A cranberry extract rich in proanthocyanidins inhibits P. aeruginosa virulence towards D. melanogaster

Treatment with cerPAC significantly inhibited the staphylolytic (LasA, F 3,8 = 21.41, p < 0.001), elastolytic (LasB, F 3,8 = 84.29, p < 0.001) and alkaline proteolytic (AprA, F 3,8 = 34.41, p < 0.001) activities of P. aeruginosa PA14 (Fig. 1a). Importantly, this inhibition was achieved without affecting bacterial growth (Fig. 1b).

Figure 1 (a) Inhibition of virulence determinants and (b) growth curves of P. aeruginosa PA14 in absence or presence of different cerPAC concentrations. LasA: staphylolytic protease, LasB: elastase and AprA: alkaline protease. Results are expressed as means and standard deviations (SD) of triplicate enzyme assays (*p < 0.001). Bacterial growth (OD 600 ) was monitored at 37 °C for 18 h in TSB medium. Error bars with average data points of growth kinetics represent the standard deviation of values obtained from four replicates. Abbreviations: cerPAC x, Cranberry extract rich in proanthocyanidins at x μg mL−1 (e.g., cerPAC 300 indicates cerPAC at 300 μg mL−1). Full size image

Next, to verify whether cerPAC can limit infection in vivo, we used a fruit fly killing assay in which we administered cerPAC to Drosophila melanogaster infected with WT P. aeruginosa PA14. As shown in Fig. 2, the median survival of D. melanogaster after exposure to P. aeruginosa was 168 h without cerPAC, but 240 h with cerPAC treatment, which is significantly (χ2 = 4.14, df = 1, p < 0.05) less virulence based on the comparison of survival curves. The survival of uninfected D. melanogaster was identical to the treatment with only cerPAC.

Figure 2 Virulence of P. aeruginosa PA14 towards D. melanogaster in absence or presence of cerPAC (200 μg mL−1). Mortality was scored daily for 14 days. Results represent measurements from experiments performed with triplicates, twice (*p < 0.05). Full size image

The difference in the treated or untreated PA14 strains’ ability to kill D. melanogaster in this feeding assay may have been due to modified survival of the bacteria on the filter papers used for exposure during incubation. To address this possibility, we analyzed the survival of PA14 on the paper discs without and with 200 μg mL−1 cerPAC under the same conditions as the fly feeding assay. There was no significant difference (F 5,30 = 0.54, p = 0.74) in culturability of the bacterium on the filter paper discs in the absence and presence of cerPAC during incubation (see Supplementary Fig. S1), indicating that an alteration in survival ability of bacteria could not account for the observed differences in fly killing. Overall, these results indicate that cerPAC protect D. melanogaster from P. aeruginosa infection.

Cranberry extract rich in proanthocyanidins modulates the AHL-mediated quorum sensing system in P. aeruginosa PA14

Since QS regulates multiple virulence determinants in P. aeruginosa, we hypothesized that cerPAC contain molecule(s) that might interfere with QS in P. aeruginosa PA14. Therefore, to determine the ability of cerPAC to modulate the production of the two principal AHL molecules by P. aeruginosa PA14, we determined AHL production kinetics in absence or presence of 200 μg mL−1 cerPAC. As shown in Fig, cerPAC significantly impairs the production of 3-oxo-C 12 -HSL (t = 7.45, df = 4, p < 0.001) and C 4 -HSL (t = 3.54, df = 4, p < 0.05), in P. aeruginosa PA14 at exponential and late stationary phase, respectively. This reduction in the production of the QS signals was observed without affecting bacterial growth (Fig. 3C).

Figure 3 cerPAC (200 μg mL−1) impairs the production of AHL-type QS molecules in P. aeruginosa PA14. Concentrations of (A) 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C 12 -HSL) and (B) butanoyl-homoserine lactone (C 4 -HSL) are shown as a function of cell growth (OD 600 ). (C) Total cell dry weight of 3 mL culture is shown as a function of cell growth (OD 600 ). Data points represent the average of triplicate experiments and the error bars show the standard deviation. Full size image

To understand the mechanism for the reduction in AHL levels, β-galactosidase transcriptional fusion reporters of lasI (3-oxo-C 12- HSL synthase) and rhlI (C 4 -HSL synthase) were assayed in P. aeruginosa PA14 bioreporter strain with the same 200 μg mL−1 cerPAC exposure. These bioassays revealed that expression of both AHL synthase genes (lasI and rhlI) is repressed by cerPAC (Fig. 4A,B). Similarly, we also investigated whether presence of cerPAC affects the expression of the two cognate transcriptional regulator genes lasR and rhlR using lacZ transcriptional fusion reporters. Expression of both regulator gene fusions was partially repressed in the presence of cerPAC (Fig. 4C,D). Thus, cerPAC inhibits both AHL synthases and partially represses the LuxR-type regulator genes associated with the production of the two AHL signals in P. aeruginosa PA14.

