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.