Re-construction of Co-expression Network of Genes Involved in Bacterial Cell Wall Synthesis and Their Role in Penicillin Resistance

Background: Peptidoglycan (Murein), which consists of disaccharide and amino acid chain subunits, has a key role in bacterial survival and ranks first in the line defense system against drug therapy. In addition, the transpeptidase enzyme plays an important role in cross-linking in bacterial cell walls. In Escherichia coli bacteria, cross-linking happens by proteins that have a D-D transpeptidase role and bond two amino acids of D-alanine together. These proteins are characterized by their affinity for and binding of penicillin thus they are called penicillin-binding proteins (PBPs). It should be noted that this bonding formation is prevented by the beta-lactam family as they have a similar structure to the above-mentioned proteins. The product of the idtD gene by characteristics such as L-D transpeptidase can catalyze the peptidoglycan structure in the bacterial cell wall in the presence of beta-lactam antibiotics. Methods: In this study, around 426 interactions were identified between genes and approximately 20 genes with a key role in the process of bacterial cell wall synthesis by the reconstruction of 44 genes involved in bacterial cell wall synthesis. Results: The idtD gene locus at the reconstructed network clearly shows that its catalytic activity is the side activity, and there won’t be a lag or disturbance in the procedure cell wall synthesis by removing it from the cycle. However, this side process causes the strengthening of the bacterial cell wall synthesis process against disorders arising by the presence of beta-lactam antibiotics. Conclusions: These five genes in E. coli that furnish L-D transpeptidase properties include IdtA, IdtC, IdtD, IdtE, and mrdA out of which, IdtD is the most important gene in this process.

the common belief, these proteins are not from the PBPs (4). Previous research on the E. coli chromosome showed that it has five genes that are responsible for the L,D transpeptidase. Among these genes, namely, YcbB and YnhG can catalyze DAP 3 →DAP 3 (5) and the other three genes (i.e., YcfS, ErfK, and YbiS) shall connect lipoproteins to peptidoglycans (6). Hugonnet

et al demonstrated that
YcbB encoded for a protein has the ability to function as a substitute for the D, D transpeptidase of the PBP family of proteins and thus causes the resistance to the beta-lactam family of antibiotics (7). Resistance to penicillin and other antibiotics from the beta-lactam family can be found in a wide range of gram-negative and gram-positive bacteria. Accordingly, the identification and study of genes involved in the resistance process can be a great help to overcome this problem and break the defense mechanism of bacteria against the beta-lactam family of antibiotics. To nail this aim using data deposited to databases and bioinformatics tools, the present study evaluated the co-expression network of genes involved in the synthesis of the bacterial cell wall and its role in antibacterial resistance.

Materials and Methods
In this study, published papers and findings and deposited data on UniProt were collected with regard to genes that were involved in the cell wall synthesis of bacteria (8), the details of which are presented in Table 1. Then, these genes were entered in the STRING database (9) and the network was expended three times. Finally, the network was obtained by 44 nodes with 426 edges. Additionally, other genes associated with this process were identified using parameters like co-expression and experiments. The obtained data from STRING analysis for the re-creation of the co-expression network purpose were uploaded into Cytoscape software (10). In addition, network topology was studied based on betweenness and closeness centrality parameters by Network Analyser software (11). An entity centrality of a complex network is named by betweenness centrality. Likewise, it is calculated based on all shortest edge pairs in a network. On the other hand, the closeness centrality is defined by the shortest distance mean from one node to all other nodes. In a simpler word, the higher  Role of Bacterial Wall Synthesis Genes in Penicillin Resistance amount of betweenness and closeness centrality for a node represents the importance of that node for the network (12,13). The ontology analysis of genes in the above network was performed by web-based software DAVID (14). Then, Benjamin P value was calculated for every single gene (15).

