Research Article | Open Access
Niharika Chandra1, Tabish Qidwai1 and Sachidanand Singh1,2
1Faculty of Biotechnology, Institute of Biosciences and Technology, Shri Ramswaroop Memorial University, Barabanki, Uttar Pradesh, India.
2Department of Biotechnology, Smt. S.S. Patel Nootan Science & Commerce College, Sankalchand Patel University, Visnagar, Gujarat, India.
Article Number: 8748 | © The Author(s). 2023
J Pure Appl Microbiol. 2023;17(3):1893-1906. https://doi.org/10.22207/JPAM.17.3.55
Received: 04 June 2023 | Accepted: 26 August 2023 | Published online: 03 September 2023
Issue online: September 2023
Abstract

We discover essential enzymes catalyzing critical metabolic reactions as potential drug targets, which may help to fight Listeria infections and their associated secondary infections extensively and effectively. A comparative metabolic pathway approach has been applied to identify and determine putative drug targets against Listeria monocytogenes. For this, enzymes unique to pathogenic pathways of L. monocytogenes EGD-e were determined using the KEGG database. They were further refined by selecting enzymes with sequences non-homologous to the host Homo sapiens and analysing their essentiality to the pathogen’s survival. We report 15 essential pathogen-host non-homologous proteins as putative drug targets that can be exploited for development of specific drug targets or vaccines against multidrug resistant strains of L. monocytogenes. Finally, four essential enzymes from the pathogen: UDP-N-acetylglucosamine 1-carboxyvinyltransferase, Acetate kinase, Phosphate acetyltransferase, and Aspartate kinase were reported as novel putative targets for vaccine and drug development against L. monocytogenes infections. Unravelling novel target proteins and their associated pathways by comparing metabolic pathway analysis between L. monocytogenes EGD-e and host H. sapiens, develops the novelty of the work towards broad spectrum putative drug targets. This research design yields putative drug target critical enzymes that turn out to be fatal to the pathogen without interacting with the host machinery.

Keywords

Listeria monocytogenes EGD-e, Homo sapiens, Comparative Metabolic Pathway Analysis, Critical Enzymes, Novel Putative Drug Targets

Introduction

Humans are victim of a large number of microbial pathogens through infection in the respiratory tract, gastrointestinal tract, or genital tract epithelium. These pathogens further migrate to secondary locations through lymph and blood to cause diseases in the liver, spleen, bones, and central nervous system (CNS).1 Listeria monocytogenes is a Gram-positive, rod-shaped, foodborne pathogenic bacterium that is associated with listeriosis, CNS infection, meningitis, sepsis, liver infection, spleen infection and premature birth/ abortions.2,3 It is also associated with immune response based diseases like Rheumatoid Arthritis (RA) and Inflammatory Bowel Disease (IBD).4-6 L. monocytogenes has been found proliferating in colon of a patient suffering from IBD, much higher than in a healthy individual. Resistant biofilms of Listeria have been reported on all forms of synthetic and biological surfaces, including gut epithelium.7,8 L. monocytogenes enters the human host through the ingestion of contaminated food and reaches the intestine. It crosses the intestinal epithelial barrier and enters the bloodstream, from where it causes secondary infection in several organs such as liver, spleen, bones, brain etc.2 In South Africa, 978 people were found positive for listeriosis infection which led to 674 hospitalizations and 183 deaths were recorded in 2018.9 Listeriosis outbreaks due to food borne infections have been recorded regularly in Turkey.10 1,763 cases of listeriosis from 27 states were recorded by European Food Safety Authority (EFSA) in 2013, leading to 191 deaths.11 The fatality rate of listeriosis is about 20-30% among various parts of the world in 2018-19.12-14 The current treatment of such Listeria infections depends on the use of antibiotics, which turn out to be ineffective and have risks of developing drug-resistant strains. At first, the most common treatment for Listeria infections was administration of antibiotics such as penicillin G and/or ampicillin along with aminoglycoside such as gentamicin or kanamycin. Subsequently, a combination of trimethoprim and a sulphonamide was also used as a therapy.3,15 Initially, the majority of the L.monocytogenes strains (isolated from various sources) were susceptible to antibiotics, active against the Gram positive bacteria. The first drug resistance was observed against the antibiotic tetracycline in 1988.16,17 A multidrug resistant strain of L. monocytogenes was also isolated in France in 1988.16,17 Consequently, several multidrug resistant strains of Listeria have been discovered from environment, food sources and human listeriosis gut/stool samples.17,18 L. monocytogenes strains isolated from several food items (including ready-to-eat food) were reported to be resistant to several antibiotics such as ampicillin, lindamycin, nalidixic acid, penicillin (100%) and oxacillin (94.1%).19 The indiscriminate use of effective antibiotics in humans and animals has resulted in an exposure to high concentrations of these antibiotics, leading to resistance development in pathogenic strains of Listeria through gene alterations and transposons.20,21 The infection of these multidrug resistant strains of L. monocytogenes through various food sources may pose a serious threat to public health.9 So, this research attempts to find enzymes catalyzing essential metabolic reactions as potential drug targets, which may help to fight L. monocytogenes infections and their associated secondary infections extensively and effectively.

