Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis.
BACKGROUND
Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet.


RESULTS
We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface.


CONCLUSIONS
Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.
Department of Biology, Federico II University, via Cinthia-MSA, 80126, Naples, Italy.
Giuliana
G
Donadio
Department of Biology, Federico II University, via Cinthia-MSA, 80126, Naples, Italy.
Mariamichela
M
Lanzilli
Department of Biology, Federico II University, via Cinthia-MSA, 80126, Naples, Italy.|School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry, CV4 7AL, UK.
Teja
T
Sirec
Department of Biology, Federico II University, via Cinthia-MSA, 80126, Naples, Italy.
Ezio
E
Ricca
Department of Biology, Federico II University, via Cinthia-MSA, 80126, Naples, Italy. isticato@unina.it.
Rachele
http://orcid.org/0000-0001-9141-4038
R
Isticato
D000327:Adsorption; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013171:Spores, Bacterial / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C101016:CotH protein, Bacillus subtilis
Bacterial spore; Display system; Heterologous protein delivery; Spore coat