Journal of Infection and Public Health 14 (2021) 160–168

Contents lists available at ScienceDirect

Journal of Infection and Public Health

journal homepage: http://www.elsevier.com/locate/jiph

Identification of potential drug targets in human pathogen Bacillus

cereus and insight for finding inhibitor through subtractive proteome
and molecular docking studies
N. Anis Ahamed a,b,c,∗ , A. Panneerselvam c , Ibrahim A. Arif a,b , M Hussain Syed Abuthakir d ,
Muthusamy Jeyam d , V. Ambikapathy c , Ashraf A. Mostafa b
a
Prince Sultan Research Chair for Environment and Wildlife, Department of Botany and Microbiology, College of Sciences, King Saud University (KSU),
Riyadh, Saudi Arabia
b
Department of Botany and Microbiology, College of Sciences, King Saud University (KSU), Riyadh, Saudi Arabia
c
Department of Botany and Microbiology, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Affiliated to Bharathidasan University, Thanjavur 620024,
India
d
Biochematics Lab, Department of Bioinformatics, Bharathiar University, Coimbatore, India

a r t i c l e i n f o a b s t r a c t

Article history: Bacillus cereus is a gram-positive, anaerobic, spore-forming bacterium related to food poisoning in
Received 6 September 2020 humans. Vomit and diarrhea are the symptoms of foodborne B. cereus infection caused by emetic toxins
Received in revised form and three enterotoxins, respectively. This bacterium is broadly present in soil and foods such as vegeta-
28 November 2020
bles, spices, milk, and meat. The antibiotics impenem, vancomycin, chloramphenicol, gentamicin, and
Accepted 2 December 2020
ciprofloxacin are used for all susceptible strains of B. cereus. But these antibiotics cause side effects in the
host due to the drug–host interaction; because the targeted proteins by the drugs are not pathogen spe-
Keywords:
cific proteins, they are similar to human proteins also. To overcome this problem, this study focused on
B. cereus
Putative drug target
identifying putative drug targets in the pathogen B. cereus and finding new drugs to inhibit the function
Network analysis of the pathogen. The identification of drug targets is a pipeline process, starting with the identification of
Foodborne targets non-homologous to human and gutmicrobiota proteins, finding essential proteins, finding other
Druggability proteins that highly interact with these essential proteins that are also highly important for protein net-
work stability, finding cytoplasmic proteins with a clear pathway and known molecular function, and
finding non-druggable proteins. Through this process, two novel drug targets were identified in B. cereus.
Among the various antibiotics, Gentamicin had showed good binding affinity with the identified novel
targets through molecular modeling and docking studies using Prime and GLIDE module of Schrödinger.
Hence, this study suggest that the identified novel drug targets may very useful in drug therapeutic field
for finding inhibitors which are similar to Gentamicin and designing new formulation of drug molecules
to control the function of the foodborne illness causing pathogen B. cereus.
© 2020 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for
Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).

Introduction diarrhea, respectively [2]. Other toxins such as phospholipases,

Bacillus cereus is an aerobic or facultatively anaerobic,
gram-positive, rod-shaped, spore-forming bacterium. It is found
throughout the environment, but primarily in soil, while also being
present in decaying organic materials, vegetables, dust, and water
[1]. It causes food poisoning in humans mainly via one emetic
and three non-hemolytic enterotoxins, producing vomiting and
∗ Corresponding author.
E-mail address: nanisahamed@gmail.com (N. Anis Ahamed).
hemolysins, and proteases make important contributions to the
pathogenicity of B. cereus; these are produced during the growth
of the organism [3].
In addition, B. cereus causes number of infections outside
the gastrointestinal tract, mostly in the immunosuppressed, drug
addicts, those with an indwelling catheter, patients with surgical
wounds, and neonates, among others [4]. B. cereus is phenotypi-
cally and genetically close to other species of Bacillus, especially B.
anthracis [5].
Imipenem, vancomycin, chloramphenicol, clindamycin, gen-
tamycin, and erythromycin are important antibiotics to which all

