Introduction
Over the last decade, the issue of antimicrobial resistance has challenged the
eradication of bacterial infections [1]
[2]. Antibiotic resistance due to mutation or
the acquisition of resistance genes may occur regardless of antibiotic exposure.
However, the presence of these agents induces a dramatic increase in the incidence
of resistant bacteria[3]. This underscores the
necessity of designing novel antibacterial agents [4]. Despite many efforts to modify the present antimicrobial drugs, only
a few have proven effective against resistant bacteria[4]. One practical approach to overcome this
problem is the identification of potential bacterial targets for developing suitable
therapeutic agents[4]. Recent studies about
the bacterial physiology and behavior have paved the way for the identification of
a
variety targets, among which Lon protease in Escherichia coli strains is an
interesting one[5].
Lon protease, the first identified ATP-dependent serine protease with a highly
conserved structure, is a homo-oligomer with ATPase and proteolytic domains [6]. Lon has a crucial role in the protein
quality control system, getting involved in the eradication of aberrant and
misfolded proteins and the selective degradation of regulatory proteins, including
those associated with cell division, capsule synthesis, and SOS response, thereby
modulating numerous metabolic and stress response pathways[6]
[7].
Lon also contributes to the cleavage of antitoxins in toxin-antitoxin (TA) systems,
where the related liberated toxins can inhibit bacterial growth by suppressing
translation or replication[7]
[8]. Activation of TA systems through antitoxin
degradation could result in a variety of phenotypes, but the most frequently
observed are those connected with growth inhibition, persistence, programmed cell
death, and biofilm formation[7]
[9]. The ubiquitousness of these systems among
clinical strains and their absence in eukaryotic organisms makes them ideal targets
for the development of suitable agents and the subsequent treatment of E.
coli infections. In general, protein-protein interactions (PPIs) play a
vital role in many biological processes and are associated with cancers and
infectious diseases, accordingly, targeting PPIs can be a promising therapeutic
strategy[10]
[11]. Considering the regulatory and central
role of Lon protease in the functionality of the TA systems, we aimed to study the
bioinformatics of Lon and its interaction with related TA systems, as well as
designing interfering peptides for restraining Lon-antitoxin interactions.
Methods
Lon and TA system network
In order to predict the interacting protein network associated with Lon and the
corresponding TA systems, the STRING’s server was used.
Conserved domains and consensus sequences of the Lon gene
The results of PSI-BLAST were introduced into the Jalview 2.8.1 and the alignment
file with the respective gap open cost and gap extension cost of 10.0 and 1.0
was created [12]. Conserved domains were
acquired from the Pfam 32.0 database by multiple sequence alignments and hidden
Markov models (HMMs) [13].
Phylogenetic study of the Lon
To study the phylogeny of the Lon protein, the results of the PSI-BLAST with at
least 60% identity were chosen. Following the omission of duplicates and
redundant sequences, molecular phylogeny was inspected using Maximum Likelihood
method by keeping bootstrap value 200. The evolutionary background was deduced
with reference to the JTT matrix-based model. Evolutionary analyses were
performed in MEGAX [14].
Lon proteases and their interactions with corresponding ATs in E.
coli
To date, numerous TA systems have been detected in E. coli however, in this study
we concentrated on the dominant TA systems controlled by the Lon protease, the
antitoxins component of which include CcdA, HipB, MazE, RelB, MqsA, and YefM
[15]. First, the TA and Lon structures
were extracted from the protein data bank (PDB) database ([Table 1]). Then, to explicate the
interaction between Lon protease and the studied ATs, molecular docking was
conducted for the proteolytic domain of Lon and ATs using the ClusPro server
[16]. To elucidate the interacting
residues, protein complexes with minimum binding energy were selected and
envisioned using the LigPlot+software [17].
Table 1 Characteristics of the predicted peptides against
Lon/antitoxin interactions.
|
TA (PDB code)
|
Complex inhibited
|
Peptide sequence
|
Interface score
|
Relative interface Score (%)
|
Binding energy
|
|
3G7Z
|
Lon/CcdA
|
EVARFIEMNGSFADEN
|
−20.630
|
44.04
|
−652.9
|
|
2WIU
|
Lon/HipB
|
TLTTFFKILQSLELSMTL
|
−16.071
|
38.68
|
−729.3
|
|
1UB4
|
Lon/MazE
|
DITPENLHENIDWGEP
|
−29.437
|
49.47
|
−704.7
|
|
4FXE
|
Lon/RelB
|
PSEALRLMLEYIADNE
|
−23.234
|
56.72
|
−595.3
|
|
3HI2
|
Lon/MqsA
|
VHCEESIMNKEESDAF
|
−12.090
|
45.89
|
−696.1
|
|
2A6Q
|
Lon/YefM
|
MSLEEYNSLEETAYLL
|
−6.267
|
54.42
|
−746.6
|
Prediction of peptide-mediated interactions
Peptides capable of obstructing Lon/AT interactions in E. coli
were designed using the Peptiderive server [18]
. This server provides linear peptides for a specific
protein-protein interaction based on “hot segments”, which
provides an interface score representative of the binding energy of the
protein-peptide complex at that particular position.