The easy accessibility of human genome sequences, complete genome sequences of several human infecting pathogens, and availability of several computational tools for their in silico analysis helps us to identify biomarkers and potential drug targets to combat these pathogens. Among these, comparative metabolic pathway analysis interpretations involve the understanding of the organism’s physiology, intracellular procedures, and networks, which can find the specific target molecules responsible for their survival, vital functions, or virulence. This can further be used to develop specific and effective drugs against the pathogens. Metabolic pathways illustrate how the bio-molecular units interact with each other to carry out the functions required for the survival, reproduction, and other organism-specific activities.22 Understanding the phenotype and function of organisms requires a detailed analysis of the metabolic pathways involved along with the study of single genes, as the expression of all the protein metabolites is a result of the action of enzymes catalyzing their interconversion. Proteins are the functional biomolecular element of the cell which converts the genetic information into practical reality. They are involved in gene regulation, cell metabolism, transport of nutrients, signal transduction, and enzymatic catalysis. Hence, the pathogen-specific proteins/enzymes can be used as broad-spectrum potential drug targets against these pathogens.

In the present study, comparative metabolic pathway analysis was implied between L. monocytogenes EGD-e (Reference genome) and H. sapiens with the aim to select enzymes unique to L. monocytogenes EGD-e, which are non-homologous to host. These enzymes can work as putative targets for the development of drugs to eradicate these pathogen without interacting with the host machinery. These enzymes were further analysed for their essential role in survival of the pathogen. As targeting the critical enzymes turn out to be fatal to the pathogen and help developing broad-spectrum drugs. Different comparative genomics and transcriptomics studies and comparative proteomic analysis have been performed between different strains of L. monocytogenes or between different species of Listeria with diverse objectives, but unravelling novel target proteins and their associated pathways by comparing metabolic pathway analysis between L. monocytogenes EGD-e and host Homo sapiens, develops the novelty of the work towards broad spectrum putative drug targets.

Materials and Methods

Metabolic Pathway Analysis of Pathogen and Host
Metabolic pathways of L. monocytogenes EGD-e and H. sapiens were retrieved from Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/).23 A manual assessment was performed to classify common and unique metabolic pathways between host and pathogen. The metabolic pathways present in pathogen but missing in the host were categorized as unique pathways, whereas the pathways present in both the pathogen and host were categorized as common pathways. The enzymes from unique metabolic pathways, having a sanctioned Enzyme Commission number (EC number), were mapped, and their protein sequences were extracted from the National Centre for Biotechnology Information (NCBI) genome database.24

Identification of Pathogen-Host Non-homologous Proteins
BLASTP25 analysis (Protein-protein Basic Local Alignment Search Tool) was performed for protein sequences of enzymes from unique metabolic pathways against the H. sapiens proteome. The protein sequences with an e value cut-off of 5e-3 were considered as homologous to the pathogen and were excluded from the study. The protein sequences without a hit under this criterion were considered to have no significant homolog in H. sapiens and selected for further analysis.26

Identification of Essential Pathogen-Host Non-homologous Proteins
BLASTP analysis was performed between the obtained non-homologous protein sequences and prokaryotic essential genes from database of essential genes (DEG; http://www.essentialgene.org/) to yield essential pathogen-host non-homologous proteins 27. The criteria of essentiality are as follows: evalue < 1e-10, bit score ≥ 100 and percentage identity ≥ 30.26

Subcellular Localization of Proteins
The exact position of these proteins in the cell was predicted using CELLO version 2.5 (subCELlular LOcalization predictor version 2.5).28 CELLO applies the amino acid composition and di-peptide composition based on physico-chemical parameters of amino acids to predict the subcellular position of the proteins.28 The gram-positive bacterial proteins have the following localization sites: the cell membrane, the cytoplasm, the extracellular space and the cell wall.

