4 DISCUSSIONS
A key part of the defense of A. baumannii to different antibiotics is a series of efflux pumps, which effectively exclude or reduce the intracellular concentration of a large number of antibiotics and other compounds, making the pathogen intrinsically more resistant to some antimicrobial agents.30 The efflux pumps are a topic of considerable interest, both from the perspective of understanding functions and as targets for adjunct therapies.45 In A. baumannii , three RND efflux systems, AdeABC, AdeFGH, and AdeIJK are reported to be primarily associated with the emergence of MDR strains.16 Other studies suggested that the AdeABC efflux pump system is the most prevalent and with the highest detection rate in clinical isolates ofA. baumannii as compared to other efflux pump genes.11 Nevertheless, the RND efflux family inA. baumannii has broad substrate specificity and can efflux a vast variety of antibiotics from the periplasm before the antibiotics even fully enter the cell.46
Despite a large number of identified efflux pumps, our understanding of the molecular mechanisms drugs effluxes remain limited and relationships between drugs and targets have been poorly understood.47 The inhibitory mechanism between amphiphilic peptide MP-B and the AdeB efflux pump remains uncharacterized. Moreover, most of the current drug targets are proteins, and the 3D structures of these proteins need to be known in target identification.48 Therefore, Computer-aided drug target identification methods can greatly reduce the searching scope of experimental targets and associated costs by identifying the drug binding sites and evaluating the druggability of the predicted protein.47
The results from this study provide novel insight into the inhibitory activity of Mastoparan-B on the RND efflux pump. The preliminary susceptibility test revealed that the organism was intrinsically sensitive to MP-B as a potent inhibitor of the membrane function. However, when the cells were grown in the presence of a sub-MIC concentration of this peptide (0.5 µg/ml), and different concentrations of antibiotics, there were significant reduction in MICs particularly against aminoglycosides, fluoroquinolones, third-generation of cephalosporins, carbapenems, and tetracycline, respectively. In contrast, the MIC against colistin, piperacillin-tazobactam, azithromycin, trimethoprim-sulfamethoxazole, and chloramphenicol remained unchanged. This led us to the understanding that, the efflux pump might be involved in this extrusion of particular antibiotics but not all. The synergic activity of MP-B with antibiotics was further supported by the 20 fold decrease in transcription of the ade B gene in the presence of sub-MIC of MP-B. Furthermore, the search for phylogenetic analysis of AdeB protein with similar sequences in the UniProtKB database revealed a high percent identity matrix using the Maximum Likelihood method by neighbor joining method. The results indicated the AdeB that investigated in this study was evolutionary-related to the majority of Acinetobacter efflux pumps. This was further confirmed by Structural neighbourhood comparison by CATH database which showed 97.9% alignment with functional family multidrug efflux transporter AcrB transmembrane domain. The study of lineage of the protein showed six functional families with the A. baumannii . Structural neighbourhood of superfamily (inside superfamily) indicated evidence they have diverged from a common ancestor.
Furthermore, we performed docking of Mastoparan-B with amphiphilic α-helical structure to the inner membrane AdeB protein via the amphiphobic amino acids. By AlphaFold 2 tool, we found that, the proteins fold into a 3D dimensional shape to bury these hydrophobic side chains in the protein interior. Since there was no reliable crystallography for a membrane protein, so we relay on computational methods. These methods are based on structural, physicochemical, and evolutionary properties that distinguish binding sites from the rest of the protein surface (e.g., amino acid composition and residue conservation). Furthermore, we investigate deep learning of artificial intelligence for the prediction of the membrane topology of transmembrane proteins. The attachment of all forms of MP-B exclusively occurred to the transmembrane helix-5 which is situated at the interior of the inner membrane domain. This was confirmed by using the BioLiP database (the structure was predicted by SPARKS-X (SPX) with template 7cz9A (z-score=33.51 and cutoff=6.9). Concurrent target-ligand interaction indicated a shift in the dihedral angles with a lower free binding energy score. Furthermore, examination of the dihedral angles changes upon ligand binding showed that, the φ and ψ changes caused a transient conformational change in the protein structure resulting in the inhibition of AdeB activity. The presence of a large amount of Phe, Ala, and Lys amino acid residues in the pore region provides a hydrophobic trap around this peptide in the AdeB molecule particularly, Phe residue was important in creating this hydrophobic microenvironment. So far, the mechanisms have not been studied in detail, therefore this statement must be further confirmed. Similarly, 3 inter domain interchange was observed in AdeB α-chains by FUpred software, utilizing contact maps prediction created by deep residual neural networks coupled with coevolutionary precision matrices.