Pseudomonas aeruginosa Biofilm Dispersion by the Human Atrial Natriuretic Peptide

Abstract Pseudomonas aeruginosa biofilms cause chronic, antibiotic tolerant infections in wounds and lungs. Numerous recent studies demonstrate that bacteria can detect human communication compounds through specific sensor/receptor tools that modulate bacterial physiology. Consequently, interfering with these mechanisms offers an exciting opportunity to directly affect the infection process. It is shown that the human hormone Atrial Natriuretic Peptide (hANP) both prevents the formation of P. aeruginosa biofilms and strongly disperses established P. aeruginosa biofilms. This hANP action is dose‐dependent with a strong effect at low nanomolar concentrations and takes effect in 30–120 min. Furthermore, although hANP has no antimicrobial effect, it acts as an antibiotic adjuvant. hANP enhances the antibiofilm action of antibiotics with diverse modes of action, allowing almost full biofilm eradication. The hANP effect requires the presence of the P. aeruginosa sensor AmiC and the AmiR antiterminator regulator, indicating a specific mode of action. These data establish the activation of the ami pathway as a potential mechanism for P. aeruginosa biofilm dispersion. hANP appears to be devoid of toxicity, does not enhance bacterial pathogenicity, and acts synergistically with antibiotics. These data show that hANP is a promising powerful antibiofilm weapon against established P. aeruginosa biofilms in chronic infections.

aeruginosa PA14 pre-formed biofilms in flow cell system. a, 3D-shadow representations of 24 h-old pre-formed P. aeruginosa PA14 biofilm structures unexposed (control condition) or exposed (30 min) to hANP (10 nM or 1 nM). b, COMSTAT image analyses of biofilms structures of P. aeruginosa PA14 control or exposed for 30 min to hANP (100 nM, 10 nM, 1 nM or 0.1 nM). Data are the result of the analysis of 9 views from three independent biological experiments (hANP 100 nM), 9 views from three independent biological experiments (hANP 10 nM), 28 measurements from nine independent experiments (hANP 1 nM), and 9 views from three independent biological experiments (hANP 0.1 nM). Statistics were achieved by ordinary one-way ANOVA followed by Dunnett's multiple-comparison test. Asterisks indicate values that are significantly different as follows: ****, P < 0.0001. aeruginosa PAK biofilm untreated (control condition) or treated to hANP at 10 nM for 2 h. b, COMSTAT image analyses of biofilms of P. aeruginosa PAK upon exposure to hANP (10 nM). c, 3D-shadow representations of the 24 h pre-formed P. aeruginosa H103 biofilm untreated (control condition) or treated to hANP at 10 nM for 2 h. d, COMSTAT images analyses of biofilm structures of P. aeruginosa H103 upon exposure to hANP (10 nM). Data are the result of the analysis of three independent biological assays (n=3) with three views each.
Statistics were achieved by a two-tailed t test. Asterisks indicate values that are significantly different as follows: ****, P < 0.0001. aeruginosa CF 8.19 and CF 9.19 clinical strains upon exposure (2h) to hANP at 100 nM and 10 nM. Data are the result of the analysis of three independent biological assays (n=3) with three views each. Statistics were achieved by ordinary one-way ANOVA followed by Dunnett's multiple-comparison test. Asterisks indicate values that are significantly different as follows: ****, P < 0.0001; ns , not significant (P ≥ 0.05). b, COMSTAT image analyses of 24 h established biofilms of P. aeruginosa PAL 0.1 and PAL 1.1 clinical isolates upon exposure (2h) to hANP at 100 nM and 10 nM. Data are the result of the analysis of three independent biological assays (n=3) with three views each. Statistics were achieved by ordinary one-way ANOVA followed by Dunnett's multiple-comparison test. Asterisks indicate values that are significantly different as follows: ****, P < 0.0001. c, COMSTAT image analyses of 24 h preformed MUC-N1, MUC-N2, MUC-P4, and MUC-P5 P. aeruginosa clinical strains untreated (control condition) or treated to hANP at 10 nM for 2 h. Data are the result of the analysis of three independent biological assays (n=3) with at least three views each unless for MUC-N1 isolate for which only two independent biological assays were performed. Statistics were achieved by a two-tailed t test. Asterisks indicate values that are significantly different as follows: **, P < 0.01; ****, P < 0.0001; ns , not significant (P ≥ 0.05). color-coded as follow: PA14 strain used as control (blue), PA14 exposed to hANP (1 µM) (orange) and PA14 exposed to hANP (0.1 µM) (grey). Results are the mean of eight replicates from three independent experiments. b, Membrane fluidity of P. aeruginosa PA14 exposed to hANP. The fluorescence anisotropy values (r) for PA14 strain used as control (blue bar) and PA14 exposed to hANP at 0.1 µM (orange bar). The error bars represent the standard error of the means (SEMs) and are the result of the analysis of three independent biological assays with eight measurements each. Statistics were achieved by a two-tailed t test. ns , not significant (P ≥ 0.05). c, d, and e, Effect of hANP on 24 h-old biofilm cell viability. Quantification of alive and dead cells was assessed using the Live/Dead BacLight kit. structures unexposed (control condition) or exposed to ANP (1 nM) for 2 h at 37°C. Bacterial cells within biofilms were stained using SYTO9. α polysaccharides were stained using Concanavalin A. Proteins were stained using SYPRO ruby. Polysaccharides and Proteins values are normalized to biofilm biomass. Data are the result of the analysis of nine views from three independent biological experiments (n=3). Statistics were achieved by a two-tailed t test.

