Impact of Osmotic Stress on Heterologous Protein Display at the Surface of Lactic Acid Bacteria Using Phage PL-1 Endolysin

GORDILLO, TANIA B.; JASTREBOW, IARA G; DE ROSSI, MARIA C; GRADASCHI, VICTORIA; BOCKOR, SABRINA S; ALLIEVI, MARIANA CLAUDIA; RUZAL, SANDRA M.; PALOMINO, MARÍA MERCEDES

Chapadmalal, Provincia de Buenos Aires, Argentina

Congreso; XVIII Congreso Argentino de Microbiología General (SAMIGE); 2023

Lactic Acid Bacteria (LAB) have been granted the “generally recognized as safe” (GRAS)status. Moreover, many LAB strains exhibit probiotic features and can survive the hostilecondition of the gastrointestinal tract, allowing them to colonize certain intestinal tissues.The endolysin of phage PL-1 of Lacticaseibacillus paracasei has a bi-modular structure,comprising a catalytic domain at its N-terminus and a cell wall binding domain (CBD) at itsC-terminus. Phage-based display systems, using the CBD of phage endolysins, haveemerged as a versatile platform for surface display in LAB.Our goal is to evaluate the CBD of the PL-1 phage endolysin as an anchoring domain.Furthermore, we explore the impact of pre-growth in NaCl on the binding capacity. To thisend, the GFP-CBDLys was firstly produced in E. coli and the recombinant protein was ableto bind to the cell wall of LAB in a binding assay. To test which component may bindCBDLys, chemical treatments were performed on LAB. Lc. paracasei was exposed todifferent chemical agents that removed cell wall components differently. CBDLys showed asignificantly higher binding in cells pretreated with 5% TCA, however this treatment affectedcell viability. Binding capacity was evaluated after growing Lc. paracasei in high saltconditions. When cells were pre-grown at 0.75 M NaCl a 3.25 fold increase in fluorescenceintensity was observed by flow cytometry, without affecting their viability. Binding capacityand Lc. paracasei morphological features were also studied using confocal and AiryScansuper-resolution microscopy. The results showed a significant increase in fluorescenceintensity of NaCl-exposed cells as well as changes in their shape compared to the controlcondition. To further investigate the changes at the surface of Lc. paracasei during itsadaptation to high salt growth, a genetic expression study on secondary cell-wall polymersgenes was conducted. Expression analysis by qPCR technique showed an inhibition in theexpression of yfnI, wchF and wcxM genes when Lc. paracasei was pre-grown in a high saltcondition compared to the control condition.To know if the cells decorated with the carrier protein as well as the surface modificationsin high salt could influence the phage-bacteria interaction, the PL-1 phage adsorptionkinetics was performed on Lc. paracasei. The results indicate that there are no differencesin the adsorption kinetics of the strain when decorated with our carrier protein. However, asignificant decrease was observed when the strain was pre grown in high salt conditions,with and without subsequent decoration.In conclusion, we propose an effective approach based on the capacity of CBDLys ofphage PL-1 using lactobacilli as a delivery display system. Additionally, a safer and innocuous alternative of pre-growth in NaCl was investigated that not only enhances thebinding capacity of our anchor protein but also preserves cellular viability.