Protective effects of a food-grade recombinant Lactobacillus plantarum with surface displayed AMA1 and EtMIC2 proteins of Eimeria tenella in broiler chickens

Abstract

Background: Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry.

Results: In this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA') from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge.

Conclusions: The novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.

Keywords: Eimeria tenella; Food-grade; Lactobacillus plantarum; Surface displayed expression.

Fig. 1

Construction process of erm-marked and asd-alr-marked plasmids for the expression of single-anchored EGFP

Fig. 2

Growth characteristics of EGFP-expressing L. plantarum strains. Bacterial cells were pre-cultured to OD₆₀₀ ≈ 0.3. MRS was divided into two equal parts, one in non-induced culture (gray bars) and another in induced culture with 25 ng/mL SppIp (white bars). OD₆₀₀ values were measured 4 h post induction. pLP-pgsA′, pLQa-pgsA′ pLp-S and pLQa-S are the empty vectors without the target proteins, and we only used the pLP-pgsA′ or pLQa-pgsA′ as empty vector control

Fig. 3

a Western blot analysis of EGFP. Same amount of total protein was loaded for each strain. Two strains containing pLP-pgsA′ (Lane 1) and pLQa-pgsA′ (Lane 2) were used as negative controls. The arrow indicates the bands of the EGFP fusion proteins. b Quantitative analysis of EGFP fusion proteins from Western blots. c Fluorescence microscopy analysis of single-anchoring expression of EGFP in L. plantarum containing asd-alr-marked or erm-marked plasmids (×100 objective). d Representative images of flow cytometry analysis of recombinants containing the following plasmids at 4 h post induction: pLp-pgsA′-EGFP (black), pLQa-pgsA′-EGFP (yellow), pLP-EGFP-S (light blue), pLQa-EGFP-S (green). pLP-pgsA′ (pink) and pLQa-pgsA′ (gray) were used as negative controls

Fig. 4

a The expression cassette for single- or dual-anchored proteins. The promoter PsppA mediated single-anchoring of the mCherryAT in pLQa-pgsA′-mCherry. Each of the co-anchoring expression plasmids (pLQa-p′m-ES and pLQa-ES-p′m) contains the inducible promoter PsppA, followed by two connected single-anchoring cassettes, which were separated by the SD sequence (AGGAAACAGACC). The difference between pLQa-p′m-ES and pLQa-ES-p′m is the splicing order of each anchoring cassette. b Western blot of EGFP using mouse anti-EGFP monoclonal antibody. c Quantitative analysis of western blot shown in b. d Western blot of mCherry using mouse anti-6 × His tag monoclonal antibody. e Quantitative analysis of western blot shown in d. f Representative images of flow cytometric analysis of L. plantarum NC8/Δalr strains containing the plasmids designed for single- or dual-anchoring of EGFP or mCherry. The strains are denoted by different colors in the flow cytometry histograms: pLQa-EGFP-S (green), pLQa-pgsA’-mCherry (red), pLQa-p’m-E (purple), pLQa-ES-p’m (blue). EGFP-A represents the EGFP-expressing strains; mCherry-A represents the mCherry-expressing strains. The L. plantarum NC8/Δalr harboring the empty pLQa-pgsA’ plasmid was used as a negative control (gray). (h) Statistical analysis of the flow cytometry results obtained by one-way ANOVA (NS: no significant difference, P > 0.05, ***P < 0.001)

Fig. 5

Fluorescence microscopy showing co-anchored EGFP and mCherry encoded by a single plasmid. L. plantarum NC8/Δalr harboring plasmids pLQa-EGFP-S or pLQa-pgsA′-mCherry designed for single-anchoring of mCherry or EGFP were used as controls (×100 objective). White arrows indicate co-localization of EGFP and mCherry in strains harboring pLQa-p′m-ES and pLQa-ES-p′m, respectively

Fig. 6

a The expression cassette for co-anchoring of AMA1 and EtMIC2 in the plasmid pLQa-AMA1S-p′EtMIC2. b Synthesis of AMA1 and EtMIC2 proteins in Lp-12 was assessed by Western blotting. Lane M: Marker; Lane 1: Lp/pLQa-pgsA′; Lane 2: Lp-12. c Immunofluorescence analysis to detect co-anchored AMA1 and EtMIC2 on the surface of L. plantarum

Fig. 7

Flow cytometry analysis of Lp-12-triggered T cell responses after vaccination. a The single cells in peripheral-blood were prepared as described and subjected to flow cytometry assay gating was done according to [57]. b Panels representing CD3⁺ CD4⁺ T cells for each group. c The percentage of CD3⁺ CD4⁺ T cells and CD3⁺ CD8⁺ T cells from peripheral-blood were detected using flow cytometry on day 10 post second immunization by Lp-12. Data were shown as mean ± S.E.M (n = 5), were compared by a one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). d Panels representing CD3⁺ CD8⁺ T cells for each group

Fig. 8

Detection of specific IgY in sera (a) and SIgA in intestinal washes (b) from chicks after boosting immunization. The data shown represent mean ± S.E.M (n = 5), which were compared by a one-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001). The samples were measured with 3 repeats. c Pathological cecum damage 7 dpi. Sample sections were stained using HE (×100 magnification); PBS: PBS control group. The villi and glands of the cecum are clearly visible and appear intact. PBS-challenge: The PBS-challenge group exhibits seriously damaged cecum villi and blurred gland was with blood cells and inflammatory cells present in the submucosa. A large number of coccidial oocysts can be found in the cecum lumen. Lp/pLQa-pgsA′: Lp/pLQa-pgsA′ immunized, followed by a challenge with E. tenella. The cecum villi were severely damaged and the glands appear blurred. Lp-12: Lp-12 immunized, followed by a challenge with E. tenella. The cecum villi were partly exfoliated, but the villus structure and the submucosal tissue remained relatively intact