Structural Classification of the Unique Identified Target Proteins
The predicted enzymes as drug targets were further analysed and classified for their three dimensional structure and presence of any best possible ligands in their binding pockets. The available structures of the predicted potential drug targets were retrieved and studied from the RCSB Protein Data Bank.29 The research design for this study has been presented in Figure.

Figure. Schematic representation of subtractive genomics methodology for identification of potential drug and vaccine targets against L. monocytogenes. KEGG pathway analysis of metabolism for the pathogen (L. monocytogenes) and the host (H. sapiens) led to the subsequent identification of essential pathogen-host non-homologous enzymes/proteins which can be targeted as possible drug/vaccine candidates. (KEGG: Kyoto Encyclopedia of Genes and Genomes; BLASTP: Protein-protein Basic Local Alignment Search Tool; DEG: Database of Essential Genes; CELLO v2.5: subCELlular LOcalization predictor version 2.5)

RESULTS AND DISCUSSION

KEGG Pathway Analysis Interpretations
KEGG database analysis revealed a total of 107 pathways associated with L. monocytogenes EGD-e. Most of these pathways were biosynthesis and metabolic pathways such as glycolysis, TCA cycle, carbohydrate metabolism, fatty acid biosynthesis and degradation, amino acid biosynthesis and degradation, nucleotide metabolism, peptidoglycan biosynthesis, vitamin metabolism, central dogma, nitrogen and sulfur metabolism etc. Also, other pathways associated with bacterial functions and virulence such as secondary metabolites biosynthesis, microbial metabolism in different environments, degradation of aromatic compounds, resistance to antibiotics such as vancomycin and beta-Lactam, cationic antimicrobial peptide (CAMP) resistance, quorum sensing, chemotaxis, NOD-like receptor signaling pathway, two-component system, and bacterial invasion of epithelial cells were reported (https://www.genome.jp/kegg/).

H. sapiens have 337 reported pathways in the KEGG database. These pathways consist of the eukaryotic biosynthesis and metabolic pathways. They also have several pathways related to drug and xenobiotics resistance and metabolism, signalling pathways, apoptosis, muscle contraction, platelet activation, circadian cycle, hormone secretion and immune responses. A number of pathways were specifically related to diseases and infections such as Alzheimer disease, Parkinson disease, Huntington disease, prion diseases, drug addiction, Vibrio cholerae infection, Pathogenic Escherichia coli infection, Salmonella infection, malaria, tuberculosis, pathways in cancer, asthma, inflammatory bowel disease, and rheumatoid arthritis etc (https://www.genome.jp/kegg/).

The comparative metabolic pathway analysis using the KEGG database resulted in 28 pathways that were unique to the pathogen L. monocytogenes EGD-e, whereas 71 pathways, common to both L. monocytogenes EGD-e and H. sapiens (Table 1 and Table 2 respectively). A total number of 180 enzymes with valid EC numbers were identified from the unique pathogen pathways (Supplementary Table 1).

Table (1):
List of metabolic pathways common to both the pathogen Listeria monocytogenes EGD-e and host Homo sapiens (KEGG: Kyoto Encyclopedia of Genes and Genomes)

