the beginning of the immunization. (A) IgM mRNA level in the head kidney; (B) IgZ mRNA level in the head kidney; (C) IgM mRNA level in the spleen; (D) IgZ mRNA level in the spleen. Naïve, basal diet group; B.s-CotC-108: basal diet plus 1 × 108 CFU/g B.s-CotC spores. B.s-CotC-CsPmy-105, 108, 1011, basal diet plus 105, 108, 1011 CFU/g B.s-CotC-CsPmy spores, respectively. *P < 0.05, **P < 0.01 (compared with naïve group); #P < 0.05, ## P < 0.01 (compared with B.s-CotC-108 group)

Fig. 4

Fig. 4

Intestinal microbiota analysis. Intestines from three fishes in the same group were homogenized for intestinal microbiota analysis. A Alpha diversity analysis. a Good’s nonparametric coverage estimator. b Chao1 index. c Shannon index. d Simpson index. B Venn graph showing the unique and shared OTUs among groups. C Scatterplot of PCA-score depicting variance of fingerprints derived from the different bacterial community. The principal components PC1 and PC2 explained 72.1% and 14.08% of the variance, respectively. D Heatmap of bacterial distribution in groups. The relative abundance of the bacterial genus is depicted by color intensity with legend indicated at the right side of the figure. Clusters based on the distance of five groups along X-axis and bacterial genus along Y-axis were showed in upper and left of the figure, respectively. Naïve, basal diet group; B.s-CotC-108: basal diet plus 1 × 108 CFU/g B.s-CotC spores. B.s-CotC-CsPmy-105, 108, 1011, basal diet plus 105, 108, 1011 CFU/g B.s-CotC-CsPmy spores, respectively

Fig. 5

Fig. 5

Protect effect in orally administrated fishes. Fishes were sacrificed at 4 weeks after the challenge infection. Fish flesh in each group was weighted and digested with artificial gastric juice (PH = 2.0) for metacercaria detection. (A) C. sinensis cercaria used for challenge infection. (B) Metacercariae found in grass carp after challenge infection. (C) Amount of metacercaria per gram fish flesh. Naïve, fishes fed with basal diet. B.s-CotC-108, fishes fed with basal diet plus 1 × 108 CFU/g B.s-CotC spores; B.s-CotC-CsPmy-1011, fishes fed with basal diet plus 1011 CFU/g B.s-CotC-CsPmy spores. **P < 0.01(compared with Naïve group), ##P < 0.05 (compared with B.s-CotC-108 group). 100 magnification for A and 200 magnification for B

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