In this study, we looked at the evolutionary relationships between a number of S. aureus genes. This is accomplished through the use of phylogenetic analysis with Mafft and IQTree2. The outcomes are shown using iTOL. The research demonstrates that the rpl genes, including rplJ, rplC, and rplL, are tightly connected to other genes, including rpoC, parE, and gyrB. ParE and GyrB are the genes with the closest connection. As seen in the phylogenetic tree, genes like tilS and rpsL, parC and gyrA, rpsG and gltX, walR and hptR, hlf and fusA are related to one another. This study offers critical new insights into the genetic relationships and evolutionary connections of these gene variations.
To further investigate the complex functional links between genes and their variations, we employed a rigorous approach that involved acquiring the protein sequences of all genes and their variants. Then, using bioinformatics tools such as the STRING and Cytoscape cytohubba, we carried out a variety of network analysis tasks. Using these techniques, we were able to investigate several networks of protein-protein interactions and create graphic depictions of these networks. We produced network stats, including Degree and Closeness, and used MCODE to identify clusters with related functions that may be involved in specific biological processes. By considering the node density and degree of connectivity inside a cluster, MCODE employs a scoring system to identify the clusters with the highest scores. Tables 2 and 3 provide summaries of the shortest path outcomes retrieved from cytohubba. Also, we performed a clustering analysis; the results are shown in Table 1 and Fig. 3, which depict one cluster and clustering scores. This research throws information on the functional similarities between closely related genes and sheds light on the evolutionary links between various strains of S. aureus.
We used ClueGo to do functional enrichment analysis on the single cluster that resulted from the clustering study. According to our study, the enriched biological process (BP) entries contained lipid metabolic process (GO:0006629) organelle organization (GO:0006996), chromosome organization (GO:0051276), DNA conformation change (GO:0071103), DNA topological change (GO:0006265). The cationic antimicrobial peptides (AMPs) are hypothesized to be resistant to phosphatidylglycerol (PG), the membrane lipid of S. aureus, through the enzymatic transfer of an l-lysine residue leading to lysyl-PG, which changes the net charge of PG from 1 to + 1(Andrä et al., 2011). A membrane protein called Multiple Peptide Resistance Factor (MprF) makes it easier to produce and transport lysyl-PG to the outer leaflet of bacterial membranes(Ernst et al., 2009). Fluoroquinolone resistance is expected to be caused by genes like gyrA, gyrB, parC, and parE in S. aureus (DNA Confirmation change, DNA Topological change, Organisation change). There haven't been many described mutations in parE/grlB or gyrB of any bacteria. The main factor contributing to the lower accumulation of these drugs is fluoroquinolone efflux(Redgrave et al., 2014). PmrA and NorA are the efflux pumps implicated in norfloxacin resistance in Streptococcus pneumoniae and S. aureus, respectively(Piddock, 1999).
Molecular functions including DNA topoisomerase activity (GO:0003916), DNA topoisomerase II activity (GO:0061505), DNA topoisomerase type II (ATP-hydrolyzing) activity (GO:0003918), and ATPase activity, linked (GO:0042623) are examples of the enrichment of biological components. The associated genes in all Molecular functions are gyrA, gyrB, parC, and parE. Resistance to the antibiotics here that target nucleic acid production is caused by a mutation in these genes. The molecular targets for fluoroquinolones in S. aureus are the DNA gyrase enzyme, which adds negative supercoils to chromosomal DNA, and topoisomerase IV, which promotes chromosome decatenation following replication. Both enzymes are heterotetramers made up of A and B subunits in DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) (Foster, 2017). Double-stranded breaks are brought on by the enzymes in a staggered manner. Each cleaved DNA strand's 5' end forms a phosphotyrosine connection with the active site tyrosine in the two A subunits. The phosphodiester connection between the backbone deoxyriboses is rebuilt, the gap is filled, and the break is crossed by a DNA strand. The fluoroquinolones form ternary complexes with Mg2 + and the A subunit of the topoisomerases at the intermediate covalent protein-DNA complex stage. As a result, the cleaved DNA strand cannot reseal, leading to irreparable double-stranded DNA breaks and a rapid bactericidal effect.
The enrichment of cellular components such as intracellular organelle (GO:0043229), intracellular non-membrane-bounded organelle (GO:0043232), and chromosomes (GO:0005694) was a significant finding from the study. The related genes with CC are rplC, parC, parE, gyrA, and gyrB. Many gram-positive bacteria have rplC gene mutations. As Linezolid is a synthetic medication, inherent drug resistance does not exist; instead, mutations are typically acquired. The peptidyltransferase center (PTC), where conserved sections of the ribosome directly interact with Linezolid, is altered as a result of mutations in the 23srRNA subunit domain V area of ribosomes. As gram-positive bacteria transmit 4 to 6 allelic copies of the 23S rRNA, it takes more than one allele to change for linezolid resistance to develop(Kumar Parthasarathy A and A. Chougale R, 2021). GyrA, GyrB, ParC, and ParE genes are engaged in cellular components (CCs) in addition to their roles in biological processes (BPs) and molecular functions (MFs).
The RNA polymerase beta subunit, which is responsible for catalyzing the synthesis of RNA during transcription, is encoded by the rpoB and rpoC genes in S. aureus. Bacteria have a highly conserved gene called rpoB, which is essential for the transcription process. Rifampin, a broad-spectrum antibiotic frequently used to treat S. aureus infections, can be resistant to mutations in the rpoB gene(Panchal et al., 2020). The antibiotic rifampin targets the beta subunit of RNA polymerase, and mutations in the rpoB gene can change the antibiotic's binding site and render it ineffective.
Elongation factor G (EF-G) is a protein that is encoded by S. aureus' fusA gene. The ribosome moves along the mRNA during the translocation phase of translation, which makes it easier for amino acids to be incorporated into the expanding polypeptide chain(Edslev et al., 2018). EF-G is involved in protein synthesis and is essential for this process. Resistance to fusidic acid, a medication frequently prescribed to treat S. aureus infections, has been linked to mutations in the fusA gene. By stopping the EF-G protein's release from the ribosome and blocking protein synthesis, fusidic acid specifically targets the EF-G protein. Fusidic acid's binding site to EF-G can change as a result of mutations in the fusA gene, which can decrease the antibiotic's effectiveness and promote resistance.
The ribosomal protein S12 is encoded by the rpsL gene in S. aureus. The ribosome, the cellular apparatus in charge of protein synthesis, is made up entirely of ribosomal proteins. The S12 protein aids in the precise identification of codons on the mRNA during the decoding phase of translation. Mutations in the rpsL gene have been linked to resistance to streptomycin, a medication frequently used to treat S. aureus infections, in the setting of antibiotic resistance. Streptomycin particularly binds to the S12 protein on the ribosome(Chen et al., 2015) to target it. Streptomycin's binding site can change as a result of mutations in the rpsL gene, lowering its efficiency and increasing resistance.