S. No.
Metabolic pathway
KEGG pathway ID
1
Glycolysis / Gluconeogenesis
lmo00010
2
Citrate cycle (TCA cycle)
lmo00020
3
Pentose phosphate pathway
lmo00030
4
Pentose and glucuronate interconversions
lmo00040
5
Fructose and mannose metabolism
lmo00051
6
Galactose metabolism
lmo00052
7
Starch and sucrose metabolism
lmo00500
8
Amino sugar and nucleotide sugar metabolism
lmo00520
9
Pyruvate metabolism
lmo00620
10
Glyoxylate and dicarboxylate metabolism
lmo00630
11
Propanoate metabolism
lmo00640
12
Butanoate metabolism
lmo00650
13
Inositol phosphate metabolism
lmo00562
14
Oxidative phosphorylation
lmo00190
15
Nitrogen metabolism
lmo00910
16
Sulfur metabolism
lmo00920
17
Fatty acid biosynthesis
lmo00061
18
Fatty acid degradation
lmo00071
19
Synthesis and degradation of ketone bodies
lmo00072
20
Glycerolipid metabolism
lmo00561
21
Glycerophospholipid metabolism
lmo00564
22
Arachidonic acid metabolism
lmo00590
23
Purine metabolism
lmo00230
24
Pyrimidine metabolism
lmo00240
25
Alanine, aspartate and glutamate metabolism
lmo00250
26
Glycine, serine and threonine metabolism
lmo00260
27
Cysteine and methionine metabolism
lmo00270
28
Valine, leucine and isoleucine degradation
lmo00280
29
Valine, leucine and isoleucine biosynthesis
lmo00290
30
Lysine degradation
lmo00310
31
Arginine biosynthesis
lmo00220
32
Arginine and proline metabolism
lmo00330
33
Histidine metabolism
lmo00340
34
Tyrosine metabolism
lmo00350
35
Phenylalanine metabolism
lmo00360
36
Tryptophan metabolism
lmo00380
37
Phenylalanine, tyrosine and tryptophan biosynthesis
lmo00400
38
beta-Alanine metabolism
lmo00410
39
Taurine and hypotaurine metabolism
lmo00430
40
Phosphonate and phosphinate metabolism
lmo00440
41
Selenocompound metabolism
lmo00450
42
D-Glutamine and D-glutamate metabolism
lmo00471
43
D-Arginine and D-ornithine metabolism
lmo00472
44
Glutathione metabolism
lmo00480
45
Other glycan degradation
lmo00511
46
Thiamine metabolism
lmo00730
47
Riboflavin metabolism
lmo00740
48
Vitamin B6 metabolism
lmo00750
49
Nicotinate and nicotinamide metabolism
lmo00760
50
Pantothenate and CoA biosynthesis
lmo00770
51
Biotin metabolism
lmo00780
52
Lipoic acid metabolism
lmo00785
53
Folate biosynthesis
lmo00790
54
One carbon pool by folate
lmo00670
55
Porphyrin and chlorophyll metabolism
lmo00860
56
Ubiquinone and other terpenoid-quinone biosynthesis
lmo00130
57
Terpenoid backbone biosynthesis
lmo00900
58
RNA polymerase
lmo03020
59
Ribosome
lmo03010
60
Aminoacyl-tRNA biosynthesis
lmo00970
61
Protein export
lmo03060
62
Sulfur relay system
lmo04122
63
RNA degradation
lmo03018
64
DNA replication
lmo03030
65
Base excision repair
lmo03410
66
Nucleotide excision repair
lmo03420
67
Mismatch repair
lmo03430
68
Homologous recombination
lmo03440
69
ABC transporters
lmo02010
70
NOD-like receptor signaling pathway
lmo04621
71
Bacterial invasion of epithelial cells
lmo05100

Table (2):
List of metabolic pathways which are unique to the pathogen Listeria monocytogenes EGD-e (KEGG: Kyoto Encyclopedia of Genes and Genomes)

S. No.
Metabolic pathway
KEGG pathway ID
No. of proteins
1
C5-Branched dibasic acid metabolism
lmo00660
7
2
Methane metabolism
lmo00680
17
3
Secondary bile acid biosynthesis
lmo00121
2
4
Lysine biosynthesis
lmo00300
17
5
Cyanoamino acid metabolism
lmo00460
5
6
D-Alanine metabolism
lmo00473
5
7
Peptidoglycan biosynthesis
lmo00550
16
8
Polyketide sugar unit biosynthesis
lmo00523
4
9
Carbapenem biosynthesis
lmo00332
2
10
Monobactam biosynthesis
lmo00261
6
11
Streptomycin biosynthesis
lmo00521
7
12
Acarbose and validamycin biosynthesis
lmo00525
2
13
Novobiocin biosynthesis
lmo00401
3
14
Benzoate degradation
lmo00362
3
15
Aminobenzoate degradation
lmo00627
3
16
Chloroalkane and chloroalkene degradation
lmo00625
1
17
Xylene degradation
lmo00622
2
18
Styrene degradation
lmo00643
3
19
Naphthalene degradation
lmo00626
1
20
Phosphotransferase system (PTS)
lmo02060
58
21
Bacterial secretion system
lmo03070
3
22
Two-component system
lmo02020
26
23
Quorum sensing
lmo02024
12
24
Bacterial chemotaxis
lmo02030
2
25
Flagellar assembly
lmo02040
1
26
beta-Lactam resistance
lmo01501
3
27
Vancomycin resistance
lmo01502
6
28
Cationic antimicrobial peptide (CAMP) Resistance
lmo01503
6

Determination of Essential Pathogen-Host Non-homologous Proteins
The unique pathogen enzymes were then compared with host proteome to identify host non-homologous proteins. Out of 180 enzymes, 120 enzyme sequences revealed d” 35% identity with human proteome, and 52 enzyme sequences did not show any hit against the human proteome (Supplementary Table 2). Enzymes which are unique to L. monocytogenes and also do not show any or significant homology to the host proteome may act as effective drug targets as these drug/vaccine candidates have reduced risk of any unwanted interaction with the host proteins. Hence, these drugs will be safe and not adversely affect the human host metabolism.

