University of Groningen
Responses of Staphylococcus aureus to mechanical and chemical stresses
Carniello, Vera
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Chapter 4
Role of Adhesion Forces in Mechanosensitive Channel Gatingin Staphylococcus aureus Adhering to Surfaces
Vera Carniello, Brandon W. Peterson, Henny C. van der Mei and Henk J. Busscher
Chapter
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ABSTRACT
Mechanosensitive channels in bacterial membranes open or close in response to changes in membrane tension invoked by changes in environmental osmolarity to al-low water fal-low across the channel. Mechanosensitive channels have also been sug-gested to play a role in drug uptake and efflux. This chapter aims to provide support for our hypothesis that gating of mechanosensitive channels cannot only be stimulated by osmotic forces, but also by the forces through which bacteria adhere to surfaces. To this end, we here relate adhesion forces to different substratum surfaces measured using single-bacterial contact probe atomic-force-microscopy of a Staphylococcus
au-reus strain and its isogenic ΔmscL mutant with the gating of mechanosensitive
chan-nels upon their adhesion to the surfaces. The percentage planktonic staphylococci or staphylococci adhering to each surface becoming fluorescent due to uptake of a fluorescent, negatively-charged molecule (calcein), increased exponentially with adhe-sion force and was higher in the parent strain (66 %) than the ΔmscL mutant (40 %), suggesting its uptake through both large and small channels. However, these uptake levels were achieved at different critical adhesion forces of 4.1 and 1.2 nN for the par-ent strain and the ΔmscL mutant, respectively. Uptake of similarly-sized but positive-ly-charged dihydrostreptomycin, monitored by staphylococcal killing upon exposure to dihydrostreptomycin, occurred in the parent strain by a reduction of 2.4 log-units CFUs, at a critical force of 3.6 nN, but remained low (reduction 1.0 log-unit CFU) in the mutant strain, independent of adhesion force. This suggests that due to attractive electro-static charge interaction between the antibiotic and membrane channel proteins, the positively-charged antibiotic may be considered as an analogue of a large molecule. Accordingly, the current observations support that adhesion to surfaces plays a role in staphylococcal channel gating. Since the majority of bacterial infections is due to bacteria in their biofilm-mode of grow, i.e. adhering to a surface, this provides better understanding of the recalcitrance of bacterial infections against antibiotic treatment.
INTRODUCTION
Mechanosensitive channels in bacteria are single proteins present in the bacterial lipid membrane surrounding the cytoplasm, that allow bacteria to interact with their envi-ronment. Mechanosensitive channels act as valves that open or close, in response to changes in membrane surface tension [1–3], for example to allow water flow across the membrane due to a decrease in environmental osmolarity [4,5], that can gener-ate a membrane force [6]. The most studied types of mechanosensitive channels are the mechanosensitive channels of large (MscL) and small (MscS) conductance [7,8]. Opening of MscLs in Escherichia coli to yield a pore with diameter of about 30 Å [9], required a critical membrane tension of around 10 mN m-1 [10], while MscSs required a
lower tension of about 5 mN m-1 [11] to open a pore of about 16 Å [10]. However,
mech-anosensitive channels do not only function to protect a bacterium against osmotic forces, but have been suggested as well to play a role in antibiotic uptake and efflux [12,13]. MscLs provide the main pathway for bacterial uptake of the antibiotic dihydro-streptomycin, which is a positively-charged molecule (731 Da) that can induce MscL opening to promote its uptake [12,13]. Dihydrostreptomycin inhibits growth of bacteria having MscLs, but not of ΔmscL mutants lacking MscLs [12]. Moreover, MscLs are also responsible for efflux of molecules like potassium [12] or cytoplasmic proteins [14,15], and in addition may facilitate efflux to enhance tolerance against selected anti-biotics, similarly to the increased nisin tolerance observed in adhering Staphylococcus
aureus [16,17].
The role of mechanosensitive channels in antibiotic uptake and efflux makes a better understanding of the nature of the forces that can lead to their opening or closing im-perative, particularly in an era of increasing antibiotic resistance among many patho-genic bacteria, including S. aureus. Realizing that the majority of bacterial infections arise from bacteria in a biofilm-mode of growth in which bacteria adhere either to each other, to mammalian cells, mineralized tissue such as bone or teeth, or biomaterial implant surfaces, this stimulates the hypothesis that opening or closing of mechano-sensitive channels cannot only be triggered by osmotic forces, but also by the forces through which bacteria adhere to a surface [18]. Surface enhanced fluorescent imag-ing of bacteria adherimag-ing to metal surfaces has clearly shown that adhesion is accom-panied by nanoscopic deformation of the bacterial cell wall [19]. The forces by which bacteria adhere to a surface and that induce cell wall deformation, may therewith equally affect lipid membrane surface tension in adhering bacteria as demonstrat-ed for osmotic forces in planktonic ones and affect the gating of mechanosensitive channels.
