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

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-?in 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.

SUBMITTER: Sun H

PROVIDER: S-EPMC7223688 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Sun Hengchang H Shang Mei M Tang Zeli Z Jiang Hongye H Dong Huimin H Zhou Xinyi X Lin Zhipeng Z Shi Cunbin C Ren Pengli P Zhao Lu L Shi Mengchen M Zhou Lina L Pan Houjun H Chang Ouqin O Li Xuerong X Huang Yan Y Yu Xinbing X

Applied microbiology and biotechnology 20200107 4

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 ...[more]

PMID: 31912200

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Project description:Clonorchiasis caused by Clonorchis sinensis has become increasingly prevalent in recent years. Effective prevention strategies are urgently needed to control this food-borne infectious disease. Previous studies indicated that paramyosin of C. sinensis (CsPmy) is a potential vaccine candidate.We constructed a recombinant plasmid of PEB03-CotC-CsPmy, transformed it into Bacillus subtilis WB600 strain (B.s-CotC-CsPmy), and confirmed CsPmy expression on the spore surface by SDS-PAGE, Western blotting and immunofluorescence assay. The immune response and protective efficacy of the recombinant spore were investigated in BALB/c mice after intragastrical or intraperitoneal immunization. Additionally, biochemical enzyme activities in sera, the intestinal histopathology and gut microflora of spore-treated mice were investigated.CsPmy was successfully expressed on the spore surface and the fusion protein on the spore surface with thermostability. Specific IgG in sera and intestinal mucus were increased after intraperitoneal and intragastrical immunization. The sIgA level in intestinal mucus, feces and bile of B.s-CotC-CsPmy orally treated mice were also significantly raised. Furthermore, numerous IgA-secreting cells were detected in intestinal mucosa of intragastrically immunized mice. No inflammatory injury was observed in the intestinal tissues and there was no significant difference in levels of enzyme-indicated liver function among the groups. Additionally, the diversity and abundance of gut microbiota were not changed after oral immunization. Intragastric and intraperitoneal immunization of B.s-CotC-CsPmy spores in mice resulted in egg reduction rates of 48.3 and 51.2% after challenge infection, respectively. Liver fibrosis degree in B.s-CotC-CsPmy spores treated groups was also significantly reduced.CsPmy expressed on the spore surface maintained its immunogenicity. Both intragastrical and intraperitoneal immunization with B.s-CotC-CsPmy spores induced systemic and local mucosal immune response in mice. Although both intragastric and intraperitoneal immunization elicited a similar protective effect, intragastric immunization induced stronger mucosal immune response without side effects to the liver, intestine and gut microbiota, compared with intraperitoneal immunization. Oral immunization with B. subtilis spore expressing CsPmy on the surface was a promising, safe and needle-free vaccination strategy against clonorchiasis.

Project description:Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes.We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome.The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores.Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic specific immune responses were elicited by oral administration of B.s-CotC-CsCP spores. The spores effectively promoted intestinal health by inducing secretion of acidic mucins, with no other side effects to the liver or intestine. Oral administration of spores expressing CsCP could provide effective protection against C. sinensis. This study may be a cornerstone for development of antiparasitic agents or vaccines against clonorchiasis based on B. subtilis spore expressing CsCP on the surface.

Project description:Bacillus subtilis is a common group of probiotics that have been widely used in the feed industry as they can increase host resistance to pathogens and balance the immune response. However, the regulatory mechanism of Bacillus subtilis on the host immune system remains unclear in teleosts. In this study, we isolated and enriched dendritic cells from white blood cells (WBCs), and then stimulated them with Bacillus subtilis. Morphological features, specific biological functions, and authorized functional molecular markers were used in the identification of dendritic cells. Subsequently, we collected stimulated cells at 0, 4, and 18 h, and then constructed and sequenced the transcriptomic libraries. A transcriptome analysis showed that 2557 genes were up-regulated and 1708 were down-regulated at 4 h compared with the control group (|Fold Change| ? 4), and 1131 genes were up-regulated and 1769 were down-regulated between the cells collected at 18 h and 4 h (|Fold Change| ? 4). Gene Ontology (GO) annotations suggested many differentially expressed genes (DEGs) (p < 0.05 and |Fold Change| ? 4) were involved in immune-related biological functions including immune system progress, cytokine receptor binding, and cytokine binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the cytokine?cytokine receptor interaction pathways were significantly enriched at both time points (p < 0.05), which may play a key role in the response to stimulation. Furthermore, mRNA expression level examination of several pro-inflammatory cytokines and anti-inflammatory cytokines genes by quantitative real-time polymerase chain reaction (qRT-PCR) indicated that their expressions can be significantly increased in Bacillus subtili, which suggest that Bacillus subtilis can balance immune response and tolerance. This study provides dendritic cell (DC)-specific transcriptome data in grass carp by Bacillus subtilis stimulation, allowing us to illustrate the molecular mechanism of the DC-mediated immune response triggered by probiotics in grass carp.

Project description:Bacillus spores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCE We show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore "age" should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

Project description:Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-? and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.

Dataset Information

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

Publications

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

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