Figure 4 Effect of cerPAC on the expression of quorum sensing genes. P. aeruginosa PA14 carrying reporter fusion plasmids (A) lasI’-lacZ (B) rhlI’-lacZ, (C) lasR’-lacZ and (D) rhlR’-lacZ were grown in TSB medium without or with 200 μg mL−1 cerPAC and expression was quantified by measuring β-galactosidase activity. Data points represent the average of triplicate experiments. The error bars show the standard deviation. Full size image

Cranberry extract rich in proanthocyanidins act as an antagonist of AHL-mediated quorum sensing in P. aeruginosa PA14

Considering that AHLs act as autoinducing ligands of LasR and RhlR, we hypothesized that cerPAC component(s) interfere with LasR/RhlR activation by AHLs. We thus investigated whether cerPAC affects LasR and/or RhlR induction by exogenous AHLs using bioreporter AHL-negative PA14 mutants with lacZ fusions. As expected, when 3-oxo-C 12 -AHL or C 4 -HSL were supplied to their respective bioreporters, they activated the expression of lasI and rhlI, respectively (Fig. 5A,B). While cerPAC had no effect on the activity of the reporters, there was a significant (p < 0.05) reduced activation by either AHLs in presence of cerPAC (Fig. 5A,B). This indicates that cerPAC partially inhibits the activation of both LasR- and RhlR-directed transcription of lasI and rhlI, respectively, the primary targets of these LuxR-type regulators. Additionally, LasR and RhlR activation titration was performed in absence and presence of three different concentrations of cerPAC, which resulted in lower activation of LasR and RhlR (Fig. 5C,D). This indicates that cerPAC can reduce the activation of both regulators by their native AHLs, likely as a potential antagonist.

Figure 5 cerPAC represses AHL induction of LasR- and RhlR-controlled regulation in P. aeruginosa PA14. (A) LasR activation of lasI-lacZ activity in ∆lasI- mutant of PA14, (B) RhlR activation of rhlI-lacZ activity in ∆rhlI- mutant of PA14. Titration for activation of (C) lasI-lacZ in ∆lasI- mutant of PA14 and (D) rhlI-lacZ in ∆rhlI- mutant of PA14 in absence and presence of cerPAC. Error bars (A,B) and shaded errors (C,D) represent SD of triplicate assays. Statistically significant differences are indicated for each sample treated with cerPAC and each autoinducer compared to the sample treated with the corresponding concentration of each autoinducer (*p < 0.05). Full size image

Cranberry extract rich in proanthocyanidins inhibits LasR activity without binding to AHL molecules and also interact with LasI

To assess a possible physical interaction between cerPAC components and either AHL molecule, we quantified C 4 -HSL and 3-oxo-C 12 -HSL in cell-free growth medium using an ethyl acetate extraction procedure followed by LC-MS analysis. If the cerPAC binds to the AHLs, we would expect to observe a reduction in AHL concentration due to compromised extraction. As shown in Supplementary Fig. S2, there was no difference in the concentration of AHLs with or without cerPAC, demonstrating that cerPAC components do not bind to the AHLs and therefore do not inhibit QS by physical interaction.

Inhibition of Las-type QS regulators’ activities by cerPAC may be due to structural interactions, important for the functional activity of transcriptional regulatory proteins. To address this possibility, in silico docking analysis was performed using protein structures of LasR (2UV046), LasI (1RO547), the monomer and dimer of epicatechin molecules (important components of cPACs48). To test our docking method, we compared the interaction energy scores (obtained using Moldock tools) of the predicted docking complex and the known crystallographic complex structures of the LasR with ligand 3-oxo-C 12 -HSL. The Moldock interaction energy score of −144.1 kcal mol−1 for the predicted complex of LasR with 3-oxo-C 12- HSL was marginally lower than the Moldock interaction energy score of −157.5 kcal mol−1 obtained for the crystallographic complex of LasR with ligand 3-oxo-C 12 -HSL (Fig. 6a and see Supplementary Table S1). The epicatecin and its dimer (proanthocyanidin) molecules were docked separately in the internal cavity of LasR (Fig. 6b,c). Ligand binding domain (LBD) of LasR with a volume of 653 Å3, exhibits sufficient space to accommodate the monomer or dimer of epicatechin with a volume of 225 Å3 or 466 Å3, respectively. The in silico docking analysis suggests that the complex formation between the epicatechin and LasR, with a Moldock interaction energy score of −127.1 kcal mol−1, is more favorable than LasR-proanthocyanidin complex with Moldock score of −68 kcal mol−1 (see Supplementary Table S1). The proanthocyanidin formed six hydrogen bonds at the internal binding cavity of LasR compared to four hydrogen bonds of the LasR-3-oxo-C 12 -HSL or LasR-epicatechin complex (Fig. 6). The increase in the Moldock score for the docking complex of LasR with proanthocyanidin compared to LasR-epicatechin complex was observed due to the steric constraints of the proanthocyanidin structure in the internal cavity space of LasR identified by the comparison of their internal energies (see Supplementary Table S1).