Chapter
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The aim of this paper is to provide evidence in support of our hypothesis that adhesion to a surface plays a role in the opening and closing of mechanosensitive channels in the important pathogen S. aureus. To this end, we assessed mechanosensitive chan-nel gating by measuring uptake of negatively-charged calcein (MW 623 Da) and dihy-drostreptomycin molecules in staphylococci adhering to different substratum surfac-es. The forces by which staphylococci adhered, as causative to cell wall deformation, were measured using single-bacterial contact probe atomic force microscopy (AFM). Gating was examined on different surfaces by comparing the behavior of a S. aureus strain with mechanosensitive channels and an isogenic ΔmscL mutant.
MATERIALS AND METHODS
Bacterial strains and growth conditions
S. aureus RN4220 and its isogenic ΔmscL mutant were grown on blood agar plates at
37 °C for 24 h. A single colony was used to inoculate 10 mL of Tryptone Soya Broth (TSB; OXOID, Basingstoke, UK), incubated at 37 °C for 24 h. This preculture was then diluted 1:20 in 100 mL of fresh TSB and incubated at 37 °C for 16 h.
Cultures were harvested by centrifugation (5000 × g) and washed twice in phosphate buffered saline (PBS: 10 mM potassium phosphate [5 mM K2HPO4, 5 mM KH2PO4], 0.15 M NaCl, pH 7.0). Staphylococci were subsequently resuspended in PBS and son-icated (3 × 10 s, 30 W) in ice-water bath (Vibra Cell Model 375, Sonics and Materials Inc., Danbury, CT, USA). The bacterial suspension was diluted in PBS to a concentra-tion of 108 bacteria mL-1 as determined in a Bürker-Türk counting chamber. Absence of
mscL genes in the mutant strain was verified by DNA sequencing using Illumina MiSeq
(Illumina Inc., San Diego, CA, USA) as previously explained [20] (Figure 1). Materials preparation and characterization
Polystyrene from 12-well culture plates (Greiner Bio-One, Frickenhausen, Germany) and gold-coated (10 nm thickness) glass slides (DLRI, St. Charles, MO, USA) were used as received after extensive rinsing with demineralized water. Borosilicate glass (Menzel-Gläser, Menzel GmbH&Co KG, Braunschweig, Germany) was cleaned with 2 % Extran, 5 min sonication with 2 % RBS35, methanol and demineralized water. For cre-ating polymer-brush-like surfaces, clean glass slides were exposed to a solution of 0.5 g L-1 Pluronic F-127 (PEO
99PPO65PEO99, molecular weight 12600; Sigma-Aldrich, St.
Louis, MO, USA) in demineralized water for 20 min. Gentle rinsing with demineralized water removed non-attached Pluronic F-127. Coupons of 1 cm2 were prepared to fit
Surface roughnesses were measured with an atomic force microscope (AFM; Bio-Scope Catalyst, Bruker, Camarillo, CA, USA), using ScanAsyst-air tips (tip curvature radius 2 nm; Bruker) to scan areas of 50 × 50 μm at a rate of 1 Hz. Contact angles were measured with water, formamide and methylene iodide, using the sessile drop technique and a home-made contour monitor. Contact angles with these liquids, hav-ing different surface tensions and polarities, allowed calculathav-ing the total surface free energy (γtot), together with its acid-base (γAB) component, which in turn can be divided
in electron-donating (γ_) and accepting (γ+) parameters, and the Lifshitz-Van der Waals
(γLW) component [21]. Surface roughness and contact angles were measured in
tripli-cate on three different material surfaces.
Contact angles on bacterial lawns and surface free energies
Hydrophobicity of bacterial cell surfaces was determined through contact angle mea-surements with different liquids on staphylococcal lawns as described in the above section. Staphylococci were deposited on 0.45 μm pore-size HA membrane fi lters (Millipore Corporation, Bedford, MA, USA) using negative pressure, and fi lters were subsequently dried until reaching constant, so-called “plateau” water contact angles, representing bacterial cell surfaces without “free” but with “bound” water. Contact an-gles were measured on six bacterial lawns from three different bacterial cultures, from which surface free energy components and parameters were then calculated as de-scribed in the above section.
Figure 1. Absence of mscL genes in the ΔmcsL mutant strain verifi ed by DNA sequencing.
Pair-wise comparison of S. aureus RN4220 (upper sequence) and S. aureus RN4220 ΔmscL (lower se-quence). Red areas represent regions with BLASTN matches of 99.9 % in the same orientations, visualized with the Artemis Comparison Tool [23,24].