The tertiary structures of the peptides were predicted using the PEP-FOLD server
[19], following which the
protein-peptide docking was performed using the Cluspro server.
Visual presentations
Protein-peptide interactions were visualized and recorded using Pymol [20] and LigPlot+software.
Results and Discussion
Due to the emergence of antibiotic resistance among many bacteria, finding new
antimicrobial targets is essential [1].
Bacteria appear to have found effective ways to neutralize antibiotics.
Therefore, the study of new targets such as vital enzymes, signaling pathways,
efflux pumps, etc. can be an alternative approach [21]. In E. coli, as one of the most
important pathogenic bacteria, Lon protease is a vital protein for bacterial
growth, metabolism, and survival [6]. One
of the interesting mechanisms of this protease is its regulatory role in
toxin-antitoxin (TA) systems [8].
TA systems have received much attention in recent years. Various studies have
shown that these systems help bacterial survival in stress conditions through
different mechanisms including biofilm and persistence development, which lead
to chronic and recurrent infections [22].
The activity of these systems is controlled by proteases such as Lon, which
break down the antitoxin component in stress conditions to liberate the toxin
component. The toxin is thereby released to inhibit bacterial growth via
different mechanisms [8].
Today, with the tremendous increase of bio-data in databases and the significant
development of bioinformatics and computational tools, it is possible to work
more quickly in the in-silico space on the screening, identification,
prediction, and design of antimicrobial compounds.
Therefore, this study has focused on the structure, evolution, and regulatory
function of the Lon protease of TA systems in E. coli as a new
antimicrobial target and finally the design of inhibitory peptides to neutralize
its regulatory effect. The results of functional connective networks of Lon and
the corresponding ATs using the STRING database ([Fig. 1]) showed that in addition to the
antitoxins CcdA, HipB, MazE, RelB, MqsA, and YefM, Lon has controlling roles
over several other TA systems in E. coli including HicB and HigA, necessitating
more studies in this field.
Fig. 1 Protein-protein interaction network associated with Lon
protease and the corresponding ATs using the STRING server. In addition
to the antitoxins CcdA, HipB, MazE, RelB, MqsA, and YefM, Lon has
controlling roles over several other TA systems in E. coli including
HicB and HigA.
Lon has a major role in controlling the functional network of the systems shown
in [Fig. 1]; along with other proteins
that may be involved in this network ([Fig.
1]). Structural analysis of the Lon protease indicated three domains,
including the substrate-binding domain, the AAA-rich domain with several
cellular activities, and the C-terminal domain with proteolytic activity ([Fig. 2]). Moreover, phylogenetic analysis
of Lon indicated its presence in a conserved manner (especially in the
C-terminal region) among the Enterobacteriaceae family ([Fig. 3]).
Fig. 2 Results of domain and phylum analysis of the Lon protease
using PhylomeDB [21]. Lon is
conserved among major lineages of the domain bacteria.
Fig. 3 Maximum likelihood tree using JTT matrix-based model.
Numbers at each node are bootstrap percentages for 200 replicates.
Evolutionary analyses were performed in MEGAX.
The results of the phylogenetic tree showed that this protease has a common
ancestor among the bacteria of this family in terms of evolutionary distance,
which has been fully protected over time. Moreover, its homologous can be found
in all bacteria known until now ([Fig.
2]) indicating the importance of this protease in bacterial homeostasis;
hence being a suitable target for antimicrobial purposes. To investigate how the
Lon protease interacts with the studied antitoxins, the docking technique was
performed by the ClusPro server and to understand the functionally interacting
residues, protein complexes with the lowest binding energy were chosen and
visualized using LigPlot+software. The amino acids involved in these
interactions are shown in [Figs. 4]
[5]
6
7
[8]
[9].
Fig. 4 Cartoon representation of the 3D structure and interactions
of the Lon protease with the CcdA antitoxin. Red color indicates Lon
protease and the green color indicates CcdA antitoxin.
Fig. 5 Cartoon representation of the 3D structure and interactions
of the Lon protease with the HipB antitoxin. Red color indicates Lon
protease the green color indicates HipB antitoxin.