Essential genes are the least number of genes which are obligatory for the existence of any organism.30 Essential genes determined for 48 bacterial species have been listed in DEG. This essential genes list can be directly extracted and BLAST analysis can be performed. Alternatively, a common list of prokaryotic essential genes can be used for analysis of other organisms which are not listed in DEG, as performed in this study (DEG; http://www.essentialgene.org/). The knockout of any bacterial essential gene can produce lethal phenotypes, so essential genes may act as significant drug targets.31 This can also be exploited for development of specific drug targets or vaccines against multidrug resistant strains such as L. monocytogenes. Some essential genes may be conserved over a number of related species and are potential targets for development of broad spectrum antibiotics.27,31 Hence, 172 host-non-homologous enzyme sequences were analyzed for essentiality of L. monocytogenes using DEG. 98 enzymes were found to be essential enzymes of L. monocytogenes with an average identity of 49% to essential protein sequences of prokaryotes (Supplementary Table 3).

Subcellular Localization of Target Enzymes/Proteins
Subcellular localization of these enzymes was determined by CELLO v.2.5, which may provide important information about the function of protein. The bacterial proteins/enzymes present in Gram-positive bacterial dataset are mostly localized in the cytoplasm and the cell membrane. Rest of the proteins are localized in the extracellular space and very few are found at the cell wall.32 CELLO categorized our essential host non-homologous enzymes of L. monocytogenes, as presented in Supplementary Table 4.

Prediction of Potential Targets/ Enzymes for Drug and Vaccine Development
Out of the 52 non-homologous enzymes (with no identity match with H. sapiens proteome), 15 were recorded as essential enzymes for L. monocytogenes by DEG analysis with an average E value ≤ 1.4 e-23 and identity ≥ 47%. Further, we propose four L. monocytogenes enzymes as putative drug targets, completely non-homologous to human and critically important to survival of pathogen (with identity ≥ 60% to the essential prokaryotic sequences), as listed in Table 3. The nature and site of action of all these four enzymes was determined to be cytoplasmic. These enzymes tend to play a central role in the pathogenesis of L. monocytogenes, causing infections in humans.

Table (3):
Potential drug target enzymes from Listeria monocytogenesEGD-e, showing their essential role in survival of the pathogen and non-homologous nature to host Homo sapiens. KEGG: Kyoto Encyclopedia of Genes and Genomes)

S.No.
Listeria monocytogenes EGD-e enzyme name
KEGG ID
Non homologous (Yes/No) and Percentage Identity (%) to Homo sapiens
Essential (Yes/No) and Percentage Identity (%) to Listeria monocytogenesEGD-e
Sub-cellular localization
1
K00790 UDP-N-acetylglucosamine 1-carboxyvinyltransferase [EC:2.5.1.7] |
lmo:lmo2526
Yes

0%

Yes

72.55%

Cytoplasmic
2
K00925 acetate kinase [EC:2.7.2.1] |
lmo:lmo1581
Yes

0%

Yes

71.61%

Cytoplasmic
3
K00625 phosphate acetyltransferase [EC:2.3.1.8] |
lmo:lmo2103
Yes

0%

Yes

63.11%

Cytoplasmic
4
K00928 aspartate kinase [EC:2.7.2.4] |
lmo:lmo1235
Yes

0%

Yes

61%

Cytoplasmic
5
K02760 PTS system, cellobiose-specific IIB component [EC:2.7.1.196 2.7.1.205] |
lmo:lmo2373
Yes

0%

Yes

57.73%

Cytoplasmic
6
K02769 PTS system, fructose-specific IIB component [EC:2.7.1.202] |
lmo:lmo0427
Yes

0%

Yes

57.29%

Cytoplasmic
7
K04041 fructose-1,6-bisphosphatase III [EC:3.1.3.11] |
lmo:lmo0830
Yes