Results and Discussion
The mechanism of antimicrobial-resistance to beta-lactam antibiotics is evaluated in 3 different ways. First, the mechanism is related to gram-negative bacteria that block the entry of antibiotics into the cell by changes in the structure of purines (pores in the bacterial cell wall). The second mechanism linked to the antimicrobial-resistant is common between gram-positive and -negative bacteria. These antibiotic-resistant bacteria have the capacity to breakdown the C-N bond in the beta-lactam ring. Finally, the third mechanism in antimicrobial-resistant bacteria to the beta-lactam family, the bacteria change the transpeptidase enzyme structure in such a way that no antibiotic can enter the bacteria. The second and third mechanisms were already reported in different strains of E. coli (16).
In general, 44 genes with a role in peptidoglycan synthesis in E. coli were selected based on our data mining and evaluation and analysis on the UniProt database (Table 2). Further, the interactions between the genes were identified using the STRING database. Furthermore, the centrality of the nodes in the protein-protein interaction network was calculated by applying the Network Analyzer tool, and finally, the co-expression network of the above genes was re-created by Cytoscape software. As shown in Figure 2, a complex network of genes was involved in the synthesis of the peptidoglycan process. The topology analysis elucidated that the murF gene by 23 connections had the highest connections with other genes in the network and then murA by 22 connections and murD, murE, murI, and ftsW genes each with 20 connections. The murA gene, along with mrcA, mrdB, mrdA, dacB, and ftsW had 0.020, 0.018, 0.0137, 0.0133, 0.0099, and 0.0092 betweenness centrality, respectively. The closeness centrality of each gene was an amount between 0 and 1. Further, 13 genes had the highest amount of closeness centrality equal to 1, including IdtD, murA, murC, murD, murE, murF, and murG. The product of the murA gene is an enzyme named 1-carboxy vinyl transferase which catalyzes the cornerstone of bacterial cell wall biosynthesis (17). As shown in Figure Table 2. Some Hub nodes in Figure 2 Networks and the Metrical Information of Networks 3, this enzyme catalyzes the biosynthesis in the presence of phosphoenolpyruvate and the uridine diphosphate N-acetylglucosamine molecule yield (1-carboxy vinyl)α-D-glucosamine. A free phosphate is also a byproduct of this reaction (18). The suppression of the murA gene by fosfomycin antibiotics prevents the cell wall synthesis and subsequently bacterial death (18). More precisely, the re-created network and the topology analysis results elucidate that the murA gene has a key role in the process. The other genes from the Mur gene family (e.g., murB, murC, and the like) all play a role in cell wall synthesis. Eventually, the gene murG catalyzes the last reaction of the process, and subsequently, causes the formation of disaccharide-pent peptide production which is a subunit of the peptidoglycan cell wall (19). Moreover, the products of mrdA and mrdB catalyze the crossed binding of peptidoglycan cell walls and have a role in the width synthesis of the peptidoglycan cell wall, and finally, preserve and protect the bacterial structure and firmness (20). Most of the genes in this network are sensitive to the beta-lactam family of antibiotics (21). As mentioned earlier, each gene has connections with a higher number of other genes in the network. For example, the IdtD gene via dacC and dacB genes is connected to the main network. Although the dacB gene does not have a direct role in transpeptidation, it exclusively catalyzes DDcarboxy-peptidase and DD-endopeptidase reactions (22). On the other hand, dacC discards the remaining of the C-terminal of D-alanine from the precursors of peptidesugar cell walls.
In E. coli bacteria, the product of IdtD AKA YcbB has the ability to catalyze the reciprocal bindings of the amino acids in the peptidoglycan structure of the bacterial cell wall in the presence of the beta-lactam family of antibiotics since the catalyzed peptidyl binding is not D-D but is of L-D type (5). Despite the activity of IdtD in the presence of the beta-lactam family of antibiotics, it remains sensitive to the carbapenem, meropenem, and imipenem family of antibiotics. Additionally, it has been determined that copper can repress the activity of IdtG (7). The IdtD gene locus in the re-created network clearly demonstrates that the catalyzed reaction is a collateral process and there will be no disruption in the process of bacterial cell wall synthesis by removing this reaction. Conversely, this collateral process has a key role in bacterial resistance during the synthesis of the cell wall against the beta-lactam family of antibiotics. For a more detailed analysis of the role of the IdtD gene in E. coli, its exclusive co-expression network has been re-created, which is shown in Figure 4.
Based on the data in Figure 4, co-expression network topology analysis shows that the IdtD gene has the most connections (n=10) in the network and other genes like ygaU, osmC, dps, and ydiZ by 9, 8, 7, and 7 connections are placed 2 to 5 in the ranking, respectively. Additionally, ycaC, osmC, and IdtD by the value of 1 have the highest closeness centrality and, from the betweenness centrality viewpoint of genes like IdtD (0.0578), IdtE (0.0236), and Note. Node size depends on betweenness centrality (lower size for a lesser amount) and the color of the node varies from blue (the least closeness centrality) to red (highest closeness centrality). The color and thickness of each line (the connection between to genes) also vary from red and thick for a higher amount and blue and thin lines for fewer amounts. Based on the results in Figure 4, the IdtD gene had an important role in the L,D transpeptidase binding process. As previously mentioned, this gene catalyzes the DAP 3 → DAP 3 . Another gene named IdtE AKA YnhG has the same function as the IdtG gene. According to a previous report, the upregulation of this gene in E. coli bacteria subsequently results in an increase in the DAP 3 → DAP 3 binding in the bacterial cell wall structure (5). In defense against oxidative stress from exposure to the organic hydro peroxidase, osmC gene plays an osmotic induction role and preferably metabolizes organic hydro peroxidases more than inorganic hydrogen peroxidase (23). The co-expression of this gene by the upregulation of the D-L transpeptidase process shall clarify that D-L transpeptidase is activated in unfavorable environmental conditions and substitutes with the D,D transpeptidase function. Nonetheless, it also has an activity of 3 to 10% in favorable environmental conditions.
The box plot ( Figure 5) displays the ontology analysis of genes in the co-expression network of bacterial cell wall synthesis. Out of 44 identified genes by the DAVID database based on molecular function, penicillin-binding with a P value of 7.2E-14 and 9 connections had the highest molecular function among all genes involved in the bacterial cell wall synthesis process. This finding proved that bacterial cell wall synthesis is extremely under the influence of the beta-lactam family of antibiotics. Binding capability to the magnesium (Mg) ion by 6 genes and the P value of 8.2E-2 is one of the active molecular functions among bacterial cell wall synthesis. These genes include D-alanine ligase A (ddlA), D-alanine ligase B (ddlB), L-alanine ligase (murC), meso-diaminopimelate ligase (murE), glucosamine 1-phosphate acetyltransferase (glmU), and finally, undecaprenyl-pyrophosphate syntheses (ispU). By considering the obtained data, the Mg enzyme was the co-factor of these enzymes and had a key role in bacterial cell wall synthesis. In addition, five genes of IdtA, IdtC, IdtD, IdtE, and mrdA had the D-L transpeptidase function and were observed in the co-expression network (Figure 4). The IdtD gene is the most key gene and responsible for D-L transpeptidase functionality in E. coli bacteria. From the locus point of view, these genes were evaluated and the results showed that all the above-mentioned genes are located in a Node size depends on betweenness centrality (lower size for a lesser amount) and the color of the node differs from blue (the least closeness centrality) to red (highest closeness centrality). The color and thickness of each line (the connection between to genes) also vary from red and thick for a higher amount and blue and thin lines for fewer amounts.  membrane or the cell wall itself. From the biopathway perspective, the cell shape regulatory and peptidoglycan biosynthesis pathways with 39 (P value of 4.6E-73) and 38 (P value of 1.2E-62) genes are the two most active biopathways, respectively. It should be noted that each gene can present in different pathways with numerous different functions.

