In silico molecular docking studies have been reported as an attractive approach to unveil the mechanism behind the antibacterial activities of nanoparticles. Here, LasI/R, RhlI/R, and PqsA/R systems, being well-known targets of QS mechanism to combat infection caused by P. aeruginosa, were selected as shown in Table S3. Molecular docking predictions revealed the binding tendency and mechanism behind the inhibition potential of silver, zinc oxide and copper oxide nanoparticles against the selected proteins.
LasR, RhlR, and PqsR, QS receptor proteins of P. aeruginosa, in cooperation with the natural signal molecules, 3-oxo-C12-HSL, C4-HSL, and PQS, respectively, trigger the virulence factor expression, bacterial motility, biofilm formation, and other factor production. It was reported that any competitive ligands with a higher binding affinity to the active sites can replace the natural signal molecule and lead to a decrease in the QS-dependent factors formation in P. aeruginosa (Mostafa et al. 2020).
To better understand the mechanism involved molecular docking analysis was performed to predict the possible sites of silver, zinc oxide and copper oxide nanoparticles binding with the QS systems LasI, RhlI and PqsA and their receptors LasR, RhlR and PqsR in P. aeruginosa. The PrankWeb server was utilized to predict potential binding sites in each structure using conservation analysis (Table S1). The active site residues obtained by Prankweb are similar to the results published in the literature (Ilangovan et al. 2013; Majumdar et al. 2020; Rex et al. 2022; L. Kumar et al. 2022). The binding energies, inhibition constants, and the active amino acids for the single metallic/oxide NPs have been reported in Table 1 and Figs. 2–7.
Table 1
Docking results of protein-ligand interaction in P. aeruginosa.
Protein name | Ligands | Hydrogen bond/amino acid residue | Hydrophobic bond /amino acid residue | Binding energy (kcal/mol) | Inhibition constant, Ki (mM) |
LasI | Ag | – | Met 54 | -0.21 | 696.14 |
ZnO | Gly163 – Ala 175 | His 89 - Phe 162 – Gly163 – Ala 175 | -2.72 | 10.09 |
CuO | Leu 87 – Phe 162 – Gly 163 – Pro 164 – Ala 175 | – | -1.85 | 44.09 |
LasR | Ag | – | Tyr 56 – Lys 34 | -0.15 | 770.33 |
ZnO | Asp 65 – Ala 70 | Glu 48 – Asp 65 – Ala 70 | -2.58 | 12.86 |
CuO | Tyr 64 - Asp 65– Tyr 69 - Ala 70 | – | -1.63 | 63.85 |
RhlI | Ag | – | Arg 71 – Glu 101 - Ser 103 | -0.19 | 720.04 |
ZnO | Leu 88 – Gly 158 – Pro 159 – Ala 170 | Leu 88 – Gly 158 –Ala 170 | -2.47 | 15.37 |
CuO | Leu 88 – Gly 158 – Leu 168 - Ala 170 | – | -1.68 | 58.72 |
RhlR | Ag | – | Tyr 43 – Tyr 64 | -0.15 | 778.18 |
ZnO | Tyr 64 | Tyr 64 | -2.51 | 14.43 |
CuO | Tyr 43 – Tyr 64 | – | -1.57 | 70.25 |
PqsA | Ag | – | Gly 173 – Trp 363 –Arg 364 | -0.22 | 686.89 |
ZnO | Gly 173 – Trp 363 – Arg 364 | Asp 98 – Gly 173 – Trp 363 – Arg 364 | -3.83 | 1.65 |
CuO | Asp 98 – Gly 173 – Trp 363 – Arg 364 | – | -2.49 | 15.00 |
PqsR | Ag | – | Ile 147 – Thr 148 | -0.19 | 729.83 |
ZnO | Gln 194 – Ile 195 – Ser 196 – Ile 236 | Gln 194 – Ile 195 – Ser 196 – Trp 234 – Ile 236 | -2.86 | 8.08 |
CuO | Gln 194 – Ile 195 – Ile 236 | – | -1.93 | 38.59 |
LasI is an HSL synthesis protein of P. aeruginosa that produces 3-oxo-C12-HSL. The protein shares 31% identity and 47% homology with RhlI, a counterpart of P. aeruginosa AHL synthase (Gould et al. 2004). The Ag was found to interact with the amino acid Met 54 of synthase LasI via electrostatic interactions (Fig. 2). The interaction energy of Ag NPs–LasI was found to be -0.21 kcal/mol (Table 1). The molecular docking analysis showed that the ZnO displayed hydrogen bond interactions with the Ala175 and Glu163 amino acids, hydrophobic interactions with His89, Phe162, Gly163 and Ala175 amino acids in the LasI active site, with a binding energy of -2.72 kcal/mol (Table 1). The docking of LasI with CuO showed a binding energy of -1.85 kcal/mol and five hydrogen bond interactions with Leu87, Phe162, Gly163, Pro164 and Ala175 amino acids (Fig. 2). This in silico docking analysis suggests that the Ag, CuO and ZnO nanoparticles have the potential to form complexes with LasI protein to compete with their native ligand S-adenosyl L methionine (SAM) and thus preventing the activation of the QS-controlled proteins like LasR, RhlR, RhlI, PqsA and PqsR.