0%

Yes

51.85%

Cytoplasmic
8
K02799 PTS system, mannitol-specific IIB component [EC:2.7.1.197]
lmo:lmo2799
Yes

0%

Yes

50.11%

Membrane
9
K02777 PTS system, sugar-specific IIA component [EC:2.7.1.-] |
lmo:lmo1017
Yes

0%

Yes

48.41%

Cytoplasmic
10
K00887 undecaprenol kinase [EC:2.7.1.66] |
lmo:lmo1464
Yes

0%

Yes

46.4%

Membrane
11
K02755 PTS system, beta-glucoside-specific IIA component [EC:2.7.1.-]
lmo:lmo2772
Yes

0%

Yes

42.11%

Membrane
12
K00425 cytochrome bd ubiquinol oxidase subunit I [EC:7.1.1.7]|
lmo:lmo2718
Yes

0%

Yes

41.45%

Membrane
13
K01624 fructose-bisphosphate aldolase, class II [EC:4.1.2.13] |
lmo:lmo0359
Yes

0%

Yes

39.1%

Cytoplasmic
14
K00003 homoserine dehydrogenase [EC:1.1.1.3] |
lmo:lmo2547
Yes

0%

Yes

38.26%

Cytoplasmic
15
K12555 penicillin-binding protein 2A [EC:2.4.1.129 3.4.16.4] |
lmo:lmo2229
Yes

0%

Yes

33.22%

Membrane

UDP-N-acetylglucosamine 1-carboxyvinyltransferase is an enzyme of class transferases that catalyzes the transfer of enolpyruvate group to UDP-N-acetyl-a-D-glucosamine which is a significant and committing reaction of cell wall formation in bacteria.33 It belongs to 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase family and subfamily of MurA. This enzyme also substantially involved in other biological processes like cell division, cell wall cycle, cell wall organization, and cell shape regulation along with peptidoglycan biosynthetic pathway.33,34 (https://www.uniprot.org/).

Acetate kinase is involved in the acetyl-CoA biosynthesis pathway by catalyzing a sub-pathway reaction of phosphorylating acetate utilizing ATP and a divalent cation such as Mg2+ or Mn2+. The reaction is summarized as: acetate + ATP = acetyl phosphate + ADP. The reverse reaction can also be catalyzed by this same enzyme. It is also involved in some metabolic intermediate biosynthesis, such as the organic acid metabolic process (https://www.genome.jp/kegg/). It has also been reported that acetyl phosphate levels in L. monocytogenes are directly involved in monitoring cell motility, chemotaxis, and resistant biofilm formation. In a study, Gueriri and co-workers developed acetate kinase mutants of L. monocytogenes with a blocked synthesis of acetyl phosphate. These mutants were reported with a phenotype of decreased ability of biofilm formation and diminished expression of flagellar protein biosynthesis and motility genes.35 Also, other studies have reported that some L. monocytogenes virulence factors such as VirR/VirS can be activated by the production of acetyl phosphate in the cells.36 The activity of acetate kinase has also been recorded in other pathogenic intestinal bacterial strains such as Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9. These pathogen bacteria have been found to be involved in causing IBD in the human host.37

The third drug target enzyme, phosphate acetyltransferase, shows transferase activity. Phosphate acetyltransferase, along with the subsequent action of acetate kinase, produces acetate from acetyl-CoA (or acetyl phosphate) and generates ATP.38

The Aspartate kinase enzyme performs kinase activity, transferase activity, and binding of ATP by the reaction: ATP + L-aspartate = 4-phospho-L-aspartate + ADP. Aspartate kinase is involved in cellular amino acid biosynthetic pathways such as lysine biosynthesis via diaminopimelate (DAP) formation, homoserine biosynthesis, and threonine biosynthetic pathway 39 (https://www.uniprot.org/). These targets have not been used for drug/vaccine development to our best knowledge.