https://doi.org/10.1016/j.jiph.2020.12.005
1876-0341/© 2020 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
N. Anis Ahamed et al.
strains of B. cereus are susceptible [6]. Ramoplanin and teicoplanin
also achieve good inhibition of gram-positive bacteria including B.
cereus [7]. However, these drugs targeted proteins of B. cereus that
are similar to human proteins. To avoid the cross-reactivity of drug
molecules with the host, the proteins targeted by a drug should be
pathogen-specific, namely, absent from the host and host gutmi-
crobiota, also it is very important for the survival of the pathogen
and targets not treated by any kinds of drugs or inhibitors [8]. The
identification of putative drug targets is important for discovering
new drugs [9]. Large amounts of data from genome sequencing and
proteomic analyses of pathogens can now be easily analyzed using
computational approaches, leading to potential drug targets in the
field of drug discovery [10].
The present study mainly focused on finding putative targets
in B. cereus using a subtractive proteomics approach and finding
inhibitors for identified novel targets to avoid cross-reactivity with
the human proteome. Specifically, the goal was to find drug targets
that are only present in B. cereus and are essential, highly interact
with other proteins of the pathogen, are cytoplasmic, not treated
with existing drugs, have clear molecular functions and pathways,
and are also absent from humans and human gut-microbiota.
Materials and methods
Retrieving protein datasets and sequence analysis
Total proteins of B. cereus strain ATCC 10987/NRS 248 were
downloaded from the UniProt database. All of these sequences were
analyzed after removing the repeated or similar sequences using
CD-hit server with a cut-off value of 0.6 (60%) [11].
Non-homologous analysis
To find non-homologous sequences among the total proteins
resulting from the CD-hit server, the proteins of B. cereus were com-
pared with the proteome of Homo sapiens using BLASTp, which is
offered in NCBI-BLAST. The thresholds applied were an e-value of
less than 0.0001 and a bit score less than 100, and BLOSUM 62
matrix was selected [12].
Essentiality analysis
The proteins with essential function were found among the
proteins resulting from the non-homology analysis. For this, the
proteins of B. cereus were compared with the essential proteins of
48 bacterial species using a BLAST search in the DEG database. The
thresholds applied were an e-value of less than 10−5 and a bit score
greater than 100, and BLOSUM 62 matrix was selected [13].
Gut-microbiota analysis
The selected essential proteins were further analyzed to find
the non-homologous proteins among them. The selected proteins
were compared with the proteome of human gut microorganisms
(Suppl. Table 1) from the literature search [14] using BLASTp search
in NCBI-BLAST. The thresholds were an e-value of less than 0.0001
and a bit score greater than 100 [15].
Protein–protein network analysis
Non-homologous proteins were further included in the
protein–protein interaction network analysis using the STRING
database. The network analysis was carried out with a high con-
fidence interaction score of 0.007 with active interaction sources,
such as text mining, experiments, databases, co-expression, neigh-
borhood, gene fusion, and co-occurrence were included to find the
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Journal of Infection and Public Health 14 (2021) 160–168
number of interacting proteins [16]. Proteins interacting with five
or more other proteins were further subjected to node attributes
analysis such as average shortest path length, betweenness central-
ity, closeness centrality and stress centrality using Cytoscape 3.8.0.
Further these proteins were included for node deletion process to
find the important proteins in the network [17].
Subcellular localization
The important drug targets selected from the network node
deletion process were further analyzed for their subcellular
localization using SVM-based prediction. Confirmation was also
performed using CELLO v.2.5 [18] to find the locality of the drug
targets.
Druggability analysis
Selected cytoplasmic proteins were further subjected to drugga-
bility analysis. DrugBank [19,20] and Therapeutic Target Database
(TTD) [21] are important databases for finding proteins that are tar-
geted by drugs and inhibitors, using BLAST search of an e-value of
10−5 .
Functional prediction
Uncharacterized proteins that are novel drug targets as pre-
dicted by the druggability analysis were further subjected to
functional prediction analysis using UniProt and InterProScan [22].
Pathway analysis
Pathway analysis of the selected novel targets was performed
using the KEGG pathway analysis database [23]. This is an impor-
tant step to determine in which pathways the proteins in the
pathogen are involved. This analysis reveals the functional contri-
butions of the proteins.
Molecular docking studies
Primarily, the 3D structures of identified targets were mod-
eled by homology modeling using Prime module of Schrödinger.
Templates were identified through BLASTp search, aligned with
sequences of drug targets and modeled. Further, the modeled
proteins were validated using Ramachandran plot analysis using
PROCHECK server. Binding site residues were identified using
sitemap, Schrödinger.
Then antibiotics Imipenem, Vancomycin, Chloramphenicol,
Gentamicin, and Ciprofloxacin were downloaded from DrugBank.
Molecular docking studies were carried out using Glide module