LasR is a transcriptional activator of several genes involved in the virulence and pathogenicity of P. aeruginosa. The target protein LasR docked with Ag demonstrated a binding energy of -0.15 kcal/mol and the main interacting amino acid residues were Tyr 56 and Lys 34 (Fig. 3). Thus, the silver nanoparticles interact with LasR at the same binding sites where its inhibitor binds. The binding energy associated with the docked conformation of LasR with ZnO was found to be -2.58 kcal/mol and the interacting residues involved were identified as Glu 48, Asp 65 and Ala 70 stabilized by hydrogen bonds formed with Asp 65 and Ala 70 amino acids. Similarly, the target protein LasR docked with CuO demonstrated binding energy of -1.63 kcal/mol, and the interactions are stabilized by hydrogen bonds. The main interacting residues involved in this binding were found to be Tyr 64, Asp 65, Tyr 69 and Ala 70. This molecular docking analysis suggests that Ag, CuO and ZnO have the capacity to form complexes with LasR protein to compete with their native ligand 3-O-C12-HSL, thus resulting in down-regulation of Las mediated virulence factor production (Pattnaik et al. 2018).
RhlI synthase is a regulatory protein of P. aeruginosa and is responsible for the production of N-butyryl-L-homoserine lactone (C4-HSL) which binds to its receptor RhlR (Yashkin et al. 2021). The target protein RhlI docked with Ag involved a binding energy of -0.19 kcal/mol and the main interacting residues were identified to be Arg71, Glu101, and Ser103 amino acids. The binding energy associated with the docked conformation of RhlI with ZnO was found to be -2.47 kcal/mol and the interacting residues involved in this binding were identified as Leu 88, Gly 158, Pro 159 and Ala 170 stabilized by hydrogen bonds formed with Gly 158, Pro 159 and Ala 170 amino acids. Similarly, the target protein LasR docked with CuO demonstrated a binding energy of -1.68 kcal/mol, and the interactions are stabilized by hydrogen bonds. The main interacting residues involved in this binding were found to be Leu 88, Gly 158, Leu 168 and Ala 170. For ZnO and CuO, our findings indicate that LasR residues Leu88, Gly158, and Ala170 are the most critical residues for complex formation (Fig. 4).
RhlR serves as a receptor for the AHL produced by RhlI synthase and then activates genes that are responsible for virulence (Cui et al. 2022). The target protein RhlR docked with Ag identified two electrostatic bonds with Tyr43 and Tyr64 amino acids (Fig. 5). The binding energy obtained for the complex Ag-RhlR was − 0.15 kcal/mol. Similarly, the target protein RhlR docked with CuO demonstrated a binding energy of -1.68 kcal/mol, and the interactions are stabilized by hydrogen bonds. The main interacting residues involved in this binding were found to be Tyr 43 and Tyr 64. Further, the protein RhlR docked with ZnO demonstrated a binding energy of -2.51 kcal/mol and stabilized by a hydrogen bond with the Tyr 64 amino acid.
The key amino acid Tyr64, was found common for all the interactions of the native ligand (C4-HSL) (Rex et al. 2022), Ag, ZnO, and CuO with the RhlR regulator. Our results indicate that the TYR64 residue is crucial in stabilizing complex formation. The binding of C4-HSL to RhlR activates the transcription of many virulent genes of P. aeruginosa (Pu et al. 2022; Mohajeri et al. 2022; Ochsner et al. 1994; Pearson et al. 1995). These docking results suggest that the use of metal/metal oxide nanoparticles to inhibit quorum sensing in P. aeruginosa may present a good strategy since these inhibitors seem to specifically compete against native ligand (C4-HSL) to interact with RhlR and ultimately reduce the expression of QS-controlled genes.
The PqsA synthase is known to be responsible for the production of PQS signaling molecules. The inhibition of such enzyme can disrupt the biosynthesis of the PQS signaling molecules and consequently the PqsR-dependent gene regulation which ultimately interrupts biofilm formation (Netrusov et al. 2022; Shaker et al. 2020). Our docking results revealed that Ag interacts with the following amino acids Gly 173, Trp 363, and Arg 364 of PqsA via electrostatic interactions. The energy of Ag-NPs–pqsA interaction was found to be -0.22 kcal/mol (Table 1). On the other hand, the binding energy associated with the docked conformation of PqsA with ZnO was determined to be − 1.9 kcal/mol and the interaction is stabilized by hydrogen interactions with the Gly 173, Trp 363 and Arg 364, hydrophobic interactions with Asp 98, Gly 173, Trp 363 and Arg 364 amino acids at the PqsA active site. Similarly, the target protein PqsA docked with CuO demonstrated a binding energy of -2.49 kcal/mol, and the interactions are stabilized by hydrogen bonds. The main interacting residues involved in this binding were found to be Asp 98, Gly 173, Trp 363, and Arg 364 amino acids. Thus, our data demonstrated that, Ag, ZnO and CuO nanoparticles interact with PqsA via common residues such as Gly 173, Trp 363 and Arg 364, which seem to be potential anti-QS target agents (Fig. 6).