Other than these four novel drug targets, most of the other 11 enzymes have also been established as potential candidates for drug targets and have been reported in several other studies. Fructose-bisphosphate aldolase (FBA) class II, is a cytoplasmic or surface exposed bacterial enzyme catalysing the cleavage of fructose-1,6-bisphosphate to D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, an important reversible step in glycolysis and gluconeogenesis.40 FBA is known to perform two or more unrelated functions in several bacterial species and hence is a moonlighting protein. FBA can play a significant role in binding to the host’s cells and to host’s proteins, subsequent generation of an immune response etc, and hence is involved in physiology and pathogenesis of the bacteria.40 The structure and sequence of FBA remains conserved among same and different bacterial species, so it can be exploited to develop broad spectrum antibiotics/vaccines against a wide group of pathogenic bacteria.41 Mendonca and coworkers reported FBA class II as a novel immunogenic surface protein and monoclonal antibody (mAb-3F8) against this protein for detection of the Listeria spp. and to distinguish Listeria from other pathogenic bacteria.42

The primary treatment for L. monocytogenes infected population is administration of b-lactam antibiotics (such as penicillins) which target a set of enzymes, the penicillin-binding proteins (PBPs) involved in peptidoglycan linking.43 Our findings also report penicillin-binding protein 2A as a putative drug target. Peptidoglycan is a foremost element of the bacterial cell wall which is integral to the cell structure and morphology. Peptidoglycan biosynthesis involves the creation of mesh like structure, which is facilitated by two steps: transglycosylation (elongation of glycan chain) and transpeptidation (peptide cross-linking the flanking glycan chains).44 High molecular weight Class A PBP catalyze both of these reactions through their N-terminal glycosyltransferase and C-terminal transpeptidase domain.44 Another research which applied several in silico approaches such as subtractive genomics and protein–protein interaction network topology, reported PDB4 along with 10 other proteins as a putative drug target in L. monocytogenes EGD-e proteome.45

We have also reported several phosphotransferase system (PTS) such as cellobiose-specific IIB component, mannitol-specific IIB component, fructose-specific IIB component, sugar-specific IIA component, and beta-glucoside-specific IIA component as putative drug targets. The PTS system is composed of a few soluble proteins and one membrane spanning protein, which are involved in the uptake/transport of PTS carbohydrates by the cell.46 It has also been reported to be actively involved in several regulatory functions such as catabolite repression, potassium transport, nitrogen and phosphate metabolism, antibiotic resistance, endotoxin production, biofilm formation, and virulence of several pathogens including L. monocytogenes.46 PTS mediated sugar transport of cellobiose, mannose, and glucose has been reported to regulate PrfA activity, which is in turn the major transcription factor regulating the virulence gene in L. monocytogenes.47 In a study based on comparative genomics of Vibrio cholera, several constituents of the PTS were described as drug and vaccine targets against the pathogen.48 Similarly, six components of the PTS system were reported as putative drug targets in Klebsiella pneumoniae MGH78578 using the in silico approach.49 Hence, the PTS system may act as an effective drug target for L. monocytogenes too.

Structural Classification of the Unique Identified Target Proteins
The available structures of the predicted potential drug targets were retrieved and studied from the RCSB Protein Data Bank.29 The structures of the predicted drug targets were available on PDB either in L. monocytogenes or in other microbes.  The minimum criteria for considering the three dimensional structure was on the basis of Identity >= 70%, Query coverage of the sequence as 80% and E Value <= 0.00. The crystal structure of PBP 4 from L. monocytogenes in the Ampicillin bound form (PDB ID: 3ZG8)50 and PBP D2 from L. monocytogenes in apo form(PDB ID: 5ZQA)51 were accessible in PDB. Both these structures were studied in expression system of Escherichia coli. Several ligands such as (2r,4s)-2-[(1r)-1-{[(2r)-2-Amino-2-Phenylacetyl]amino}-2-Oxoethyl]-5,5-Dimethyl-1,3-Thiazolidine-4-Carboxylic acid, glycerol and Di(hydroxyethyl)ether have been reported to bind to the A and B chains of this enzyme. Structures for other drug target proteins were available for different other organisms. For instance, Apo structure of fructose 1,6-bisphosphate aldolase from Bacillus anthracis str. ‘Ames Ancestor’ (PDB ID: 3Q94) can be studied at PDB.52 This crystal structure was determined through X-ray diffraction experiment and the enzyme was found to be composed of A and B chains with two reported ligands (1,3-Dihydroxyacetonephosphate and acetate ion) interacting with the A chain of the protein. Similarly, the structure of Cytochrome BD-I ubiquinol oxidase from Escherichia coli (PDB ID: 6RX4) 53 and Homoserine Dehydrogenase from Saccharomyces cerevisiae (PDB ID: 1EBU) 54 were available. Two ligands (3-Aminomethyl-Pyridinium-Adenine-Dinucleotide and L-Homoserine) interacting with the D chain of the Homoserine Dehydrogenase enzyme and four ligands (Heme b/c, Cis-Heme d hydroxychlorin gamma-Spirolactone, 1,2-Dioleoyl-Sn-Glycero-3-Phosphoethanolamine, and Ubiquinone-8) binding with A and B chains of Cytochrome BD-I ubiquinol oxidase have been reported so far. Also, the structures of several enzymes reported as drug targets from the PTS system were retrieved for different organisms from PDB. Such as crystal structure of PTS System Cellobiose-specific Transporter Subunit IIB from Bacillus anthracis (PDB ID: 4MGE),55 and structure of IIB domain of the mannitol-specific permease enzyme II from Escherichia coli (PDB ID: 1VKR)56 can be studied from PDB. The crystal structure of the fructose specific IIB subunit of PTS system was available for Bacillus subtilis (PDB ID: 2R48).57 Similarly, the closest structures available for sugar-specific IIA component and beta-glucoside-specific IIA component of the PTS system were studied from PDB. The details of all these structures including the classification, expression system, mutations, gene names and ligand interactions have been compiled in Supplementary Table 5.