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Zeta potentials
To measure bacterial zeta potentials, bacteria were resuspended in 10 mM potassium phosphate buffer at different pH values (pH 2, 3, 4, 5, 7). Using the Helmholtz-Smo-luchowski equation [22], zeta potentials were derived from electrophoretic mobilities obtained with particulate microelectrophoresis (Zetasizer Nano ZS, Malvern Instru-ments, Worcestershire, United Kingdom). Experiments were performed in triplicate with different bacterial cultures.
Microbial adhesion to hydrocarbons (kinetic MATH assay)
The combined effects of surface hydrophobicity and charge on staphylococcal ad-hesion to a hydrophobic ligand were determined, as previously described [25]. Briefly, staphylococci were resuspended in 3 mL of 10 mM potassium phosphate buffer at different pH values (pH 2, 3, 4, 5, 7) to an optical density at 600 nm between 0.4 and 0.6 (initial absorbance at time zero [A0]) containing 1:20 hexadecane as spectropho-tometrically measured (Spectronic 20 Genesys, Spectronic Instruments, Rochester, NY, USA). The suspension was vortexed for 10 s and allowed to settle for 10 min, and optical density was measured again (absorbance at time t [At]). This procedure was repeated for five more times to enable calculation of an initial rate of bacterial removal from the aqueous phase defined as
(1) where t is the vortexing time. The experiment was performed in triplicate with different bacterial cultures.
Atomic force microscopy (AFM)
Single-bacterial contact AFM probes were prepared by immobilizing bacteria on NP-O10 tip-less cantilevers (Bruker), as described previously [26]. Briefly, cantilevers were calibrated by the thermal tuning method displaying spring constants in the range of 0.03 - 0.12 N m-1 and mounted on a micromanipulator (Narishige International, Tokyo,
Japan) under microscopic observation (Leica DMIL, Wetzlar, Germany). The cantilever apex was then dipped into a droplet of 0.01 % poly-L-lysine (molecular weight 70,000 to 150,000, Sigma-Aldrich) for 1 min, dried in air for 2 min and dipped into a bacterial suspension droplet (3 × 106 mL-1 in 10 mM potassium phosphate buffer, pH 7.0) for
2 min. Imaging a calibration grid (HS-20MG BudgetSensors, Innovative Solutions Bul-garia Ltd., Sofia, BulBul-garia) with the bacterial probe confirmed single-bacterial contact with the surface [16], and probes yielding double contour lines were discarded (which seldom or never happened).
Formulas Chapter 4
Rate of initial removal= limt → 0 dtdlog (At
A0 x 100) (1) Y = Y0+(Yplateau- Y0)(1- e- F Fc) (2) Formulas Chapter 5 Re = (w+h)v U (1) ∂C ∂t + ∇∙J = Q (2) jo* = 0.538 D∞ C r ( h Pe x ) 1/3 (3) P(zt- 〈zt〉) = A exp (-G(zkt- 〈zBTt〉)) (4) β (t- τ) = β∞- (β∞- β0) exp (-(t- τ)τc ) (5) ΔD (t- τ) = ΔD∞- (ΔD∞- ΔD0) exp (-(t- τ)τc ) (6)
AFM force measurements were done on a BioScope Catalyst AFM (Bruker), at room temperature in 10 mM potassium phosphate buffer. Force-distance curves were ob-tained under a loading force 3 nN at approach and retraction velocity 2 μm s-1, and
taken without and with 10 s bond-maturation. To verify that measurements did not disrupt bacterial integrity, five force-distance curves at a loading force of 3 nN and 0 s contact time were acquired on clean glass at the onset and end of each exper-iment. When adhesion forces differed more than 1 nN from the onset to the end of an experiment, the probe and its last data set obtained were discarded and the probe was replaced by a new one. For each strain, AFM measurements were performed with three probes prepared from three different bacterial cultures. With each probe, three different spots on each material surface were measured, recording five force-distance curves in each spot for each contact time.
Calcein uptake
For measuring the gating of small mechanosensitive channels (MscS), bacterial up-take of the fluorescent dye calcein (Sigma-Aldrich, MW 623) was monitored. First, bac-teria were allowed to sediment from a suspension in PBS (108 mL-1) onto the different
surfaces for 90 min, after which PBS was removed and gentle rinsing with PBS was applied to remove non-adhering bacteria. Subsequently, the bacteria were exposed to calcein 4 mM for 15 min, followed by fixation with 4 % formaldehyde (VWR Interna-tional, Breda, the Netherlands) for 15 min to prevent removal of intracellular calcein. Extensive rinsing in ultrapure water removed the excess extracellular calcein.
As a control, planktonic bacteria were used. Calcein (4 mM) was added to a staph-ylococcal suspension for 15 min, followed by fixation with 4 % formaldehyde for 15 min. Excess extracellular calcein was removed by filtration with 0.45 μm pore-size HA membrane filters (Millipore Corporation). Bacteria were then collected from the filters by vortexing and resuspended in PBS.