Fig. 8 Cartoon representation of the 3D structure and interactions
of the Lon protease with the MqsA antitoxin. Red color indicates Lon
protease and the green color indicates MqsA antitoxin.
Fig. 9 Cartoon representation of the 3D structure and interactions
of the Lon protease with the YefM antitoxin. Red color indicates Lon
protease and the green color indicates MqsA antitoxin.
In recent years, peptide drugs such as natural or synthetic interfering peptides
have received much attention due to their physical and chemical properties, and
ease of synthesis and handling [11]
[23]
[24]. Online servers and computational soft wares have been used in
this study to design interfering linear peptides (Peptide drive server) to
interfere with the Lon/antitoxin interactions. The sequence of these
peptides is shown in [Table 1].
To evaluate the binding energy of these peptides, the 3-D structure of the
peptides was first modeled using the PEP-FOLD server (Fig. 10) and then docked with the Lon
protease using the ClusPro server. The results of this section showed that the
linear peptide EVARFIEMNGSFADEN has 16 amino acids in length and can bind to 26
residues of the Lon protease with a binding energy of -652.9, interfering the
Lon/CcdA interaction ([Fig.
11a]).
Fig. 11 Molecular docking (left panels) and interacting residues
(right panels) between the derived peptides and the Lon protease
residues.
CcdA is the antitoxin component of the CcdA/B TA system that is involved in the
maintenance of plasmids and the death of plasmid-deficient cells in E.
coli (Post-segregational killing) [25]. Another peptide designed in this study had the
TLTTFFKILQSLELSMTL sequence that could bind to the interface of the
Lon/HipB, with a binding energy of -729.3 ([Fig. 11b] ). The hipB antitoxin
gene is located on the upstream of the hipA/B operon. Studies have shown
that this antitoxin plays a role in the formation of E. coli biofilm,
such that its removal reduces the ability to biofilm formation [26]. MazE/F TA is one of the most
well-known and conserved TA systems among bacteria which, in stress conditions,
is involved in programmed cell death as well as biofilm formation [27]. To inhibit the regulatory effect of
the Lon protease on this system and interfere with the Lon/MazE
interaction, the 16-amino acid linear peptide DITPENLHENIDWGEP was predicated by
the Peptide Drive server. It should be noted that the linear peptides designed
in this study are derived from the amino acid structure of the studied
antitoxins, which mimic the behavior of antitoxins in binding to the Lon
protease. Information on other peptides designed to interfere with the
interaction of the Lon protease with RelB, MqsA, and YefM antitoxins are shown
in [Table 1] and [Figs. 11a–f]. In general, the
docking results of the designed peptides to the Lon protease and their binding
energy have proven encouraging as means of interfering with these TA
systems.
The vital roles of Lon and its homologues among bacteria have made this protease
an attractive antimicrobial target for researchers. In a study in 2019, M. Babin
et al. examined the effects of different hybrid peptides on the Lon protease
inhibition in E. coli. They screened various peptide compounds and showed
that boronic acid has efficient Lon-binding and -inhibitory capacity. Their
results showed that interfering with this protease accelerates the UV induction
of bacterial filamentous structure and also reduces bacterial tolerance to the
antibiotic ciprofloxacin [28].
In 2020, in an in silico study on TA systems and ClpP regulatory protease
in Listeria monocytogenes, Mohammadzadeh et al. showed that the
interaction between the studies TA systems and the ClpP regulatory protease
could be a new target for antimicrobial peptides. In that study, they predicted
linear peptides of 10 to 16 amino acids with ClpP-binding energies of
−455 to −907 and stated that these peptides could eventually
inhibit or reduce the formation of persister cells in L.
monogytogenes
[29].
In another study, Suredr et al. designed the linear peptides ELAAIRHRCA and
AYPYESEAER to inhibit the TA systems VapB/C and MazE/F in
Mycobacterium tuberculosis, respectively. They declared that these
peptides could be new therapeutic compounds against this bacterium given that TA
systems are not present among Eukaryotic cells [30].
Peptide-based therapies are being developed because of their ease of design and
production, and their encouraging properties such as being highly efficient,
selective and well-tolerated by the host [11]. In general, the results of this study showed useful information
about the structure and binding properties of the Lon protease and its
corresponding antitoxins. Lon, as a central regulatory protease, plays crucial
roles in bacterial survival and has characteristics that make it a suitable
therapeutic target against antibiotic-resistant bacteria including E.
coli. The design and use of peptides to interfere and inhibit PPIs in
bacteria can be an interesting platform for investigating and outlining new
antimicrobial approaches.