of Schrödinger, protein preparation process such as adding miss-
ing hydrogen atoms, protein optimization and minimization was
carried out with protein preparation wizard. OPLS3e force field
was used for minimizing energy of the protein molecule. Protein
grid was generated covering the binding site residues. Drugs opti-
mized for docking process by OPLS3e force field in Ligprep. Finally,
XP docking process was performed using identified novel targets
with the drugs [24]. The workflow of identification of putative drug
targets in B. cereus was shown in Fig. 1.
Results and discussion
Sequence analysis
The total of 5821 proteins of Bacillus cereus strain ATCC
10987/NRS 248 were downloaded from the UniProt database. The
total proteins wer subjected to various analyses. Primarily, the
N. Anis Ahamed et al.
Fig. 1. Workflow for identifying putative drug targets in Bacillus cereus.
proteome of B. cereus was subjected to the removal of repeated
proteins showing more than 60% sequence similarity along with
smaller proteins using the CD-hit server. This server grouped all of
the sequences into 5626 clusters, which included only 157 clus-
ters having more than one sequence. Proteins shorter than 100
amino acid residues were also deleted manually, as smaller pro-
teins are less likely to be essential [25]. As a result, 1327 proteins
were eliminated; the remaining 4494 proteins were included in
further analysis.
Non-homology analysis
The selected 4494 sequences were subjected to non-
homologous analysis to find protein targets that are not
present in the host (Homo sapiens). The reason for tar-
geting proteins in B. cereus that do not have homologs in
humans is to avoid cross-reactivity of drugs with human
metabolism, by binding to the active site of similar human
proteins. This approach is also very useful to design exclu-
sively pathogen-specific drug molecules [26]. The proteins of
B. cereus were compared with the proteome of Homo sapi-
ens. From this analysis, 1050 proteins matched with human
proteins, while the remaining 3444 (Suppl. Table 2) proteins
showed no significant similarity, so they were considered to be
non-homologous.
Essential protein analysis
Essential protein analysis is important for developing drugs
against pathogens [27]. Essential proteins are important for the sur-
vival, growth, replication, and adaptability of the pathogen. These
proteins also have evolutionary relationships with other proteins
and these proteins have the same function in different organisms.
Targeting essential proteins may lead to death of the pathogen [28].
These 3444 non-homologous proteins were further subjected to
essentiality analysis. From the BLAST results in DEG, 933 proteins
(Suppl. Table 3) were matched with existing essential proteins of
various organisms and they were considered to be essential. The
remaining 2511 proteins showed no hits and were eliminated from
further analysis.
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Journal of Infection and Public Health 14 (2021) 160–168
Gutmicrobiota analysis
Human gutmicrobiota is important for the metabolism of
humans by fermenting indigestible food, while also helping to pre-
vent gut colonization by pathogenic bacteria [29]. According to
Kärnell et al. [30], normal healthy humans have 1014 microor-
ganisms in their gastrointestinal tract. In this study, the selected
933 essential proteins were compared with the proteome of gut
microorganisms. From this analysis, only 39 proteins (Suppl. Table
4) were fully non-homologous relative to gut-microbiota. These
proteins were selected for further analysis.
Protein network analysis
The protein network analysis was carried out with a high confi-
dence score (0.007) and the proteins in the network with only low
and medium confidence scores were eliminated to avoid false pos-
itives and negatives. Protein network analysis was carried out on
39 selected drug targets, in order to analyze the interactions with
other proteins. Out of these 39 proteins, 6 showed no interactions,
19 showed fewer than 5 interactions, 10 showed 5–10 interactions,
and 4 proteins showed more than 10 interactions (Table 1). The
proteins in these latter two groups were taken for further analysis
because proteins that interact with more proteins are particularly
important for drug targets because they are metabolically active
[31]. As such, 14 proteins were included in the subsequent node
deletion process, while 25 proteins were excluded. From the node
attributes analysis, the selected proteins are very important in the
network because all those proteins have lowest average short-
est path length value, highest closeness centrality, betweenness
centrality and stress centrality values (Suppl. Table 5). The low-
est average shortest path length value indicates the efficiency of
network is good. Higher values of closeness centrality, between-
ness centrality and stress centrality indicates the target proteins
closely to other proteins for fast communication, target proteins
maximum time lies on the shortest path to contact the other pro-
teins and target proteins have higher shortest paths, respectively
[32,33].
The selected 14 proteins (Fig. 2) and their importance were
studied by a node deletion process in STRING database. In this
process, each node was deleted, followed by confirmation of its
importance by analyzing certain parameters. The importance of the
protein network was analyzed using nodes (interacting proteins),
N. Anis Ahamed et al. Journal of Infection and Public Health 14 (2021) 160–168

