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)

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

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)

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.