Fig. 6. Ligplot image representing the interaction between hANP and AmiC.
Interactions between the human ANP peptide and the AmiC P. aeruginosa protein were determined using LigPlot+ (Laskowski and Swindells, 2011). Key: green dotted lines: hydrogen bonds between a side chain and main chain; pink dotted lines: salt bridge; pink dashed arc: hydrophobic interactions (hANP); red dashed arc: hydrophobic interactions (AmiC). The direction of the arcs indicates the residues on the other partner that the hydrophobic interactions are formed between.

Percentage of lactate dehydrogenase (LDH) released by A549 lung cells after 5 h infection with
P. aeruginosa PA14 unexposed (control; blue bar) or exposed to various concentrations of hANP (orange bars). Data are the result of the analysis of three independent biological assays with four replicates each. Statistics were achieved by ordinary one-way ANOVA followed by Dunnett's multiple-comparison test. ns , not significant (P ≥ 0.05). b, HCN/CNproduction in PA14 control condition (dark blue bar), PA14 exposed to hANP at 1 µM (brown bar), 100 nM (orange bar), and 10 nM (light orange bar). Data are the result of the analysis of three independent biological assays with three replicates each. Statistics were achieved by ordinary one-way ANOVA followed by Dunnett's multiple-comparison test. ns , not significant (P ≥ 0.05). c, Kaplan-Meier survival plots of C. elegans challenged by PA14 unexposed to hANP (black squares; n = 194) or exposed to hANP at 1 µM (red diamonds; n = 228), 0.1 µM (inversed green triangle; n = 192) or 10 nM (blue diamonds; n = 173). For each condition, the median survival was obtained at 6 h.

Caenorhabditis elegans virulence assay
The Caenorhabditis elegans wild-type Bristol strain N2 was obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA) which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). C. elegans were maintained under standard culturing conditions at 22°C on nematode growth medium agar plates with Escherichia coli OP50 as a food source (Sulston and Hodgkin, 1988). Synchronous worm cultures were generated as previously described (Blier et al. 2011). Bacterial virulence was evaluated using the liquid killing assay as previously described with slight modifications (Chua et al., 2014).
Briefly, from an overnight bacterial growth, P. aeruginosa PA14 was inoculated at OD580 nm = 0.08 into LB supplemented with hANP as appropriate. After two hours, bacteria were collected by centrifugation (2 min at 8 000 g), cells were resuspended into CELKA medium to OD580nm = 0.1 and used for virulence assay on C. elegans (Chua et al., 2014). Twenty to twenty-five L4 (48 hours old) synchronized worms, harvested with M9 solution, were added to 80 µL of P.
aeruginosa PA14 previously exposed to hANP (at different concentrations) and non-exposed controls and incubated at 22°C. Worm survival was scored at 1 hour and each following hour using an Axiovert S100 optical microscope (Zeiss, Oberkochen, Germany). The worms were considered dead when they remained static without grinder movements for 20 s or did not respond to light flashes. Results are expressed as a percentage of living worms. The results are the average of three independent assays. For the C. elegans killing assay, nematode survival was calculated by the Kaplan-Meier method (GraphPad Prism version 9.0; GraphPad Software, San Diego, California, USA).