Table 1
Protein–protein network analysis of selected targets from gut-microbiota study.

S. no. Protein name UniProt ID String ID Number of Interacting proteins

interactions (at
score ≥7.00)

1. Uncharacterized protein Q73BF1 DJ87 3326 1 DJ87 3327

2. Response regulator Q74NU6 DJ87 4944 6 DJ87 2940, DJ87 1948, DJ87 4517, DJ87 4513, DJ87 392,
DJ87 4943
3. Uncharacterized protein Q72YT1 DJ87 88 1 spollB 1
4. Uncharacterized protein Q73AY7 BG03 3625 19 petB, ccsB 1, cccA 1, cccA 2, ccsB, ctaG, DJ87 926, BG03 946,
DJ87 1286, BG11 4268, DJ87 5309, AN402 556, ctaF, DJ87 694,
BG03 1260, DJ87 3157, petC, BG11 4268, BG11 4
5. Lipoprotein Q734V7 DJ87 1662 – –
6. Uncharacterized protein Q733H0 DJ87 1284 9 DJ87 1282, DJ87 5672, DJ87 2153, DJ87 1875, DJ87 1876,
DJ87 2685, Ctc, DJ87 1796, DJ87 4124
7. Uncharacterized protein Q730H4 catD1 1 catE 1
8. Heptaprenyl diphosphate Q73AY3 hepS 1 6 menG, hepT, polA, uppS, xseB, DJ87 685
synthase component I
9. Uncharacterized protein Q737J5 DJ87 2247 – –
10. Uncharacterized protein Q73EA2 ywrJ 2 DJ87 4611, DJ87 4612
11. Uncharacterized protein Q738I8 DJ87 2457 2 dhbB, sfp 1
12. ABC transporter, permease Q72X50 DJ87 3802 3 yxiF 5, lipC 2, ung
protein
13. Uncharacterized protein Q73AU5 DJ87 5414 12 DJ87 5418, dnaD, nth, DJ87 3177, DJ87 3519, DJ87 587,
DJ87 3863, DJ87 586, DJ87 2537, ysxE, ypjB, DJ87 5313
14. Membrane protein Q737B3 DJ87 2173 1 DJ87 2174
15. Uncharacterized protein Q730Y3 DJ87 655 29 DJ87 1948, DJ87 2940, DJ87 1371, spoIIB 1, DJ87 4513,
DJ87 5524, DJ87 3214, DJ87 3310, DJ87 776, DJ87 3960,
DJ87 3518, DJ87 3874, DJ87 2620, ythB 2, DJ87 1368,
DJ87 392, DJ87 1444, DJ87 5705, DJ87 4517, DJ87 2540,
DJ87 3779, DJ87 3459, DJ87 3776, DJ87 1577, DJ87 4179,
DJ87 4180, safA, phaR, DJ87 842
16. Conserved domain protein Q72ZJ6 DJ87 328 8 isdE, yusV 2, isdF, isdE 1
srtB, isdA, isdG, hemE
17. Acetoin utilization protein Q72Z67 acuA 1 5 guaB 1, acuC 1, acsA 1, cvfC 1, DJ87 5026
AcuA
18. Response regulator Q73CK9 DJ87 1444 8 DJ87 1443, DJ87 4517, DJ87 1371, DJ87 2940, DJ87 1948,
DJ87 4513, DJ87 655, DJ87 392
19. Non-hemolytic enterotoxin Q73A16 hbla 1 1 thrC
C
20. Uncharacterized protein Q73BC6 DJ87 3299 2 DJ87 3300, DJ87 3301