Phase-contrast and fluorescent images were acquired with a fluorescence micro-scope (Leica, Wetzlar, Germany) on both adhering and planktonic bacteria, to evalu-ate total number of adhering bacteria or in suspension. The percentage of bacteria that displayed fluorescence in suspension or adhering to a surface was taken as a measure for calcein uptake, according to a protocol previously described for gating in mechanosensitive channels reconstructed in liposomes [27] and validated here for use in bacteria (Table 1). For validation, channels were closed by 15 min exposure to 4 % formaldehyde [28] or closure was induced by 15 min exposure to 100 μM gadolin-ium [29] before calcein exposure.
Chapter
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The increase in the percentage fluorescent staphylococci (Y) as a function of their adhesion forces (F) on the different substratum surfaces was fitted to an exponential function to yield a plateau level of the percentage of fluorescent staphylococci and a critical adhesion force, according to
(2) in which Y0 is the percentage fluorescent staphylococci in suspension (zero adhesion force) and Fc is the critical adhesion force. Yplateau is the plateau level of the percentage fluorescent staphylococci reached.
To allow more accurate fitting, the plateau level was mathematically confined to the maximum percentage of adhering, fluorescent staphylococci observed for each strain. Experiments were performed in triplicate with different bacterial cultures.
Table 1. Validation of calcein uptake protocol for assessing staphylococcal channel gating.
Fluo-rescence of planktonic staphylococci and staphylococci adhering in polystyrene wells due to up-take of the fluorescent molecule calcein prior to and after exposure of bacteria to formaldehyde [28] as a channel blocker or gadolinium to induce channel closure [29–32]. ± signs represent standard deviations over 9 different fluorescence images obtained from three different bacterial cultures.
Bacterial state Exposure to % fluorescent staphylococci
S. aureus RN4220 S. aureus RN4220
ΔmscL
Planktonic Calcein Formaldehyde 3 ± 4 7 ± 7
Planktonic Formaldehyde Calcein 9 ± 8 13 ± 4
Adhering Calcein Formaldehyde 45 ± 11 45 ± 11
Adhering Formaldehyde Calcein 6 ± 3 9 ± 5
Adhering Gadolinium Calcein 9 ± 3 6 ± 2
Dihydrostreptomycin uptake
First, minimal inhibitory (MIC) and bactericidal concentrations (MBC) of the staph-ylococci for dihydrostreptomycin were determined. To this end, bacterial cultures (105 mL-1 in TSB) were dispensed in a 96-well microtiter plate with
dihydrostreptomy-cin sesquisulfate (Sigma-Aldrich, MW 731) in TSB with known concentrations and ap-plying a step factor dilution of 2 starting from 512 μg mL-1. Incubation was done at
37 °C for 24 h. After incubation, MIC was taken as the lowest antibiotic concentra-tion not generating visible turbidity. Then, 10 μL of bacterial suspensions of each well Formulas Chapter 4
Rate of initial removal= limt → 0 dtdlog (At
A0 x 100) (1) Y = Y0+(Yplateau- Y0)(1- e- F Fc) (2) Formulas Chapter 5 Re = (w+h)v U (1) ∂C ∂t + ∇∙J = Q (2) jo* = 0.538 D∞ C r ( h Pe x ) 1/3 (3) P(zt- 〈zt〉) = A exp (-G(zkt- 〈zBTt〉)) (4) β (t- τ) = β∞- (β∞- β0) exp (-(t- τ)τc ) (5) ΔD (t- τ) = ΔD∞- (ΔD∞- ΔD0) exp (-(t- τ)τc ) (6)
showing no turbidity, were plated on TSB agar plates and incubated at 37 °C for 24 h. The MBC was taken as the lowest concentration at which no colonies were visible on the plate. Experiments were performed in triplicate with different bacterial cultures. To evaluate the differential uptake of dihydrostreptomycin through MscL channels [12] in adhering and planktonic staphylococci, bacterial suspensions (108 mL-1 in PBS)
were allowed to sediment on different surfaces for 90 min or maintained planktonical-ly in suspension. After subsequent exposure to dihydrostreptomycin (512 μg mL-1) in
PBS for 120 min, bacteria were collected by 1 min sonication in water bath and serially diluted in PBS. Exposure to PBS was applied as a control. Next, 10 μL aliquots were plated on TSB agar plates and incubated for 16 h at 37 °C. The number of colonies formed on the plates was manually counted.
The reduction in the number of CFUs per mL as a function of their adhesion forces F on the different substratum surfaces was fitted to an exponential function to yield a plateau level of the reduction in the number of CFUs per mL and a critical adhesion force, similar as done for the percentage fluorescent staphylococci (Equation 2). To allow more accurate fitting, the plateau level was mathematically confined to the max-imum percentage of reduction in the number of CFUs per mL observed for each strain. Experiments were performed in triplicate with different bacterial cultures.