Hydrogen cyanide assay
Hydrogen cyanide concentration in P. aeruginosa PA14 cultures, unexposed (control condition) or exposed to various concentrations of hANP, was determined by the polarographic technique described by Blier et al. (2012). Bacterial cultures were centrifuged 10 min at 8000 g and the supernatant was passed through a 0.22 µm filter to remove all bacterial bodies. The filtered supernatant was then diluted in 0.2 mol.L -1 borate electrolyte (pH=10.2). The solution was purged with N2 for 3 min to remove dissolved oxygen and then for 20 s more between each addition of cyanide potassium (KCN) that was used as an internal standard. A scan of the electric potential was carried out in the cathodic negative sense from -0.1 V to -0.5 V with a sweep rate of 10 mV/s. The pulse amplitude was 0.05 V with a pulse duration of 0.04 s. The peak height of cyanide was measured at -200 mV in a differential pulse mode and the cyanide concentration was determined by the addition of 4 successive aliquots of 10 mg.L -1 KCN standards.

Cell culture
The human A549 lung epithelial cell type II line (ATCC-CCL185TM) (American Type Culture Collection Manassas, VA) was grown at 37°C in 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM, Lonza) supplemented with 10% fetal calf serum (Lonza) and 1% (w/v) penicillin and streptomycin (Penistrep, Lonza). Routinely, cells were seeded in a 25 mL flask and used at 80% confluence. For cytotoxicity assays, cells were seeded in 24 well plates at a final density of 3x10 5 cells per well and grown for 48 h before use. A minimum of 24 h before infection assays, cells were deprived of antibiotics and fetal calf serum by addition of a fresh serum-free medium.

Measurement of the release of cytosolic lactate dehydrogenase (LDH) by A549 cells
Lactate dehydrogenase (LDH) is a stable cytosolic enzyme released into the culture medium after cell lysis. The amount of LDH released by eukaryotic cells in the presence of the bacteria, exposed to various concentration of hANP, was determined using the Cytotox 96 enzymatic assay (Promega, Charbonnières, France). A549 cells were incubated for 6 h with control or pre-treated P. aeruginosa PA14 at a multiplicity of infection of 10. A lysis buffer, consisting of a solution of Triton X-100 (9 % (v/v) in water), was employed to determine the maximum LDH release by A549 cells in our experimental conditions (100 % LDH release). A background level was established using culture medium alone, and defined as 0 % LDH release, to eliminate the contribution of the culture medium. The percentage of LDH release in the cell population was then calculated using the equation: The assay was sufficiently sensitive to measure a concentration of LDH equivalent to the lysis of 1 % of the cell population.

Determination of hANP binding on human ether-a-go-go (hERG) channel
Affinity of hANP for the hERG channel was determined using the Predictor™ hERG fluorescence polarization (FP) assay kit (Thermo Scientific) following the manufacturer's

Membrane fluidity
Fluorescence anisotropy analysis of P. aeruginosa PA14 unexposed (control condition) or exposed to hANP was performed as described by Vincent et al. (Vincent et al., 2004) with a few modifications. P. aeruginosa PA14 strain was grown in 10 mL LB broth at 37°C under agitation (180 rpm). After 2 h growth, the bacterial cultures were untreated (control condition) or hANP-treated at the required concentration. The bacterial cultures were allowed for an additional 3 h incubation. Bacterial cells were then harvested by centrifugation (5 min, 7500 g), washed twice in 10 mM MgSO4 and resuspended in the same wash solution to an OD580 of 0.1.
Next, 1 µL of a 4 mM stock solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) (Sigma-Aldrich) in tetrahydrofuran was added to a 1 mL aliquot of the resuspended cultures and incubated in the dark for 30 min at 37°C to allow the probe to incorporate into the membrane bilayer.
Measurement of the fluorescence polarization was performed using the Spark 20M multimode Microplate Reader (Tecan) equipped with the Te-Cool TM active temperature regulation system.
Excitation and emission wavelengths were set to 365 and 425 nm, respectively, and the anisotropy (r) was calculated according to Lakowicz (Lakowicz, 2006). Data were recorded using SparkControl TM software (Version 2.1, Tecan). Fluorescence polarization and membrane fluidity are inversely related since increasing anisotropy values correspond to a less fluid membrane environment and vice versa. All values reported for each sample are means of eight measurements of at least three independent experiments.