21. Uncharacterized protein Q732X7 DJ87 1184 1 DJ87 516
22. Response regulator Q733Y4 DJ87 5705 7 DJ87 5706, DJ87 4513, DJ87 1371, DJ87 1948, DJ87 4517,
DJ87 655, DJ87 392
23. Uncharacterized protein Q74NX7 DJ87 4915 1 DJ987 4916
24. Response regulator Q735S3 DJ87 5250 3 DJ87 4517, DJ87 4513, DJ87 5249
25. Ribosomal protein L5 Q73B52 DJ87 3233 – –
domain protein
26. Uncharacterized protein Q72Y69 DJ87 4971 – –
27. Uncharacterized protein Q733G9 DJ87 1285 2 DJ87 1282, DJ87 5672
28. Competence transcription Q73C31 Comk 2 7 Comk 1, mecA, comEA, mecB, sbrB 1, codY, DJ87 4682
factor
29. Uncharacterized protein Q735B1 DJ87 1712 – –
30. Uncharacterized protein Q730T8 DJ87 614 2 DJ87 612, DJ87 613
31. SCO1/SenC family Q738W5 ypmQ 1 5 coxB, ctaA, ctaG, petB, TQ94 20310
lipoprotein
32. Lipoprotein Q739V7 DJ87 2887 1 dksA
33. Poly(R)-hydroxyalkanoic Q73BI8 phaC 4 phaJ 1, phaQ, phbB, phaR
acid synthase, class III,
PhaC subunit
34. Uncharacterized protein Q732A0 DJ87 894 5 DJ87 895, DJ87 3357, DJ87 908, BG03 567, BG03 3933
35. Uncharacterized protein Q73C53 yhdL 1 4 sigM 1, DJ87 3031, DJ87 523, 2502
36. Uncharacterized protein Q737S4 DJ87 2312 1 DH87 2313
37. Non-hemolytic enterotoxin Q73A17 DJ87 2940 24 DJ87 1371, DJ87 1948, DJ87 655, spoIIB 1, DJ87 2620,
B DJ87 1368, thrC, DJ87 2941, nprE 2, DJ87 4517, DJ87 4944,
DJ87 1444, DJ87 4513, DJ87 2938, DJ87 2540, DJ87 776,
DJ87 5524, DJ87 3214, DJ87 3310, DJ87 3518, DJ87 3776,
DJ87 4179, Plc, DJ87 3779
38. Uncharacterized protein Q737B9 DJ87 2178 – –
39. Uncharacterized protein Q73E08 BG11 3251 1 B4079 4175

edges (interactions), average clustering coefficient, connected com-
ponents, and average node degree. Based on this analysis, all 14
proteins were shown to be very important for the network integrity
and communication between nodes. Specifically, if any of these pro-
teins were absent from the network, the numbers of nodes and
edges decreased, values of average clustering coefficient and aver-
age node degree decreased, and connected groups increased (Suppl.
Table 6). The clustering coefficient is defined as the degree of con-
nectivity with neighboring proteins. The value of this coefficient
and the average node degree decreased when the query node was