Statistical analysis
GraphPad Prism, version 7 (San Diego, CA, USA) was employed for statistical analysis. Data were tested for normal distribution with Shapiro-Wilk normality test. If data were normally distributed, one-way analyses of variance (ANOVA) with Tukey’s HSD post-hoc test or a two-tailed Student’s t-test were employed. When data were not normally distributed, Kruskal-Wallis test with Dunns’ approximation replaced ANOVA. p < 0.05 was used as significance for all tests.
RESULTS
Physico-chemical characterization of substratum and bacterial cell surfaces Physico-chemical properties of the substratum surfaces used, as well as of the parent strain, S. aureus RN4220 and its isogenic ΔmscL mutant, were first evaluated. Sub-stratum surfaces had slightly different roughnesses that were all in the nm-range be-tween 3 nm for gold to 13 nm for polystyrene (Table 2; one-way ANOVA, p < 0.0161, F = 6.238, df = 16), which can be considered sufficiently smooth not to influence contact angles with liquids [33]. Water contact angles were significantly (p < 0.001,
Chapter
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F = 463, df = 17) smaller on glass and glass with a Pluronic-coating, as compared to more hydrophobic polystyrene and gold. The differences in contact angles among the three liquids led to higher (p < 0.001) electron-donating parameters and total surface free energies for glass and Pluronic-coated glass as compared to polystyrene and gold (Table 2).
Both S. aureus strains showed extremely small initial removal rates from an aqueous phase by hexadecane (Figure 2A), which indicates either a hydrophilic bacterial cell surface, electrostatic double-layer repulsion between staphylococci and hexadecane, or both [34]. To assess the contribution of electrostatic double-layer interactions, bac-terial zeta potentials were measured as a function of pH (Figure 2B). Both strains had negative zeta potentials between pH 2 to 7. Note that bacterial zeta potentials became closer to zero towards pH 2, concurrent with a slight increase in removal rate by hexa-decane due to decreased electrostatic double-layer repulsion. At pH 7, used in our gating experiments, both strains had a similar (two-tailed Student’s t-test, p = 0.7415, t = 0.3535, df = 4) zeta potential of -26 mV (Figure 2B). Water contact angles on bac-terial lawns indicated that all strains were hydrophilic with water contact angles less than 60 degrees. Surface thermodynamic analysis of the contact angles measured with the three different liquids on each bacterial strain, generated a higher electron-do-nating component for the ΔmscL mutant compared to the parent strain (p = 0.0254, t = 2.551, df = 12), and minor differences (Figure 2C; p > 0.05) in other surface free energy parameters and components, ultimately resulting in a similar total free energy for both strains (Figure 2C; p = 0.8941, t = 0.1359, df = 12). In line with these physi-co-chemical properties of the parent and mutant strain, initial adhesion forces of the different staphylococci, i.e. measured before bond-maturation, were comparable (Ta-ble 3). Taken together, the physico-chemical properties of the surfaces of S. aureus RN4220 and its isogenic mutant can be considered highly similar with negligible dif-ferences of no biological signifi cance. This implies that the cell surface in both staph-ylococcal strains is identical and that deletion of the mscL gene did not affect the outermost cell surface.
Figure 2. Physico-chemical properties 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 (θw), formamide (θf), methylene iodide (θm) and staphylococcal surface free energy parameters and components. Total surface free energy γtot
results from Lifshitz-Van der Waals γLW and acid-base γAB 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 different bacterial cultures.
Table 2. Physico-chemical surface properties of the substratum materials. Surface roughness,
contact angles measured with water (θw), formamide (θf), and methylene iodide (θm), and surface free energy parameters and components. Total surface free energy γtot results from Lifshitz-Van
der Waals γLW and acid-base γAB components, and electron-donating γ_ and electron-accepting
γ+ parameters. ± signs represent standard deviations over measurements on three different
sur-faces.
