Role of adhesion forces in mechanosensitive channel gating in Staphylococcus aureus adhering to surfaces

Abstract

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials.

Fig. 1. Physico-chemical characteristics of staphylococcal cell surfaces.

a Initial bacterial removal rates from an aqueous phase (10 mM potassium phosphate buffer) by hexadecane as a function of pH. b Zeta potentials of staphylococci in 10 mM potassium phosphate buffer as a function of pH. c Contact angles on bacterial lawns with water (θ_w), formamide (θ_f), methylene iodide (θ_m), and staphylococcal surface free energy parameters and components. Total surface free energy γ^tot results from Lifshitz–Van der Waals γ^LW and acid-base γ^AB components, and electron-donating γ⁻ and electron-accepting γ⁺ parameters. Error bars in panels (a) and (b) represent standard deviations over measurements on three different bacterial cultures. ± signs in panel (c) represent standard deviations over measurements on six bacterial lawns, prepared from three separate bacterial cultures.

Fig. 2. Staphylococcal uptake of calcein.

Percentage fluorescent planktonic and adhering staphylococci due to uptake of fluorescent calcein as a function of adhesion force. Lines indicate an exponential fit used to derive plateau levels and critical adhesion forces. Percentage fluorescent staphylococci were calculated with respect to the total number of planktonic bacteria or bacteria adhering to each substratum surface. Dashed lines indicate the 95% confidence band. Horizontal error bars represent standard deviations over at least 45 force–distance curves, comprising for one probe three different spots recording five force–distance curves in each spot. Three probes prepared from three separate bacterial cultures. Vertical error bars represent standard deviations over nine different fluorescence images obtained from three different bacterial cultures.

Fig. 3. Staphylococcal uptake of dihydrostreptomycin.

Reduction in CFUs (log mL⁻¹) of planktonic and adhering staphylococci after 2 h exposure to dihydrostreptomycin, expressed relative to exposure to PBS, as a function of adhesion force. Lines indicate an exponential fit used to derive plateau levels and critical adhesion forces. Dashed lines indicate the 95% confidence band. Horizontal error bars represent standard deviations over at least 45 force–distance curves, for one probe comprising three different spots recording five force–distance curves on each spot. Three probes prepared from three separate bacterial cultures. Vertical error bars represent standard deviations over three measurements from three different bacterial cultures.

Fig. 4. Schematic presentation of the effect of adhesion and adhesion forces on the bacterial cell wall and mechanosensitive channel gating.

Planktonic bacteria in the absence of osmotic stress do not experience adhesion forces causing cell wall deformation and changes lipid membrane tension that are accompanied by opening of mechanosensitive channels.