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Fig. 2. Protein network analysis of selected proteins.
absent from the network. Proteins are important in the pathway
when their average clustering coefficient and average node degree
are particularly high among all of the proteins [34]. Connected com-
ponent is one important factor for understanding the strength of
the protein network. When some proteins are removed from the
network, if they destroy the single largest connected component,
the removed nodes are considered collective influencers [35]. Also,
the decreased value of network density indicates the importance
of the selected proteins in the network [36] (Fig. 2).
Subcellular localization analysis
Prediction of the subcellular localization of proteins is a key
process because protein localization is strongly connected to bio-
logical function [37]. Generally, proteins are found at five major
sites, namely, plasma membrane, outer membrane, extracellular
region, cytoplasm, and periplasm. The localization of protein scan
help to identify them as drug or vaccine targets: membrane pro-
teins are targets for vaccines and cytoplasmic proteins are targets
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Journal of Infection and Public Health 14 (2021) 160–168
for drugs [38]. The 14 proteins selected in this study were further
subjected to subcellular localization analysis. From the results, out
of the 14 proteins, 10 proteins were present in the cytoplasmic
region, 3 in the extracellular region, and 1 in the membrane region
(Table 2). Cytoplasmic proteins can be used as drug targets, so the
10 cytoplasmic proteins were included for further analysis, while
the other 4 proteins were eliminated.
Druggability analysis
The other significant property for potential therapeutic targets
is druggability [39]. Proteins that are already targeted by drugs are
druggable targets, while those that are not targeted are called novel
targets [19,20]. From the results, seven proteins were not matched
with the drug targets of DrugBank and TTD, while the remaining
three were matched with the drug targets of TTD but have an e-
value less than 0.00001, so they are not druggable targets and are
considered as novel drug targets (Table 3).
N. Anis Ahamed et al. Journal of Infection and Public Health 14 (2021) 160–168

Table 2
Subcellular localization of selected drug targets.

S. no. UniProt ID Protein name Subcellular localization

1. Q74NU6 Response regulator Cytoplasmic

2. Q73AY7 Uncharacterized protein Cytoplasmic
3. Q733H0 Uncharacterized protein Extracellular
4. Q73AY3 Heptaprenyl diphosphate synthase component I Cytoplasmic
5. Q73AU5 Uncharacterized protein Cytoplasmic
6. Q730Y3 Uncharacterized protein Cytoplasmic
7. Q72ZJ6 Conserved domain protein Membrane
8. Q72Z67 Acetoin utilization protein AcuA Cytoplasmic
9. Q73CK9 Response regulator Cytoplasmic
10. Q733Y4 Response regulator Cytoplasmic
11. Q73C31 Competence transcription factor Cytoplasmic
12. Q738W5 SCO1/SenC family lipoprotein Cytoplasmic
13. Q732A0 Uncharacterized protein Extracellular
14. Q73A17 Non-hemolytic enterotoxin B Extracellular

Table 3
Druggability analysis of selected drug targets using DrugBank and TTD.

S. no. Drug target Drug bank TTD

Matched target Drugs used Matched target E-value Drug/inhibitor used

1. Response regulator (Q74NU6) No match – No match – –

2. Uncharacterized protein (Q73AY7) No match – No match – –
3. Heptaprenyl diphosphate synthase component I (Q73AY3) No match – No match – –
4. Uncharacterized protein (Q73AU5) No match – Apolipoprotein B-100 0.67 SPC4955
5. Uncharacterized protein (Q730Y3) No match – Hormone sensitive lipase 0.25 Benzyl benzoate
6. Acetoin utilization protein AcuA (Q72Z67) No match – No match – –
7. Response regulator (Q73CK9) No match – No match – –
8. Response regulator (Q733Y4) No match – Bifunctional aminoacyl 0.55 HT-100
tRNA synthetase
9. Competence transcription factor (Q73C31) No match – No match – –
10. SCO1/SenC family lipoprotein (Q738W5) No match – No match – –

Pathway analysis

Pathway analysis was carried out on the five identified novel

drug targets (Q73AY7, Q73AU5, Q730Y3, Q72Z67 and Q738W5)
using the KEGG database. Acetoin utilization protein AcuA
(Q72Z67) is involved in enzymes-acyltransferases while SCO1/SenC
family lipoprotein (Q738W5) is involved in mitochondrial biogen-
esis (Table 5).