Conclusions
In this study, about 426 interactions were identified between genes and approximately 20 genes with a key role in the process of bacterial cell wall synthesis by the reconstruction of 44 genes involved in bacterial cell wall synthesis. E. coli bacteria has the resistance machinery and mechanism to the penicillin (beta-lactam) family of anti-biotic. This process can proceed via different pathways. However, the cell wall structure changes, and the alteration and prevention of beta-lactam antibiotic efficacy in the bacterial cell wall synthesis process is one of the main mechanisms in bacterial resistance. In E. coli bacteria, the product of the IdtD gene has the ability to catalyze the reciprocal bindings of amino acids in the presence of the beta-lactam family of antibiotics. In addition, the IdtD gene locus in the re-created network clearly suggests that catalyzing activity is a collateral process, and there will be no disruption to the bacterial cell wall synthesis by removing it from the network. Nevertheless, this collateral process causes strengthening the synthesis pathway against disruption by the presence of the beta-lactam family of antibiotics. It seems that the D-L transpeptidase mechanism is activated in unfavorable environmental conditions, replacing the D, D transpeptidase mechanism. However, the amount of activity goes up to 10% in favorable environmental conditions. Further, the magnesium co-factor has a key role in bacterial cell wall synthesis. Overall, 5 genes in E. coli have D-L transpeptidase function, including IdtA, IdtC, IdtD, IdtE, and mrdA. Finally, the IdtD gene has an essential role in the D-L transpeptidase function in E. coli.