No abstract

Most microorganisms possess a negative surface charge under physiological conditions due to the presence of anionic carboxyl and phosphate groups. Cell surface charge plays an important role in controlling cell adhesion and aggregation phenomena, as well as antigen−antibody, cell−virus, cell−drug, and cell−ions interactions. We have used atomic force microscopy (AFM) with chemically functionalized probes to investigate the surface charges of yeast cells. Force−distance curves and adhesion maps recorded with probes terminated with ionizable carboxyl groups were strongly influenced by pH:  while no adhesion was measured at neutral/alkaline pH, multiple adhesion forces were recorded at pH ≤ 5. Three pieces of evidence indicated that these changes were related to differences in the ionization state of the cell surface functional groups. First, the adhesion force vs pH curve was correlated with microelectrophoresis data, the pH of the largest adhesion force corresponding to the cell isoelectric point, i.e., pH 4. Second, treating the cells with Cu(II) ions caused a reversal of the cell surface charge at neutral pH and promoted the adhesion toward the negatively charged probe. Third, control experiments using nonionizable hydroxyl-terminated probes indicated that the changes in adhesion forces were not simply due to the titration of the probe surface charges. This study shows that AFM with chemically modified probes is a valuable approach in microbiology and biophysics for probing the local electrostatic properties of microbial cell surfaces.

Knowledge of the molecular interactions between lectins and carbohydrates is a key to understand cellular interactions and to develop new bioanalytical applications. We have used atomic force microscopy (AFM) imaging and force measurements to probe the specific interactions between the lectin concanavalin A (Con A) and oligoglucose saccharides. To this end, gold-coated substrates were first functionalized with Con A and thiol-terminated hexasaccharide molecules. The functionalization procedures were validated by means of X-ray photoelectron spectroscopy and AFM. AFM images recorded in aqueous solution revealed that the hexasaccharide-terminated substrates interact specifically with the lectin. Force-distance curves were then recorded between hexasaccharide-terminated AFM probes and Con A-terminated substrates. About half of the retraction curves showed unbinding forces of 96 ( 55 pN (n ) 100), along with elongation forces and rupture lengths ranging from 0 to 200 nm. These features were not observed when the measurements were performed in the presence of mannose or with a hydroxyl-terminated probe. These results, together with the AFM images, indicate that the measured unbinding forces originate from specific lectin-carbohydrate interactions. The carbohydrate AFM probes designed here offer promising prospects for mapping lectin receptors at cell surfaces.