Polystyrene Gold Glass Pluronic-coated glass Roughness (nm) 13 ± 5 3 ± 1 4 ± 1 8 ± 3 θw (degrees) 80 ± 2 82 ± 3 10 ± 1 9 ± 3 θf 64 ± 2 65 ± 4 10 ± 1 14 ± 4 θm 35 ± 1 54 ± 2 34 ± 3 45 ± 2 γ– (mJ m-2) 10 ± 4 5 ± 2 55 ± 1 56 ± 2 γ+ 0 ± 0 0.3 ± 0.4 0.9 ± 0.2 2 ± 0.1 γAB 0 ± 0 2 ± 1 14 ± 1 19 ± 0.4 γLW 42 ± 0.3 32 ± 1 43 ± 1 37 ± 1 γtot 42 ± 0.3 33 ± 2 57 ± 0.2 56 ± 1
Chapter
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94
Bacterial adhesion forces
Adhesion forces between staphylococci and substratum surfaces were measured with single-bacterial contact probe AFM, allowing 10 s for the bond between adher-ing staphylococci and substratum surfaces to mature after initial contact. Among the different substratum surfaces, gold generated the strongest adhesion forces for both strains, while the weakest forces were recorded on Pluronic-coated glass (Figure 3 and Table 3). Note that the range of force values observed over the different substrata after bond-maturation was relatively small in S. aureus RN4220 (1.6 nN on Pluronic-coated glass to 5.4 nN on gold), while being largest in the ΔmscL mutant (0.5 to 14.6 nN). Uptake of calcein in staphylococci
Uptake of calcein through mechanosensitive channels in adhering and planktonic staphylococci was evaluated by exposing bacteria to the relatively small fluorescent dye calcein, and enumerating the number of fluorescent bacteria, as previously de-scribed for liposomes [27]. To ensure similar action of calcein in staphylococcal gating as in its liposome uptake [27], it was first assessed that all channels of small and large conductance were closed in planktonic staphylococci and calcein uptake was absent. Indeed, after staphylococcal exposure to calcein in the planktonic state and subsequent channel blocking by formaldehyde [28] to prevent calcein outflow, low per-centages (3 to 7 %) of fluorescent staphylococci were observed (Table 1). Incidentally, also blocking of channels with formaldehyde prior to calcein exposure of bacteria in the planktonic and adhering state, yielded a low percentage of fluorescent staphylo-cocci (6 to 13 %). Exposure of staphylostaphylo-cocci adhering to polystyrene to calcein on the other hand, yielded 45 % fluorescent bacteria, in the parent strain and ΔmscL mutant, indicating calcein uptake regardless of the possession of large conductance channels suggesting that calcein uptake can take place equally well through MscS as through MscL. Inducing channel closure with gadolinium, as common in liposomes [32,35] and bacteria [30,31], prior to exposure to calcein, prevented uptake of the fluorescent dye, as evidenced by low percentages (6 to 9 %) of adhering, fluorescent staphylococci (see also Table 1).
Considering planktonic staphylococci as bacteria that experience a zero adhesion force, it can be seen that the percentage bacteria becoming fluorescent due to uptake of calcein in suspension or adhering to the different surfaces increased exponentially with the adhesion force exerted by the substratum surface on the adhering bacteria, regardless of the strain considered. An exponential fit of the data yielded plateau levels of fluorescent staphylococci and critical adhesion forces that differed for the parent strain and the ΔmscL mutant (Figure 3). Plateau levels of calcein fluorescence were achieved at different critical adhesion forces (see Equation 2; Table 4). The parent
strain achieved the highest plateau level of fluorescent bacteria (66 %) at the highest critical adhesion force (4.1 nN), likely because both small and large channels were available for calcein uptake. The plateau level of fluorescent staphylococci of the par-ent strain was therewith signifi cantly higher (p = 0.0007) than of the ΔmscL mutant in which only small channels are available for calcein uptake, which was reached at a signifi cantly (p = 0.0026) higher critical adhesion force than observed for the ΔmscL mutant.
Table 3. Staphylococcal adhesion forces before and after 10 s bond-maturation. Initial ad-hesion forces Fi between staphylococci and substratum surfaces, and adhesion forces after
10 s bond-maturation F10 s . ± signs represent standard deviations over at least 45 force-distance
curves, comprising 9 different spots on each surface and three probes prepared from three dif-ferent bacterial cultures. * indicates signifi cant differences from S. aureus RN4220 (Kruskal-Wal-lis test, * p < 0.05, ** p < 0.01). S. aureus RN4220 RN4220 ΔmscLS. aureus Fi (nN) F10 s (nN) Fi (nN) F10 s (nN) Polystyrene 0.4 ± 0.4 3.1 ± 2.3 0.3 ± 0.2 3.0 ± 2.4 Gold 1.0 ± 0.8 5.4 ± 2.7 2.2 ± 1.5 ** 14.6 ± 8.6 * Glass 0.9 ± 0.6 3.5 ± 2.4 0.6 ± 0.3 3.4 ± 1.9 Pluronic-coated glass 0.3 ± 0.2 1.6 ± 1.0 0.2 ± 0.2 0.5 ± 0.6 **
Figure 3. Uptake of calcein in S. aureus RN4220 and the mutant S. aureus RN4220 ΔmscL. Percent-age fluorescent planktonic and adhering staphylococci due to uptake of the fluorescent molecule calcein as a function of adhesion force. Lines indicate an exponential fi t 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 surface. Dashed lines indicate the 95 % confi dence band. Horizontal error bars represent standard deviations over at least 45 force-distance curves, comprising 9 different spots on each surface and three probes prepared from three different bacterial cultures. Vertical error bars represent standard deviations over 9 different fluorescence images obtained from three different bacterial cultures.
