ITRAQ-based Proteomics Analysis Of Antibacterial Mechanisms Of Action Of Juglone Against Staphylococcus Aureus

Staphylococccus aureus was a gram-positive microbial causes various symptoms, including food poisoning, skin and blood infection, owing to production of staphylococcal enterotoxins, mainly type A and B. It was often detected in the food safety incidents and become an important public health hazard. In recent years, due to the over- and frequent-use of atibiotics, resulting in the birth of’Super bacterial’directly, and promote the drug reisitant problem to a unprecedented hight. There wer two main reasons to explain the occurrence of drug resistance. Firstly, long-term drug pressure lead to the genome mutation, and making the change of original targets’ structure and location accordingly. Secondly, strain evoluted its stress resistance system, and discharge or degradate the antibiotic through efflux pump and degrading enzymes respectively before its binding.Therefore, development of new natural products with antimicrobial activity and tap new antibacterial mechanism are especially important. Juglone (5-hydroxy-1,4-naphthoquinone), a natural product found in the roots, leaves, nut-hulls, bark, and wood of Juglans that has been used for centuries in folk medicines to cure acne, allergies, gastrointestinal disorders, intestinal parasitosis, cancer, fungal infections, bacterial infections, and viral infections. In daily life, it was also commonly used as hair dyes and skin coloring. In recent years, research on juglone mainly focused on antitumor activity and related mechanisms but lacking research on its antibacterial activity and mechanisms discovering.Due to the limitations, our research focused on detecting the antibacterial mechanisms of juglone against S. aureus using iTRAQ proteomics technology. On the other hand, we give a new drug target in S. aures based on bioinformatics analysis and structural biology, and brought it to virtual drug screening to addressed the staphylococc drug resistan problem.After treatment with juglone at 1/2 MIC (18.75μg/mL), our proteomic analysis results (6 groups, three biological replicates) showd that there were 9834 unique peptides identified (FDR≤0.1), corresponding to 1379 protein groups including 1376 proteins that were quantified in each channel. Differentially expressed proteins were classified using the following scale:more than 1.2-fold (P< 0.05) or less than 0.83-fold (P< 0.05). Compared to the control group,53 (22 up-regulated,31 down-regulated) of the 1376 proteins were found to be differentially expressed, and performed annotation analysis using Blast2Go. Combined with our previous research results, we found juglone mainly inhibit S. aureus cell membrane synthesis by oxidative damage, destruction of membrane ion channels, thereby inhibiting transmembrane transport and increased membrane permeability accrodingly. Meanwhile, juglone can also hinder cell wall synthesis, cell division, destruction ribosome structure and inhibition of protein synthesis accrodingly. In addition, juglone can also damage the cells’ stress response system, so that cells can not make a response and repair when facing to oxidative and DNA damage. Most importantly, we found that juglone can inhibit bacterial DNA replication and transcription, it may be bonded to the double-stranded DNA directly and break it. Accoroding to our protein interaction network analysis, we found that juglone show its bacteriostatic effect through multi-channel rather than binding to one target and cause other proteins’expression change accrodingly.Also, we summarized and classified the drug targets’infrmation from 54 kinds anti-staphylococc antibiotics, and found that there were three main antibacterial mechanisms of actions, including inhibition of cell wall formation, DNA replication and transcription, and protein synthesis. Subsequently, we annotated the pathyway of S. aureus NCTC8325 global and known drug target protein using KEGG. After removing the target enrichment pathway, the remaining proteins were analyzed using protein interaction network per pathway. The results suggested that FhuC and AtpB play a major role in ABC transporters system and oxidative phosphorylation process respectively, which wer never reported potential drug targets. Subsequent homology modeling and virtual drug screening indicated that Purealin and its derivatives are potential FhuC inhibitor, with assay significance.