Novel drug targets

Acetoin utilization protein AcuA (Q72Z67)

Fig. 3. Overall result of identification of putative drug targets in B.cereus.
Protein functionality analysis
Molecular function, biological process, and structural informa-
tion of protein are very important for the process of new drug
discovery [9]. Out of 10 drug targets, 3 proteins (Q73AY7, Q73AU5,
and Q73AY3) are uncharacterized or hypothetical proteins. A search
for information on biological process, molecular function, family,
and domains for uncharacterized proteins was performed in the
UniProt database and InterProScan server, but no such informa-
tion found on these proteins. From the similarity search analysis,
similar proteins with more than 40% identity were found for novel
uncharacterized proteins but those proteins also have not known
functional domain and molecular function (Table 4)..
In addition, UniProt indicated four (Q74NU6, Q73CK9, Q733Y4,
and Q73AY3) novel targets predicted from this study as putative
proteins. Uncharacterized proteins and reported existing putative
proteins were eliminated, while the remaining two proteins were
identified as novel drug targets.
AcuA belongs to the acetyltransferase family, functions of this
protein as Elp3-related proteins [40], and is encoded by the acuA
gene. It is part of the acuABC operon, which is possibly involved in
the breakdown of acetoin and butanediol. This protein is involved
in acetoin degradation, which is part of ketone degradation. Acetoin
dehydrogenase and N-acetyltransferase activities are important
molecular functions of the AcuA protein. N-Acetyltransferase activ-
ity is involved in catalyzing the transfer of an acetyl group to a
nitrogen atom on the acceptor molecule. This protein is 214 amino
acid residues in length and has a single domain, Acetyltransf 1;
the region of this domain extends from residue 31 to 141. Binding
site residues of the AcuA protein include Ile105, Glu106, Val107,
Gln117, and Lys118.
SCO1/SenC family lipoprotein (Q738W5)
This protein belongs to the copper chaperone SCO1/SenC fam-
ily and is encoded by the BCE 2278 gene. This family is involved in
the biogenesis of respiratory and photosynthetic systems. SCO1 is
required for a post-translational step in the accumulation of sub-
units COXI and COXII of cytochrome c oxidase [41]. SenC is required
for optimal cytochrome c oxidase activity and maximal induction
of genes encoding light-harvesting and reaction center complexes

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Table 4
Functional prediction of uncharacterized protein.

S. no. Protein name and ID Similar protein name and Id % of Identity Functional domain Molecular function

1. Uncharacterized protein (Q73AY7) Putative lipoprotein (D5DRG7) 58.9 – Not predicted

2. Uncharacterized protein (Q73AU5) GTPase (A0A073K287) 51.7 – Not predicted
3. Uncharacterized protein (Q730Y3) NlpC/P60 family protein (A0A3S9PXW4) 40.4 Domain: NLPC P60 (unknown function) Not predicted

Table 5
Pathway analysis of selected drug targets.

S. no. Protein name and ID Gene name Identified pathway

1. Acetoin utilization protein AcuA (Q72Z67) acuA (BCE 4802) Enzymes – acyltransferases
2. SCO1/SenC family lipoprotein (Q738W5) BCE 2278 Mitochondrial biogenesis

Fig. 4. Modeled proteins (a) Acetoin utilization protein AcuA and (b) SCO1/SenC family lipoprotein.

Fig. 5. Ramachandran plot analysis modeled proteins (a) Acetoin utilization protein AcuA and (b) SCO1/SenC family lipoprotein.

Table 6
Predicted binding site residues of novel drug targets using sitemap.

S. no. Protein name Binding site residues

1. Acetoin utilization protein AcuA D42,I44,A45,F46,R47,Q52,A55,I59,T81,L83,D86,E89,E101,G103,A104,I105,E106,V107,P109,F111,

R112,G113,C114,A115,V116,G117,K118,T138,E139,Y140,W142,H143,W144,D145,Q148,T149,L151,
Y156,V159,M160,M163,E178
2. SCO1/SenC family lipoprotein D36,L37,E38,T39,F40,T52,L55,K56,W60,N168,G169,K170

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Table 7
Docking result of antibiotics compound with AOU protein.

