A Fast, Efficient and Easy to Implement Method to Purify Bacterial Pili From Lacticaseibacillus rhamnosus GG Based on Multimodal Chromatography.

Raphael Dos Santos Morais,Sofiane El-Kirat-Chatel,Sarah Barrau,Cédric Paris,Blandine Simard,Frédéric Borges,Jennifer Burgain,Claire Gaiani

doi:10.3389/fmicb.2020.609880

Abstract

Pili are polymeric proteins located at the cell surface of bacteria. These filamentous proteins play a pivotal role in bacterial adhesion with the surrounding environment. They are found both in Gram-negative and Gram-positive bacteria but differ in their structural organization. Purifying these high molecular weight proteins is challenging and has certainly slowed down their characterization. Here, we propose a chromatography-based protocol, mainly relying on multimodal chromatography (core bead technology using Capto Core 700 resin), to purify sortase-dependent SpaCBA pili from the probiotic strain Lacticaseibacillus rhamnosus GG (LGG). Contrary to previously published methods, this purification protocol does not require specific antibodies nor complex laboratory equipment, including for the multimodal chromatography step, and provides high degree of protein purity. No other proteins were detectable by SDS-PAGE and the 260/280 nm ratio (∼0.6) of the UV spectrum confirmed the absence of any other co-purified macromolecules. One can obtain ∼50 μg of purified pili, starting from 1 L culture at OD600nm ≈ 1, in 2–3 working days. This simple protocol could be useful to numerous laboratories to purify pili from LGG easily. Therefore, the present work should boost specific studies dedicated to LGG SpaCBA pili and the characterization of the interactions occurring with their protein partners at the molecular level. Moreover, this straightforward purification process might be extended to the purification of sortase-dependant pili from other Gram-positive bacteria.

Highlights

Pili, or fimbriae, are length-variable proteinaceous appendages found at the surface of bacteria and archaea where they play a key role in interaction and/or adhesion with the surrounding environment (Berne et al, 2018)
Polymerization occurs through the reaction of a peculiar Lys of a pilin motif and a specific Thr found in the LPXTG motif which is common to the three pilins (Krishnan, 2015; von Ossowski, 2017)
Sortase-dependent pili were thought to be exclusive to pathogens, but in the late 2000s, they were observed by transmission electron microscopy (Kankainen et al, 2009) on the non-pathogenic probiotic bacterium Lactobacillus rhamnosus GG (LGG) (Capurso, 2019), which was recently reclassified as Lacticaseibacillus rhamnosus GG (Zheng et al, 2020)

Summary

Introduction

Fimbriae, are length-variable proteinaceous appendages found at the surface of bacteria and archaea where they play a key role in interaction and/or adhesion with the surrounding environment (Berne et al, 2018) These proteins exhibit a high structural organization diversity (Proft and Baker, 2008). LGG SpaCBA pili can reach up to 1 μm in length and 10 to 50 copies are found per cell (Kankainen et al, 2009) They are composed of a basal pilin (SpaB, ∼20 kDa), a major pilin (SpaA, ∼30 kDa) and a tip pilin (SpaC, ∼90 kDa), whose genes are clustered (Kankainen et al, 2009). Chemically-induced derivatives of LGG (non-GMO) have been generated in order to improve adhesion and the associated putative probiotic role through SpaCBA pili (Rasinkangas et al, 2020)

Methods

Results

Conclusion