HETEROLOGOUS PROTEINS DISPLAY ON LACTIC ACID BACTERIA BY USING THE Lacticaseibacillus paracasei PHAGE PL-1 ENDOLYSIN

BOCKOR, SABRINA SOL; PALOMINO, MARÍA MERCEDES; ALLIEVI, MARIANA CLAUDIA; GORDILLO, TANIA BELÉN; RUZAL, SANDRA MÓNICA

Buenos Aires

Congreso; SAIB - SAMIGE Joint Meeting 2021 on line; 2021

For centuries, Lactic Acid Bacteria (LAB), many of which have been granted the ?generally recognized as safe? (GRAS) status, have been used for the production of fermented food and their preservation. Additionally many LAB strains have probiotic features, can survive the hostile condition of the gastrointestinal tract (low pH, high bile concentration, protease resistance), a feature that allows them to colonize certain intestinal tissues, have intrinsic adjuvant response, and can interact with human immune cells, making them attractive vehicles for vaccine delivery. The endolysin from Lacticaseibacillus paracasei phage PL-1 has a typical modular structure with a cell wall binding domain (CBD), at the C-terminus and one catalytic domain, at the N-terminus. The aim of the present work was to evaluate the CBD of phage PL-1 endolysin as a potential anchor domain to bind functional proteins of non-genetically modified LAB. For this purpose, the CBD region was fused with GFP and the GFP-CBDLys was heterologously produced in E. coli. Several LAB strains were incubated with a whole lysate containing excessive GFP-CBDLys and also with the purified protein. The maximum level of binding retention, which was evaluated by flow cytometry, was found in L. paracasei 27092, L. paracasei 27139, L. casei BL23 and Lactiplantibacillus plantarum BL8. We further determined how GFP-CBDLys-decorated Lactobacilli could impact cell viability when cells were exposed to hostile gastrointestinal tract conditions. For this purpose, the survival rates of native and decorated cells of L. paracasei 27092 were compared with the input after treatments simulating gastrointestinal conditions (low pH, concentrations of bile salts and pancreatin). Decorated cells showed a significant lower decrease in survival rate compared with native cells, suggesting the display of heterologous protein could offer a protective role against the adverse conditions of the gastrointestinal tract. To determine which component CBDLys binds, we studied the effects of different chemical pretreatments (TCA, Mutanolysin, EDTA, SDS) to remove cell wall components in a differential manner. Compared to non pretreated cells, TCA treatment showed a significant increase in fluorescence intensity in Lactobacilli strains, indicating that this pretreatment is efficient in enhancing the CBDLys binding capacity. On the other hand, pretreatment with Mutanolysin showed a significant decrease in binding capacity. Further studies are being performed to explore the potential use of nongenetically modified and GRAS microorganisms for the delivery of biomolecules mediated by the CBD of PL-1 endolysin as anchor protein.