S. no. Compound Dock score (kcal/mol) Interacting residues Bond length (Å) HBond value

1. Gentamicin −12.0 Arg47, Glu58, Asp86, Glu89, Glu101 2.07, 2.21, 1.89, 2.21, 1.90 −3.4
2. Chloramphenicol −7.3 Val107(2), Tyr156 1.68, 2.06, 2.20 −2.8
3. Imipenem −7.0 Glu139(3) 1.89, 2.10, 2.50 −1.6

Fig. 6. Gentamicin docked with the novel drug targets (a) Acetoin utilization protein AcuA and (b) SCO1/SenC family lipoprotein.

Table 8
Docking result of antibiotics compound with SCOII protein.

S. no. Compound Dock score (kcal/mol) Interacting residues Bond length (Å) HBond value

1. Imipenem −7.0 Asp36, Leu37, Lys56 1.69, 1.86, 1.82 −2.8

2. Gentamicin −7.0 Asp36(3), Leu37, Lys56, Gly169, Lys17 (2) 1.83, 1.97, 2.04, 1.94, 1.81, 2.02, 1.75, 2.22 −2.6
3. Chloramphenicol −4.1 Leu37(2), Lys56, Asn168, Lys170 1.90, 1.96, 2.13, 2.15, 1.90 −1.5

[42]. This protein contains 194 amino acid residues and has a single new drug molecules specifically for novel drug targets for inhibiting
domain named SCO1-SenC; this region extends from residue 37 to the function and growth of foodborne causing pathogen B. cereus.
171 The result of putative drug target identification in B. cereus was
shown in Fig. 3. Conclusion

This study was identified 2 novel drug targets, Acetoin uti-

Molecular docking studies
Protein structure prediction
Both structures were modeled using Prime suite, 2Q04 and
4HDE were identified as templates for novel targets Acetoin uti-
lization protein AcuA and SCO1/SenC family lipoprotein with the
sequence identity of 56.04% and 90.81%, respectively. These mod-
eled proteins (Fig. 4) were further validated which resulted with
both proteins having good quality with more than 90% of residues
in the favored region (Fig. 5). Proteins binding site residues were
shown in Table 6.
From the docking analysis, the antibiotic Gentamicin had good
binding affinity with two novel targets than other antibiotic drugs
vancomycin, imipenem, chloramphenicol, and ciprofloxacin. It had
dock score −12.0 kcal/mol (Table 7) and interacted with the binding
site residues Arg47, Glu58, Asp86, Glu89, Glu101 of Acetoin uti-
lization protein AcuA (Fig. 6). Also, it had dock score −7.0 kcal/mol
(Table 8) and interacted with the binding site residues Asp36,
Leu37, Lys56, Gly169, Lys170 of SCO1/SenC family lipoprotein
(Fig. 6).
The Gentamicin may be influence the novel targets especially
with Acetoin utilization protein AcuA. It will be confirmed through
in vitro and in vivo studies. But, Gentamicin and other antibiotics
may cause adverse side effects in the host; particularly it is toxic
to kidney [43]. Not only Gentamicin also the antibiotics which are
using for B. cereus has side effects due to drug-host interaction. But
this binding affinity of Gentamicin with the novel targets gives new
insights to find good inhibitors from natural sources or synthesized
which are similar to Gentamicin. Also, this study suggests designing
lization protein AcuA (Q72Z67) and SCO1/SenC family lipoprotein
(Q738W5) from B. cereus which is causing foodborne infection in
human using subtractive proteomics approach. These targets are
only present in B. cereus and not in human and gut-microbiota
also these targets are not treated with any kind of existing drug
molecules. These targets may very helpful in the field of pharma-
cotherapy to produce new drugs for inhibiting the function and
growth of organisms in human without any cross-reactivity to
human proteins.
Funding
No funding sources.
Competing interests
None declared.
Ethical approval
Not required.
Acknowledgment
This project was supported by King Saud University, Deanship
of Scientific Research Chair. We are very grateful to Prince Sultan
Research Chair for Environment and Wildlife & Saudi Biological
Society. We thank the Department of Botany & Microbiology, Col-

167
N. Anis Ahamed et al.
lege of Sciences, King Saud University (KSU), Riyadh, Saudi Arabia,
for encouragement and support for funding this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.jiph.2020.12.005.
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