Detection and localization of single LysM-peptidoglycan interactions

Abstract

The lysin motif (LysM) is a ubiquitous protein module that binds peptidoglycan and structurally related molecules. Here, we used single-molecule force spectroscopy (SMFS) to measure and localize individual LysM-peptidoglycan interactions on both model and cellular surfaces. LysM modules of the major autolysin AcmA of Lactococcus lactis were bound to gold-coated atomic force microscopy tips, while peptidoglycan was covalently attached onto model supports. Multiple force curves recorded between the LysM tips and peptidoglycan surfaces yielded a bimodal distribution of binding forces, presumably reflecting the occurrence of one and two LysM-peptidoglycan interactions, respectively. The specificity of the measured interaction was confirmed by performing blocking experiments with free peptidoglycan. Next, the LysM tips were used to map single LysM interactions on the surfaces of L. lactis cells. Strikingly, native cells showed very poor binding, suggesting that peptidoglycan was hindered by other cell wall constituents. Consistent with this notion, treatment of the cells with trichloroacetic acid, which removes peptidoglycan-associated polymers, resulted in substantial and homogeneous binding of the LysM tip. These results provide novel insight into the binding forces of bacterial LysMs and show that SMFS is a promising tool for studying the heterologous display of proteins or peptides on bacterial surfaces.

FIG. 1.

Strategy for measuring the LysM-peptidoglycan interaction forces by use of AFM. (A) Schematic representation of the recombinant AcmA cell wall-binding domain. (B) Schematics of the surface chemistry used to functionalize AFM tips and supports with LysM and peptidoglycan. AcmA LysM modules terminated with cysteine residues were attached onto gold-coated AFM tips, while peptidoglycan was covalently attached onto carboxyl-terminated surfaces via NHS/EDC chemistry. The blue boxes represent the GlcNAc and MurNAc disaccharide repeating units of peptidoglycan and are cross-linked by pentapeptides. (C and D) AFM images, and cross-sections taken in the middle of the images (graphs below images), of the biologically modified supports in PBS, confirming the presence of smooth, homogeneous LysM (C) and peptidoglycan (D) layers. To determine the layer thicknesses, small square areas were first scanned under large forces (>10 nN), and this was followed by recording 5-μm by 5-μm images of the same areas under smaller forces.

FIG. 2.

Force spectroscopy of the LysM-peptidoglycan interaction. (A and B) Representative retraction force curves (A) and adhesion force histograms (n = 750) (B) measured in PBS between a LysM tip and a peptidoglycan surface. Data were obtained using five independent samples and eight different tips. All curves were obtained using a retraction speed of 1,000 nm·s⁻¹. (C and D) Force curves (C) and adhesion force histograms (n = 250) (B) obtained after the injection of free peptidoglycan (10 μg·ml⁻¹).

FIG. 3.

Dynamics of the LysM-peptidoglycan interaction. (A) Dependence of the adhesion force on the loading rate applied during retraction (mean ± standard error of the mean), measured between a LysM tip and a peptidoglycan surface at a constant approach speed (1,000 nm·s⁻¹). (B) Dependence of the adhesion frequency on the interaction time, measured at a constant approach and a retracting speed of 1,000 nm·s⁻¹.

FIG. 4.

AFM imaging of single Lactococcus lactis cells, either in the native state or after treatment with TCA. (A) Schematic representation of the cell wall of L. lactis. Abbreviations: TA, teichoic acids; PR, proteins; PS, polysaccharides; PG, peptidoglycan; PM, plasma membrane. (B) Schematic of the cell wall after treatment with TCA, which is expected to remove peptidoglycan-associated polymers. (C) AFM deflection image in Tris-maleate buffer showing two dividing L. lactis cells trapped into a porous polymer filter for in situ imaging. (D) AFM deflection image in Tris-maleate buffer showing dividing L. lactis cells after treatment with TCA.

FIG. 5.

Detecting single LysM-peptidoglycan interactions on L. lactis cells. (A and B) Low-resolution AFM images with Tris-maleate buffer obtained for native (A) and TCA-treated (B) L. lactis cells. (C to F) Adhesion force maps (gray scale, 300 pN) (C and D) and adhesion force histograms (n = 1,024) (E and F) together with representative retraction force curves recorded with a LysM tip on the native (C and E) and TCA-treated (D and F) cell surfaces by use of a constant retraction speed (1,000 nm/s). The bright pixels, observed frequently on top of the treated cell but very rarely on the native cell or on the filter, document substantial binding of the LysM tip due to the exposure of peptidoglycan. Approach and retraction force curves were similar near the contact region.