Heterologous expression of antigenic peptides in Bacillus subtilis biofilms

Abstract

Background: Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins.

Results: We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties.

Conclusions: In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Keywords: Antigen; Bacillus subtilis; Biofilm; E. granulosus; Endospores; Heterologous protein; Paramyosin; TasA; Tropomyosin; mCherry.

Fig. 1

Spatial distribution of TasA within the biofilm. Vertical thin sections of biofilms harboring a yfp reporter fusion to P_tapA (a) or a TasA-mCherry fusion protein (b). Biofilms were frozen at 72 h of development prior to cryosectioning and fixation. Images represent partial colonies, the edge is shown at the left and the agar surface is at the bottom. Transmitted light images were overlaid with fluorescence images that were false-colored for each reporter. Bar 50 µm

Fig. 2

a Top view of biofilm architecture from wild type, tasA and tasA/sinR strains with negative (left panels) and positive expression of TasA-mCherry (right panels) after 72 h of incubation. A magnification of the edges of the colony is presented at the right of each the whole colony top view picture. Scale bar is 1 cm. b Immunoblotting of biofilm extracts from wild type (lane 1), sinR (lane 2), tasA::TasA-mcherry (lane 3) and tasA/sinR::TasA-mCherry (lane 4) strains; TasA and TasA-mCherry were detected using specific anti-TasA (upper panel) and anti-dsRed2 (lower panel) antibodies. The red arrows indicate the position of TasA and TasA-mCherry. The red bracket indicates an anti-TasA reactive band with a lower molecular weight of TasA, presumably TasA degradation. The molecular weights (kDa) of the proteins are indicated. c Red fluorescence quantification of 24, 48 and 72 h biofilms. Data is presented as the mean ± SEM of four independent experiments. Asterisks denote significant differences in TasA-mCherry red fluorescence between tasA and tasA/sinR strains expressing TasA-mCherry (t test, **p < 0.001, n ≥ 4). d Viable spore counts comparing sinR, tasA::TasA-mCherry and tasA/sinR::TasA-mCherry to wild type percent of spores in biofilms. Error bars indicate SEM (t-test, *p < 0.05, n = 4)

Fig. 3

a Immunoblotting of biofilm extract at 72 h for wild type, tasA, sinR and tasA/sinR (single antibiotic selection) strain for the detection of TasA using an anti-TasA antibody. b Schematic representation of tapA operon carrying E. granulosus antigenic peptides, EgTrp and EgA31, fused in frame at the 3′ end of tasA. tapA, anchoring and assembly protein; sipW, signal peptidase and tasA, major protein matrix. The amino acid region corresponding to each antigenic peptide is indicated. For simplicity of the figures, TasA-(102-207)EgTrp, TasA-(102-278)EgTrp, TasA-(170-369)EgA31 and TasA-(370-583)EgA31 are named as (102-207)EgTrp, (102-278)EgTrp, (170-369)EgA31 and (370-583)EgA31, respectively. Diagram not to scale. c Top view of biofilm architecture from wild type, tasA/sinR, TasA-(102-207)EgTrp, TasA-(102-278)EgTrp, TasA-(170-369)EgA31 and TasA-(370-583)EgA31 strains at 72 h. Scale bar is 1 cm. d Immunoblotting of biofilm extract for wild type (lanes 1 and 5), tasA/sinR (lanes 2 and 6), TasA-(102-207) EgTrp (lanes 3 and 7) and TasA-(102-279)EgTrp (lanes 4 and 8) strains detected with anti-TasA (left panel) and anti-EgTrp (right panel) antibodies. The white arrowhead indicates the position of the TasA-EgTrp antigenic peptides and the red arrow indicates the position of TasA. e Immunoblotting of biofilm extracts for wild type (lanes 1 and 4), tasA/sinR (lanes 2 and 5), TasA-(170-369)EgA31 (lane 3) and TasA-(370-583)EgA31 (lane 6) strains detected with anti-TasA (upper panel) and anti-EgA31 (lower panel) antibodies. The white arrowhead indicates the position of the TasA-EgA31 antigenic peptides and the red arrow indicates the position of TasA. The protein molecular weights marker (kDa) is indicated

Fig. 4

Detection of TasA fusion protein in biofilms by immunohistochemistry. Biofilms were grown for 72 h and then were formalin-fixed, paraffin-embedded and treated for immunohistochemistry. Images correspond to partial section of the colonies. TasA and TasA fused to E. granulosus antigenic peptides for EgTrp and EgA31 were detected using a specific anti-TasA antibody followed by a secondary antibody conjugated to Alexa-594 (red). Transmitted light images were overlaid with fluorescence images. The agar surface is at the bottom of the each image. Scale bar is 50 µm

Fig. 5

Wild type and recombinant spores display equal performances. Spores were tested under different conditions as: a heat resistance, b acidic medium and c shelf-life storage. Data represent the mean ± SEM from three independent experiments (t-test, not significant, p value >0.05). Error bars indicate SEM. d Transmission electron microscopy of wild type, tasA/sinR, TasA-(102-207)Egtrp, TasA-(102-278)EgTrp, TasA-(170-369)EgA31 and TasA-(370-583)EgA31 spore strains. Spores were frozen with liquid nitrogen, fixed with glutaraldehyde, counterstained and photographed. Black arrowhead, spore coats; white arrowhead, spore cortex peptidoglycan; star, spore protoplast. Scale bars are 50 and 100 nm, as indicated