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Bacterial susceptibility and uptake of dihydrostreptomycin in staphylococci
To evaluate the uptake of dihydrostreptomycin through mechanosensitive channels, adhering and planktonic staphylococci were exposed to 512 μg mL-1 of
dihydrostrep-tomycin for 2 h, well above the minimal inhibitory (MIC) and bactericidal (MBC) con-centrations of dihydrostreptomycin for S. aureus RN4220 (MIC and MBC are 8 μg mL-1)
and S. aureus RN4220 ΔmscL (MIC and MBC are 16 μg mL-1). High concentrations of
dihydrostreptomycin were applied to provoke a fast reduction in the number of viable staphylococci. The reduction in the number of CFUs per mL in planktonic staphylococ-ci due to antibiotic exposure was directly compared to a buffer control, while adher-ing bacteria were dispersed in buffer immediately after antibiotic or buffer exposure before CFU determination and calculation of the reduction in the numbers of CFUs achieved. Similar as for staphylococcal fluorescence due to calcein uptake, staphy-lococcal killing due to dihydrostreptomycin uptake also increased exponentially with adhesion force (Figure 4). The parent strain exhibited a signifi cantly different behavior than the mutant strain. In the parent strain, an exponential increase in staphylococcal killing due to dihydrostreptomycin uptake was observed and, after fi tting, a stationary CFU reduction of 2.4 log-units was achieved, which is signifi cantly (p = 0.0073) more than the killing observed for the ΔmscL mutant, suggesting uptake of dihydrostrepto-mycin requires large channels, despite its similar size as calcein that is being taken up through large and small channels. Interestingly, whereas killing in the ΔmscL mu-tant was independent of the forces by which they adhered (“infi nite” critical adhesion force), the critical adhesion force with respect to killing due to dihydrostreptomycin uptake in the parent strain (3.6 nN) was similar as observed for critical adhesion force with respect to gating allowing calcein uptake (Figure 4 and Table 4).
Figure 4. Uptake of dihydrostreptomycin in S. aureus RN4220 and the mutant S. aureus RN4220
ΔmscL. Reduction in CFUs (log mL-1) of planktonic and adhering staphylococci after 2 h
expo-sure to dihydrostreptomycin, expressed relative to expoexpo-sure to PBS buffer, as a function of ad-hesion force. Lines indicate an exponential fi t used to derive plateau levels and critical adad-hesion forces. Dashed lines indicate the 95 % confi dence band. Horizontal error bars represent standard deviations over at least 45 force-distance curves, comprising 9 different spots on each surface and three probes prepared from three different bacterial cultures. Vertical error bars represent standard deviations over three measurements from three different bacterial cultures.
S. aureus
RN4220 RN4220 ΔmscLS. aureus parameters (t; df)Student‘s t-test
Calcein molecule uptake
Plateau fl uorescence (%) 66 ± 16 40 ± 9 *** 4.171; 16
Critical force Fc (nN) 4.1 ± 1.4 1.2 ± 2.0 ** 3.564; 16
Dihydrostreptomycin uptake Plateau killing
(reduction in log-units CFUs) 2.4 ± 0.4 1.0 ± 0.2 ** 5.027; 4 Critical force (nN) 3.6 ± 4.8 independent of
adhesion forcea
-amathematically indicating an “infi nite” critical adhesion force.
DISCUSSION
This chapter demonstrates that mechanosensitive channels in S. aureus are not only opened by changes in membrane surface tension due to osmotic forces but also by the forces involved in their adhesion to surfaces. Assessing channel gating by the uptake of the negatively-charged molecule [36] calcein and similarly-sized but positive-ly-charged dihydrostreptomycin, it was found that calcein can be transported through both small and large channels, while dihydrostreptomycin can only be transported through large channels. Considering the range over which electrostatic attraction can occur as being equal to the Debye-Huckel length (7.5 Å in PBS), this implies that pos-itively-charged dihydrostreptomycin will be adsorbed to the walls of channels with a diameter of around 15 Å, i.e. the small channel diameter reported for E. coli [10], pre-venting its transport through small channels. Transport through the middle of large channels can occur without such adsorption. Therewith, dihydrostreptomycin can be taken as a large molecule analogue and its uptake indicative of large channel opening. It was found that small channels typically open upon exposure to a critical adhesion
Table 4. Plateau uptake levels and critical adhesion forces derived from exponentially fi tting the
relation between bond-matured adhesion forces and uptake of calcein and dihydrostreptomycin. Plateau levels for the percentage of fluorescent staphylococci after calcein uptake and the CFU reduction due to dihydrostreptomycin uptake and critical adhesion forces for each of the two staphylococcal strains used. ± signs represent standard deviations over 9 different fluorescence images obtained from three different bacterial cultures for calcein uptake, and three measure-ments from three different bacterial cultures for dihydrostreptomycin uptake. * indicates signif-icant differences from S. aureus RN4220 (two-tailed Student’s t-test, ** p < 0.01, *** p < 0.001). T-values and degrees of freedom (df) of the Student’s t-test are indicated in the last column.
