1 INTRODUCTION
Computation chemistry has greatly increased our knowledge about protein
conformation and drug interaction and helped us better understand
microbial infection treatment.1 Biomolecular
simulation with models allows for investigations of target proteins’
structure and function features, which are useful for identifying
drug-binding sites and elucidating drug action
mechanisms.2,3 Recently a breakthrough in the 3D
construction of various proteins occurred with the invention of the
AlphaFold Protein Structure Database in cooperation with EMBL-EBI.
AlphaFold can accurately predict 3D models of protein structures and is
accelerating research in nearly every field of biology such as
drug–target interaction.4
In this regard, A. baumannii is a Gram-negative hospital-acquired
pathogen to blame for high morbidity and mortality within the medical
care units such as ICUs of various hospitals across the
world.5 Following the increasing emergence of strains
that are not susceptible to any clinically used antibiotics, the WHO has
ranked carbapenem-resistant strains of this bacterium in first place in
its global priority pathogen list.6 A.
baumannii has different resistance mechanisms against antimicrobial
agents such as β-Lactam inactivation, biofilm formation, efflux pumps,
alterations of the affinity for antibiotics, etc.7-9One of the most important mechanism of resistance to antibiotics is the
efflux pump which plays a key role in the intrinsic resistance to
several antibiotics.10,11 The efflux pumps are divided
into five superfamilies: ATP-binding cassette (ABC), small multi-drug
resistance (SMR), multi-antimicrobial extrusion (MATE), major
facilitator (MFS), and resistance/nodulation/division
(RND).12 The best-characterized multi-drug efflux
system in A. baumannii is the RND
superfamily.13 These tripartite complexes comprise an
inner membrane that acts as a secondary active H+/drug
antiporter (AdeB), extruding a vast spectrum of structurally unrelated
drugs through a periplasmic membrane fusion protein channel (AdeA),
connected to an outer membrane funnel shape channel
(AdeC).14 The primary RND efflux pump of A.
baumannii (AdeABC) was first described in MDR strain
BM4454.14 The Ade abc locus has consisted of
three tandemly linked genes encoding AdeA, AdeB, and AdeC proteins and
showed resistance to several antibiotic classes such as aminoglycosides,
tetracyclines, and fluoroquinolones, respectively.15The AdeB is an inner membrane protein and transports various drugs
through the central pore.16 The pore is opened to the
periplasm via three vestibules located at subunit interfaces and a
central cavity.17 More researchers demonstrated that,
the ade B is the most important gene in the adeABC efflux
system.17,18 Genetic studies suggested that
discrimination between the substrates in A. baumannii occurs
mainly within the periplasmic region instead of the transmembrane
region.19 Cryo-Electron Microscopy (CEM) of the
monomers of the inner membrane protein suggested that AdeB can adopt
three different states: access (loose), binding (tight), and extrude
(release) to provide essential dynamics for the efflux
process.20 The AdeB trimer primarily adopts mainly a
resting state (absence of antibiotics) with all promoters in a
conformation devoid of transport channels or antibiotic binding
sites.21
Transmembrane proteins are relevant for drug development since they make
up more than 50% of all human drug targets given that approximately
25% of all the proteins.22 Membrane proteins are
difficult to study due to their partially hydrophobic surfaces,
flexibility, and lack of stability.21 Consequently
creates the need for bioinformatics tools that can accurately identify
these types of proteins by predicting their topology, position, and
orientation as well as their N-terminal utilizing the contact map with
coevolutionary precision matrices.23 Several methods
have been developed over the last decades that predict protein topology
with high accuracy. Many of these methods are freely available as web
servers, both individually or combine with several other bioinformatics
tools.24
In the past 20 years, scientists were unable to produce effective
antibiotics against Gram-positive or Gram-negative pathogens.
Furthermore, the use of nanoparticles, gene therapy, phage therapy, and
immunotherapy are either costly or still in their
infancy.25 In contrast, the amphiphilic antimicrobial
peptides like Mastoparan B (a cationic, tetradecaeptide
LKLKSIVSWAKKVL-CONH2 isolated from the venom of the hornet Vespa
basalis) at low concentrations has potent antimicrobial activity and can
be used along with antibiotics for treatment of various
diseases.26 These compounds frequently destabilize
biological membranes by dual amphiphilic activity resulting in membrane
perforation.27 In recent years, numerous efflux pump
inhibitors have been discovered and tested, including natural products,
antibiotics, and synthetic molecules.28 The
combination of antibiotics with natural products circumvents the
resistance phenomenon and consequently decreases the dose and drug side
effects. The activity of these peptides in the combination with
antibiotics on Escherichia coli AcrAB-TolC and thePseudomonas aeruginosa MexAB-OprM complexes were well
delineated.29 In contrast, there is no information
about the effect of Mastoparan-B on the RND efflux in A.
baumannii . It has been reported that the phenylalanine-arginine
β-naphthylamide (PAβN) a broad-spectrum efflux pump inhibitor interacts
with AdeB strongly through the distal binding
pocket.30 In another investigation, computational
screening suggested that Strictamin (Akuammilan-17-oic acid methyl ester
from Alstonia scholaris) and Limonin (7, 16-dioxo-7, 16-dideoxylimondiol
from Citrus spp.) exhibited binding to RND efflux pump in MDR strains ofA. baumannii .31
Therefore, in the present study, for the first time, we are reporting
transcriptional analysis of the ade B gene in the presence of
Mastoparan-B, the detailed stereochemical structure of AdeB protein
before and after binding to this peptide, superfamily analysis by CATH
database, Gene Ontology, molecular docking, prediction of structure
conformation and helix folding by AlphaFold Protein Structure Database
(AlphaFold 2). The emerging picture using this peptide in combination
with antibiotics further suggests opportunities for drug designs.