Nanoscale mapping and functional analysis of individual adhesins on living bacteria

Abstract

Although much progress has been made in the identification and characterization of adhesins borne by pathogenic bacteria, the molecular details underlying their interaction with host receptors remain largely unknown owing to the lack of appropriate probing techniques. Here we report a method, based on atomic force microscopy (AFM) with tips bearing biologically active molecules, for measuring the specific binding forces of individual adhesins and for mapping their distribution on the surface of living bacteria. First, we determined the adhesion forces between the heparin-binding haemagglutinin adhesin (HBHA) produced by Mycobacterium tuberculosis and heparin, used as a model sulphated glycoconjugate receptor. Both the adhesion frequency and adhesion force increased with contact time, indicating that the HBHA-heparin complex is formed via multiple intermolecular bridges. We then mapped the distribution of single HBHA molecules on the surface of living mycobacteria and found that the adhesin is not randomly distributed over the mycobacterial surface, but concentrated into nanodomains.

A nanomechanical interface to rapid single-molecule interactions

“…Single-molecule force spectroscopy (SMFS) experiments with the atomic force microscope (AFM) 7 have demonstrated the potential of intermolecular forces to be used for chemically specific nanoscale imaging 8 , manipulation 9 , directed assembly 10 , and biophysical studies of deformation and failure of biomolecules 11 . Forces measured during the rupture of molecular bonds are affected by thermally activated dissociation, which results in loading-rate-dependent characteristics.…”

Towards a nanoscale view of fungal surfaces

“…Control experiments (blocking with mannose, use of a non-flocculating strain) led to the conclusion that the measured adhesion forces reflected individual lectin-carbohydrate interactions involved in yeast flocculation. This type of single-molecule experiment will have an important impact on medicine by allowing the measurement of the minute forces driving host-pathogen interactions, as recently demonstrated with mycobacteria (Dupres et al, 2005). An alternative approach to probing molecular interactions associated with microbial cells is to immobilize them on the AFM cantilever and to measure the forces between the obtained cell probe and solid surfaces.…”

Characterization of heparin–living bacteria interactions by chemiluminescence electrophoretic mobility shift assay

“…With this purpose, scintillation counting of radioisotope radiation [12] and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [8] were used to assess the binding of heparin to living bacteria. More recently, atomic force microscopy was successfully used to investigate heparin-living bacteria interactions and producing quantitative data [13]. However, these methods are qualitative [8], require radioisotope labeling [12], an instrument, which may not be readily available in common laboratories [13].…”

“…More recently, atomic force microscopy was successfully used to investigate heparin-living bacteria interactions and producing quantitative data [13]. However, these methods are qualitative [8], require radioisotope labeling [12], an instrument, which may not be readily available in common laboratories [13].EMSA has been widely employed to detect DNA-protein interactions since its first application [14]. In this study, CL EMSA is introduced to complement the existing methods to study heparin-bacteria interactions.…”

“…More recently, atomic force microscopy was successfully used to investigate heparin-living bacteria interactions and producing quantitative data [13]. However, these methods are qualitative [8], require radioisotope labeling [12], an instrument, which may not be readily available in common laboratories [13].…”