Out of the four novel drug targets predicted, the structures of two enzymes: UDP-N-acetylglucosamine 1-carboxyvinyltransferase and phosphate acetyltransferase were available for L. monocytogenes.58,59 The crystal structure of UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) from L. monocytogenes EGD-e (PDB ID: 3R38) was determined through X-ray diffraction experiments in the Escherichia coli BL21 expression system. Two ligands have been determined for this protein (Sulfate ion and Chloride ion) which bind to the chain A of the protein.58 Similarly, the crystal structure of phosphate acetyl/butaryl transferase (from L. monocytogenes EGD-e) in complex with CoA (PDB ID: 3U9E) has been determined in Escherichia coli BL21(DE3) by X-ray diffraction studies. Four ligand molecules (Coenzyme A, Arginine, Glycerol, Chloride) have been known to interact with A and B chains of the protein59 (Supplementary Table 5).

The structure for the third novel protein acetate kinase was found for the organism Salmonella enterica subsp. enterica serovar Typhimurium (PDB ID: 3SK3) in the RCSB Protein Data Bank. Citric acid and 1,2-Ethanediol ligands interact with the A and B chain of the enzyme.60 Similarly, the structure for aspartate kinase was found for the organism Pseudomonas aeruginosa PAO1 (PDB ID: 5YEI) which was determined by X-ray diffraction in expression system Escherichia coli BL21(DE3). Three ligand molecules: Threonine, Lysine, and Glycerol are known to interact with the protein61 (Supplementary Table 5). Unpinning the structure categorization and identifying the inhibitors for these target proteins opened different methods of research towards drug design and reverse vaccinology approach.

CONCLUSION

The comparative metabolic pathway analysis approach of L. monocytogenesH. sapiens resulted in four novel putative target proteins: UDP-N-acetylglucosamine 1-carboxyvinyltransferase, acetate kinase, phosphate acetyltransferase, and aspartate kinase, which were very high in essentiality index with the pathogen and non-homologous nature with the host. Other 11 enzymes on the list are also significant putative drug targets and some of them have been reported by prior studies as well. The predicted potential drug target enzymes from L. monocytogenes will not interact with host machinery and also perform essential functions such as peptidoglycan biosynthesis, cell motility, chemotaxis, resistant biofilm formation, virulence, bacterial pathogenicity, amino acid biosynthesis, cell division, and cell wall organization. Therefore, drug development against these targets to combat L. monocytogenes infections will be very promising. Unravelling novel target proteins and their associated pathways by comparing metabolic pathway analysis between Listeria monocytogenes EGD-e and host Homo sapiens, develops the novelty of the work towards specific and broad spectrum putative drug targets. In addition to this, a detailed further analysis of these potent target proteins in terms of in-vivo and in-vitro approach will attain new and unique generation of biomolecules against the diseases caused by L. monocytogenes.

Supplementary Information

Additional file: Additional Table S1- S5.

Declarations

ACKNOWLEDGMENTS
The authors would like to thank Faculty of Biotechnology, Shri Ramswaroop Memorial University, Lucknow-Deva Road, Barabanki, India, for their support.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
NC and SS designed the experiments. NC performed the experiments. NC analyzed the data and wrote the manuscript. NC and TQ revised the manuscript. All authors read and approved the final manuscript for publication.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript and/or in the supplementary files.

ETHICS STATEMENT
Not applicable.

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