Figure 6 Molecular docking analysis of the LasR protein with AHL molecule and two main components of the cerPAC. (a) Left panel represents full view of the ribbon structure of LasR protein with the ligand binding cavity (highlighted in golden color) between four β-sheets (β1, β2, β4 and β5) and two α-helixes (α3 and α4). Upper right panel represents the inset view of docked complex with known binding position (reported crystallographic structure) of ligand 3-oxo-C12-HSL (shown in magenta color) and the predicted binding position of 3-oxo-C12-HSL (shown in black color) during in silico docking. The docking complexes of LasR with (b) the monomer of epicatechin (shown in blue color) and (c) the dimeric form of the epicatechin (proanthocyanidin, shown in green aqua color) are shown in the presence of 3-oxo-C12-HSL (shown in magenta color) for the comparison of binding positions. All possible hydrogen bonds are shown using black lines and binding residues shown in bright green color. Full size image

Due to the lack of crystallographic structure of LasI protein bound with its natural substrates or functional analogues, we performed in silico docking analysis to predict a complex of LasI with its natural substrate S-adenosyl L methionine (SAM) (Fig. 7a). This putative complex with LasI was used as a reference for both docking analyses of epicatechin and proanthocyanidin. The best five structural positions of SAM with higher interaction energies occupied the same binding cavity on the LasI protein. The docking analysis showed the formation of hydrogen bonds of SAM with residues that surround the putative binding cavity with Moldock interaction energy score of −126.2 kcal mol−1 (Fig. 7a and see Supplementary Table S1). The binding cavity known for the second substrate of LasI, the acyl-acyl carrier protein (acyl-ACP) was not identified as a potential binding site for either of the tested cerPAC components (epicatechin or proanthocyanidin). The LasI-epicatechin complex showed single hydrogen bond with Moldock interaction energy score of −106.8 kcal mol−1 (Fig. 7b and see Supplementary Table S1). The docking complex of the LasI protein with the proanthocyanidin molecule suggests the more favorable complex formation with five hydrogen bonds and Moldock interaction energy score of −153.6 kcal mol−1 compared to the LasI-SAM complex (Fig. 7c). This in silico docking analysis suggests that both main components of cerPAC have the potential to form complexes with LasR and LasI proteins to compete with their native ligands 3-oxo-C 12 -HSL and SAM, respectively.

Figure 7 Molecular docking analysis of the LasI protein with substrate S-adenosyl L methionine (SAM) and two main components of the cerPAC. (a) Left panel represents full view of the ribbon structure of LasI protein with its substrates binding cavities and right panel represents the inset view of docked complex with substrate SAM (shown in magenta color). The docking complexes of LasI with (b) the monomer of epicatechin (shown in blue color) and (c) the dimeric form of the epicatechin (proanthocyanidin, shown in green aqua color) are shown with predicted binding residues (shown in bright green color). The surface structures are shown in red and blue for hydrophobic and hydrophilic attributes, respectively and possible hydrogen bonds are depicted using black lines. Full size image

Cranberry extract rich in proanthocyanidins impairs AHL production in other pathogenic strains

According to our data, cerPAC can act as a general QS inhibitor by interfering with the binding of the AHL ligand to LuxR-type transcriptional regulators. To verify that cerPAC is able to impede QS in other bacterial species, we performed an AHL production kinetics assay to examine the effect of administering cerPAC to wild type strains of Burkholderia ambifaria and Chromobacterium violaceum. The addition of cerPAC to growth medium significantly impairs the production of the two main AHLs (C 8 -HSL and C 6 -HSL) in B. ambifaria (Fig. 8A) and C 6 -HSL in C. violaceum (Fig. 8B). Since the primary target of LuxR regulators are luxI homologues, these observations validate the capacity of cerPAC to interfere with AHL-mediated QS in different bacterial species.