Date Published: March 24, 2016
Publisher: Public Library of Science
Author(s): Mickaël Castelain, Marie-Pierre Duviau, Alexis Canette, Philippe Schmitz, Pascal Loubière, Muriel Cocaign-Bousquet, Jean-Christophe Piard, Muriel Mercier-Bonin, Etienne Dague.
Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.
Many bacteria, especially pathogens, produce long polymeric cell-surface organelles, called pili, that initiate bacterial attachment to host tissues, facilitating colonization and invasion [1–3]. The biogenesis, structure, and properties of these extended hair-like structures are well characterized in Gram-negative bacteria, especially for type 1, IV and P pili [1–5]. In contrast, pilus-like structures on the surface of Gram-positive bacteria were first detected in the 1960’s in Corynebacterium renale by electron microscopy  but have only recently been characterized at the molecular level . A number of studies has reported the occurrence of filamentous structures in Gram-positive bacteria (see, for example, ref.  for review) and demonstrated significant morphological and structural differences between the pili of Gram-positive and Gram-negative bacteria [9,10]. A generic model for the assembly of pili in Gram-positive bacteria [7,11] has proposed that they are assembled from hundreds of copies of a single major pilin that forms the shaft along with one or two ancillary pilins i.e. an adhesive pilin located at the pilus tip and an anchoring pilin at the base of the pilus. Pilin subunits are secreted extracellularly and are assembled linearly by a pilus-specific class C-sortase, a transpeptidase that links the C-terminus of one subunit to the amino side chain of a lysine residue from the next pilin subunit through a covalent isopeptide bond [7,12]. Once a pilus is assembled, another sortase (usually a housekeeping sortase) ligates the anchoring pilin to an amino group of the cell wall peptidoglycan , highlighting the fact that these pili are entirely covalent assemblies. This architecture, typical of Gram-positive sortase-assembled pili, has been reported for several pathogens, such as Streptococcus agalactiae , Streptococcus pyogenes  or Streptococcus pneumoniae . In contrast, data available for pili of non-pathogenic Gram-positive bacteria, such as Lactic Acid Bacteria (LAB), are scarce and restricted to Lactobacillus rhamnosus GG pili consisting of 1-μm long linear ultrastructures, resulting from the assembly of several copies of major pilin SpaA along with ancillary pilins . The distribution of the SpaC ancillary pilin along the shaft confers the ability of the L. rhamnosus GG pilus to adhere via several attachment sites [18,19].
We analyzed the persistence length of pili produced by L. lactis reflecting their morphological and biomechanical properties. We have proposed a technical and analytical methodology to provide a reliable interpretation of certain aspects of their structural/nanomechanical relationship. This methodology consisted of using Force-Measuring Optical Tweezers to apply minute forces on several pili until some detached leaving a single pilus, followed by the exploitation of an experimental procedure, i.e. directly at the single-pilus scale using an interaction-screening protein (bovine serum albumin). Our methodology allowed us to investigate the functionality of the different pilins needed to form a pilus. We have clearly established the combined role exerted by sortase C and the backbone pilin PilB. In contrast, the tip pilin PilA was shown to be nonessential for pilus nanomechanics.