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force of 1.2 nN, while large channels opened upon exposure to a significantly higher force of 3.6 nN. Interestingly, plateau levels of the percentages of fluorescent bacteria in parent and isogenic mutant populations were less than 100 %, consistent with the observation that the number of mechanosensitive channels in each bacterial cell var-ies remarkably within the same culture [37]. Bacteria having more channels need lower critical membrane tension to open at least one channel, compared to bacteria having less channels [37]. Higher plateau levels of the percentages of fluorescent bacteria observed in the parent compared to the mutant strain indicate that, with increasing ad-hesion forces between bacteria and surfaces, calcein uptake occurs through MscSs, in addition to uptake through MscLs.
Adhesion forces modulate mechanosensitive channel gating by provoking deforma-tion of staphylococcal cell wall upon bacterial adhesion on a surface. Stronger ad-hesion forces generate larger cell wall deformation [38]. As a component of the cell wall, the cytoplasmic lipid membrane is also deformed, and the density of lipids in the membrane decreases [19], producing a surface tension change in the membrane that triggers mechanosensitive channels opening (Figure 5). Translation of critical adhesion forces into critical membrane surface tension changes is impossible, be-cause the necessary rigidity data of the cell wall are not available and the influence of the “cushioning” effect [39] of extracellular cell surface components on deformation [19] is unknown. Moreover, direct comparison of critical adhesion forces with critical membrane surface tensions required to open mechanosensitive channels as report-ed in the literature dependreport-ed on whether derivreport-ed from channels in whole bacteria or channels reconstructed in liposomes [11] or spheroplasts [40]. Importantly, gating in reconstructed channels depended on membrane properties like membrane thickness [7], hydrophobicity [41] and type of lipids [7]. In whole E. coli and S. aureus, the critical gating tension for MscS was two-fold smaller than for MscL [41]. Our results indicate that the critical adhesion force for MscS gating is three-fold smaller than for MscL gating in S. aureus, which might be considered comparable.
The critical adhesion forces required for small and large conductance channel gating differed significantly in the ΔmscL mutant, with the critical force required to open small channels being 1.2 nN, and the larger channels requiring an infinitely high force to open due to the absence of the mscL genes. By comparison, these results show that ΔmscL mutant cell wall becomes more readily deformed already at lower adhesion forces than the parent strain cell wall, because of the absence of MscL channel pro-teins, which can affect membrane stiffness [42].
Collectively, our results demonstrate that mechanosensitive channels opening in-creases with increasing adhesion forces, that were varied in this work by allowing the staphylococci to adhere to different material surfaces. Staphylococcal adhesion forc-es after bond maturation for 10 s and as rforc-esponsible for cell wall deformation, were stronger on hydrophobic surfaces than on hydrophilic surfaces due to hydrophobic interactions with hydrophobic groups located on the outermost cell surface, such as proteins [43], fatty acids [44] or eDNA [45]. In a more generalized approach towards infection, the role of adhesion forces experienced by the staphylococci as arising from their adhesion to a material surface, is taken over by the forces by which they adhere to tissue cells, or other microbial cell surfaces. In conclusion, it has been demonstrated that channel gating in S. aureus is not solely triggered by osmotic forces, but also by the forces by which they adhere to surfaces, as occurring in infections of various kinds.
Figure 5. Schematic presentation of the effect of adhesion and adhesion forces on the bacterial
cell wall and mechanosensitive channels gating. Planktonic bacteria in the absence of osmotic stress do not experience adhesion forces and lipid surface tension is such that mechanosensi-tive channels are closed. Upon adhesion to a surface, the cell wall deforms under the influence of adhesion forces arising from a substratum surface and cell wall deformation causes changes in lipid membrane surface tension that are accompanied by opening of mechanosensitive channels.
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ACKNOWLEDGEMENTS
This study was entirely funded by the University Medical Center Groningen, Groningen, The Netherlands. Prof. dr. Armagan Kocer, Department on Neuroscience, University Medical Center Groningen, is gratefully acknowledged for the useful discussions on mechanosensitive channels. We are grateful to Prof. dr. Jan M. van Dijl, Department of Medical Microbiology, University Medical Center Groningen, for providing the strains S.
aureus RN4220 and S. aureus RN4220 ΔmscL, and Dr. John W.A. Rossen, Department
of Medical Microbiology, University Medical Center Groningen, for his contributions in genetic sequencing to verify the ΔmscL mutant. H.J.B. is also a director of a consulting company, SASA BV. We declare no potential conflicts of interest with respect to author-ship and/or publication of this article. The opinions and assertions contained herein are those of the authors and are not construed as necessarily representing the views of the funding organization or the authors’ employers.
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