Display of Heterologous Proteins on the Surface of Lactic Acid Bacteria Using Phage PL-1 Endolysin from Lacticaseibacillus paracasei

GORDILLO, TANIA B.; BOCKOR, SABRINA S; ALLIEVI, MARIANA CLAUDIA; RUZAL, SANDRA M.; PALOMINO, MARÍA MERCEDES

Virtual

Congreso; Congreso Conjunto SAIB -SAMIGE 2021; 2021

Sociedad Argentina de Investigación Bioquímica y Sociedad Argentina de Microbiologia General

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 probioticfeatures, can survive the hostile condition of the gastrointestinal tract (low pH, high bile concentration, protease resistance), afeature that allows them to colonize certain intestinal tissues, have intrinsic adjuvant response, and can interact with humanimmune cells, making them attractive vehicles for vaccine delivery. The endolysin from Lacticaseibacillus paracasei phage PL-1has 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 functionalproteins of non-genetically modified LAB. For this purpose, the CBD region was fused with GFP and the GFP-CBDLys washeterologously produced in E. coli. Several LAB strains were incubated with a lysate containing excessive GFP-CBDLys and thepurified protein. The maximum level of binding retention, which was evaluated by flow cytometry, was found in L. paracasei27092, 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 hostilegastrointestinal tract conditions. For this purpose, the survival rates of native and decorated cells of L. paracasei 27092 werecompared with the input after treatments simulating gastrointestinal conditions (low pH, concentrations of bile salts andpancreatin). Decorated cells showed a significant lower decrease in survival rate compared with native cells, suggesting thedisplay 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, Mutanolizyn,EDTA, SDS) to remove cell wall components in a differential manner. Compared to non pretreated cells, TCA treatment showeda significant increase in fluorescence intensity in Lactobacilli strains, indicating that this pretreatment is efficient in enhancingthe DBDLys binding capacity. On the other hand, pretreatment with Mutanolysin showed a significant decrease in bindingcapacity.Further studies are being performed to explore the potential use of GRAS microorganisms for the delivery of biomoleculesmediated by the CBD of PL-1 endolysin as anchor protein.