Microbial adhesion and ultrastructure from the single-molecule to the single-cell levels by Atomic Force Microscopy

Abstract

In the last decades, atomic force microscopy (AFM) has evolved towards an accurate and lasting tool to study the surface of living cells in physiological conditions. Through imaging, single-molecule force spectroscopy and single-cell force spectroscopy modes, AFM allows to decipher at multiple scales the morphology and the molecular interactions taking place at the cell surface. Applied to microbiology, these approaches have been used to elucidate biophysical properties of biomolecules and to directly link the molecular structures to their function. In this review, we describe the main methods developed for AFM-based microbial surface analysis that we illustrate with examples of molecular mechanisms unravelled with unprecedented resolution.

Keywords: Adhesion; Atomic force microscopy; Interaction; Single-cell force spectroscopy; Single-molecule force spectroscopy; Tip functionalization.

Fig. 1

Different methods for proper physical entrapment of microorganisms. (A) Microorganisms suspensions can be filtered through a porous membrane whose pore size corresponds to that of the cells. (B) AFM deflection images of the yeast Candida albicans trapped in a pore, allowing high resolution images where a cell bud scar is visible. (C) Schematics of the immobilisation methods using PDMS microstructured stamps as developed by Formosa et al. (2015a). Living cells are assembled into the stamps using convective and capillary assembly. (D) AFM images of such PDMS cells arrays filled with C. albicans. (E) Immobilization in microfluidic device as developed by Peric et al. (2017). The microfluidic chip with bacterial traps is mounted to a square opening in the silicon holder. The underside of the device is transparent to allow simultaneous AFM and optical microscopy measurements. (F) AFM image of Escherichia coli bacterium trapped in such trap. (G) Immobilization in micro-tube arrays with an open-up structure as developed by Chen et al. (2014). SEM Images of the mold use to create PDMS micro-tubes structures. (H) AFM imaging of the bacteria dividing along the micro-channel. The design of the set-up allows for simultaneous AFM - fluorescent imaging. Fig. 1B, right, has been reproduced from (Dufrene, 2015) with permission from Elsevier Reprints. Fig. 1C-D have been reproduced from (Formosa et al., 2015a) with permission from Springer Nature. Fig. 1E, F have been reproduced from (Peric et al., 2017) with permission from Springer Nature. Fig. 1 G-H have been reproduced from (Chen et al., 2014) with permission from John Wiley and Sons.

Fig. 2

High resolution imaging of peptidoglycan. (A) Deflection images of sacculi from the Gram positive bacteria Bacillus subtilis (left) (Hayhurst et al., 2008) and Lactococcus lactis (right) (Andre et al., 2010). (B) Different structural organisations observed on the side wall peptidoglycan from B. subtilis in mid-exponential phase (left) compared to that in stationary phase (right) (Li et al., 2018). (C) High resolution images taken on living Group B Streptococcus reveal a nanoscale net-like surface architecture (left, middle), and circular arrangement of bands around the pole (right) (Dover et al., 2015). (D) Images of L. lactis living cells also show circular arrangement of the peptidoglycan at the bacterial pole (left) and periodic bands running parallel to the short cell axis on the bacteria longitudinal side (middle, right) (Andre et al., 2010). (E) Direct visualisation of glycan strand arrangement in the Gram negative bacteria E. coli envelope, obtained by mounting peptidoglycan fragments on poly-l-ornithine (Turner et al., 2018). Fig. 2A has been reproduced from (Andre et al., 2010) and (Hayhurst et al., 2008) with permission from Springer Nature and the National Academy of Sciences. Fig. 2B has been reproduced from (Li et al., 2018) with permission from Frontiers. Fig. 2C has been reproduced from (Dover et al., 2015) with permission from Springer Nature. Fig. 2D has been reproduced from (Andre et al., 2010) with permission from Springer Nature. Fig. 2E has been reproduced from (Turner et al., 2018) with permission from Springer Nature.

Fig. 3

Similitude of adhesion mechanism through the expression of adhesins for the bacteria Pseudomonas fluorescens (A–C) and the pathogenic yeast Candida albicans (D–F). (A, D) Optical images (A: Phase, D: DIC) showing the microscopic adhesion of microorganisms to hydrophobic substrates after several hours of incubation. (B, E) AFM deflection images of single microorganisms immobilized by trapping in porous membrane or hydrophobic interactions. (C, F) Representative force-distance curves obtained during the unfolding of adhesins using functionalized antibody tips. Some curves represent single weak epitope recognition and others feature sawtooth patterns documenting repeated-regions unfolding. Fig. 3A–C have been reproduced from (El-Kirat-Chatel et al., 2014a) and Fig. 3D–F have been reproduced from (Beaussart et al., 2012) with permission from the American Chemical Society.

Fig. 4

Single-cell force spectroscopy method to decipher bacterial adhesion mechanisms. The main surface components involved in the interactions are (A) bacterial polysaccharides, (B) adhesins, (C, D) pili, (E) multiple biomolecules in the case of two interacting microbes and (F) tethers formed by host cell membrane elongation in the case of host/microbes adhesion.