To stick or not to stick? Pulling pili shed some light on biofilm formation

Using a single laser beam opens the doors of nano-world. It has been possible from the last decade to manipulate and pull a single macromolecule on the surface of a living bacteria until it breaks. Such experiment produces relevant information regarding biomechanical properties such as elasticity or conformation when exposed to an external force in picoNewton (pN, one millionth of millionth of picoNewton). Such results have been recently published at PlosOne and Biofouling journals.

 

Researchers of LISBP, in collaboration with MICALIS Institute (INRA, Jouy-en-Josas) have focused on the structure of pili (sing. pilus). That is, a pilus is a protein secreted by a bacterium at its surface and strongly involved in adhesion process. In this work, pili were produced by Lactococcus lactis - a widely used species in dairy industry - that has the ability to form communities so called biofilms. Battling biofilm formation in agro-food industry is challenging for food safety.

 

A pilus is an appendage emanating from the cell surface of the majority of Gram-positive bacteria (fig.1). Pili biogenesis is a machinery that involves a sortase (srtC), an enzyme that assembles covalently protein subunits (pilins) and in head-to-tail series to form the backbone (PilB) in L.lactis IL1403 with a tip pilin (PilA) at the distal end of the pilus (fig.2). This assembly is firmly anchored to the peptidoglycan wall using PilC anchor pilin.

 

It has been demonstrated that under static conditions pili are involved in self-aggregation and modify the architecture of a growing biofilm.1 To provide a deeper understanding of the role played by pili in biofilm formation, teams of LISBP has focused on the influence of different pilins and sortase C on (i) adhesion of cells to surfaces under dynamic conditions2 and (ii) nanomechanical properties of pili using single-molecule force spectroscopy3 (fig.3).

 

It has been reported that the presence of pili increased drastically the adhesion properties of L .lactis on polymeric surfaces  and that sortase C was mandatory for cells to sustain the shear flow.2 Force spectroscopy experiments confirmed such requirement and provided data showing extreme flexibility of the pili, much more than DNA strands.3 Such discovery opens up new assumptions about homotypic interactions between two cells in particular and about self-assembly in general. Such work is ongoing.

 

A better understanding of the conditions that drives the bacterial colonization to biotic (e.g. intestinal tract) or abiotic surfaces (e.g. milking machine) remains a major socio-economic issue. Deciphering nanomechanics of pili and their importance in the adhesion of lactococci allows to bring elements on the possible role played in the formation and structuration of biofilms about relevant sites in food-processing industry as milking machines or wood aging boards for cheese. Eventually, such type of biofilm would allow to struggle against pathogens (e.g. Listeria) but also against spoilage flora in order to propose in fine new solutions for food biopreservation.

 

Figure 2: Pil operon harboring the genes coding for the production of proteins surbnuit and sortase required for pilus assembly. Topology of the pilus.

 

Figure 3: Experimental multiscale approach using shear stress flow chamber to determine adhesion properties of a population of piliated individuals and optical tweezers developed by M. Castelain to characterize the nanomechanical properties of individual pili.

 

 

Figure 1: SEM micrograph of IL1403 L.lactis Pil strain producing pili (black arrows). Scale bar, 500 nm. Reprinted figure from PLoS One journal (http://dx.doi.org/10.1371/journal.pone.0152053.g003

 

 

Figure 4 : Mickaël Castelain setting up a sample before calibrating the force of the optical tweezers applied on micrometric beads using a custom-made control/acquisition software developed in LISBP. - ©Ch. Maitre/Inra.

 

 

REFERENCES

 

1.    Oxaran, V.; Ledue-Clier, F.; Dieye, Y.; Herry, J.-M.; Péchoux, C.; Meylheuc, T.; Briandet, R.; Juillard, V.; Piard, J.-C., Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation. PLoS ONE 2012, 7 (12), e50989.

2.    Castelain, M.; Duviau, M. P.; Oxaran, V.; Schmitz, P.; Cocaign-Bousquet, M.; Loubiere, P.; Piard, J. C.; Mercier-Bonin, M., Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow. Biofouling 2016, 32 (8), 911-23.

3.    Castelain, M.; Duviau, M. P.; Canette, A.; Schmitz, P.; Loubiere, P.; Cocaign-Bousquet, M.; Piard, J. C.; Mercier-Bonin, M., The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin. PLoS One 2016, 11 (3), e0152053.