The PqsR is the third transcriptional regulator of P. aeruginosa that controls the expression of virulent genes. PqsR is activated by the binding of the 2-heptyl-4-quinolone (HHQ) and Pseudomonas quinolone signal (PQS) (Wade et al. 2005). PqsR controls the polycistronic operon (pqsABCDE) where major genes encoding for the synthases responsible for PQS and HHQ production are located (Qais et al. 2019). The docking results showed that the PqsR protein interacts with the Ag nanoparticles via two electrostatic bonds involving the Ile 147 and Thr 148 amino acids (Fig. 7). The binding energy obtained for the complex Ag-PqsR was − 0.19 kcal/mol. On the other hand, the protein PqsR docked with ZnO demonstrated a binding energy of -2.86 kcal/mol. In addition, this interaction is stabilized through both hydrogen and hydrophobic bonds involving Gln 194, Ile 195, Ile 236, and Gln 194, Ile 195, Ser 196, Trp 234 amino acids respectively. The binding energy associated with the docked conformation of PqsR with CuO was estimated to be -1.93 kcal/mol and the interaction is stabilized via hydrogen bonds. The main interacting residues involved in binding CuO nanoparticles were found to be Gln 194, Ile 195, and Ile 236. Thus, The Gln 194 amino acid residue, was found common during the interaction of native ligand (PQS) (Yin et al. 2022; Soukarieh et al. 2021), ZnO, and CuO nanoparticles with the regulator PqsR. This finding indicates that the Gln 194 is crucial in stabilizing the complex formation. In addition, this data demonstrates that the CuO and ZnO nanoparticles possess a strong binding affinity to the cognate receptor (PqsR) which can affect the natural ligand binding and thus resulting in down-regulation of Pqs mediated virulence factor production.
Based on the binding energy data presented in Table 1, the binding energy of ZnO is slightly lower compared to Ag and CuO. Thus, the binding strength of ZnO to the QS-controlled proteins is higher than Ag and CuO NPs. In addition, the PqsA-ZnO complex shows the highest affinity with a binding energy of -3.83 kcal/mol (Table 1).
Our molecular docking results of the interaction and bindings of Ag NPs QS-controlled proteins (LasI and RhlI) autoinducer synthases as well as with QS-controlled transcriptional regulatory proteins (LasR and RhlR) demonstrated that Ag NPs establish an electrostatic interaction with these proteins involving binding energies (energy loss resulting from the electrostatic interaction) of -0.21, -0.19, -0.15 and − 0.15 kcal/mol, respectively. These data are not in agreement with those reported in previous studies performed by Vyshnava et al. (2016). We believe that the molecular docking software used in a given study can lead to different types of interactions). Ali et al. (2017), reported that Ag NPs have the potential of blocking the production of signaling molecules by inhibiting LasI/RhlI synthase. Moreover, interference of transcriptional regulator proteins inactivated the LasR/RhlR system, thus ultimately blocking the production of QS-controlled virulence. Although the interaction and binding of Ag NPs with PqsA and PqsR have not been previously reported in the literature, herein for the first time, we have shown that the binding energies of Ag NPs with PqsA and PqsR receptors are comparable to those of LasR and RhlR (Table 1).
The molecular docking analysis of CuO NPs indicates that these nanoparticles establish hydrogen interactions with the QS-controlled proteins (lasI, lasR, RhlI, RhlR, PqsA, and PqsR) involving low binding energies of -1.85, -1.63, -1,68, -1,57, -2,49, and − 1,93 kcal/mol respectively (Table 1). Our results are consistent with previous findings by Mishra and Mishra.( 2021). They showed that copper nanoparticles can bind AHL synthase (LasI/RhlI) as well as the receptor proteins (LasR/RhlR) and suppress the virulence and biofilm formation. However, our present study is the first in silico report of CuO NPs interaction with QS-controlled proteins.
Finally, our molecular docking study indicated that ZnO NPs establish hydrogen and hydrophobic interactions with synthase proteins, LasI, RhlI, and PqsA as well as with receptor proteins of lasR, RhlR, and PqsR involving higher binding energies compare to Ag and CuO nanoparticles (-2.72, -2,47, -3,83, -2,58, -2,51 and − 2,86 kcal/mol, respectively) (Table 1). However, these data are in disagreement with the findings reported by Ali et al. (2021). Here, again we believe that the choice of the molecular docking software can revealed different types of interactions between the complex entities. On the other hand, to our knowledge, this is the first in silico investigation of the interaction between ZnO nanoparticles (NPs) and PqsA and PqsR proteins (Table 1). As a result, it is not possible to compare these findings with those reported in previous literature. As shown in Table 1, The ZnO NPs demonstrate a higher inhibitory effect and lower binding energy compared to Ag and CuO nanoparticles which make them the most effective nanoparticles that have the potential to affect the QS in P. aeruginosa bacteria.