Insights into the Effects and Mechanism of Andrographolide-Mediated Recovery of Susceptibility of Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

The frequent resistance associated with ß-lactam antibiotics and the high frequency of mutations in ß-lactamases constitute a major clinical challenge that can no longer be ignored. Andrographolide (AP), a natural active compound, has been shown to restore susceptibility to ß-lactam antibiotics. Fluorescence quenching and molecular simulation showed that AP quenched the intrinsic fluorescence of ß-lactamase BlaZ and stably bound to the residues in the catalytic cavity of BlaZ. Of note, AP was found to reduce the stability of the cell wall (CW) in methicillin-resistant Staphylococcus aureus (MRSA), and in combination with penicillin G (PEN), it significantly induced CW roughness and dispersion and even caused its disintegration, while the same concentration of PEN did not. In addition, transcriptome sequencing revealed that AP induced a significant stress response and increased peptidoglycan (PG) synthesis but disrupted its cross-linking, and it repressed the expression of critical genes such as mecA, blaZ, and sarA. We also validated these findings by quantitative reverse transcription-PCR (qRT-PCR). Association analysis using the GEO database showed that the alterations caused by AP were similar to those caused by mutations in the sarA gene. In summary, AP was able to restore the susceptibility of MRSA to ß-lactam antibiotics, mainly by inhibiting the ß-lactamase BlaZ, by downregulating the expression of critical resistance genes such as mecA and blaZ, and by disrupting CW homeostasis. In addition, restoration of susceptibility to antibiotics could be achieved by inhibiting the global regulator SarA, providing an effective solution to alleviate the problem of bacterial resistance. IMPORTANCE Increasingly, alternatives to antibiotics are being used to mitigate the rapid onset and development of bacterial resistance, and the combination of natural compounds with traditional antibiotics has become an effective therapeutic strategy. Therefore, we attempted to discover more mechanisms to restore susceptibility and effective dosing strategies. Andrographolide (AP), as a natural active ingredient, can mediate recovery of susceptibility of MRSA to ß-lactam antibiotics. AP bound stably to the ß-lactamase BlaZ and impaired its hydrolytic activity. Notably, AP was able to downregulate the expression of critical resistance genes such as mecA, blaZ, and sarA. Meanwhile, it disrupted the CW cross-linking and homeostasis, while the same concentration of penicillin could not. The multiple inhibitory effect of AP resensitizes intrinsically resistant bacteria to ß-lactam antibiotics, effectively prolonging the use cycle of these antibiotics and providing an effective solution to reduce the dosage of antibiotics and providing a theoretical reference for the prevention and control of MRSA.

Potential Mechanisms of Quercetin Influence the ClfB Protein During Biofilm Formation of Staphylococcus aureus.

This study aimed to establish the mode of binding between Quercetin (QEN) and an essential protein called ClfB in forming biofilm in Staphylococcus aureus (S. aureus). In this study, the raw data of GSE163153 were analyzed for quality control, alignment, and gene counts, and the differential analysis detected the key differentially expressed genes (DEGs) assisting in the formation of the S. aureus biofilm. Then, the protein-protein interaction (PPI) and gene function enrichment analyses of the target genes, identified a gene called clfB to be closely related to biofilm formation. ClfB was structurally characterized, molecularly docked, and kinetically simulated to unravel the mode of binding of QEN to ClfB. Meanwhile, the growth curve and transmission electron microscopy methods examined the effect of QEN on the S. aureus growth. Results indicated that the clfB gene was increasingly expressed during biofilm formation and was involved in cell adhesion, pathogenicity, and infection. We identified 5 amino acid sites of ClfB (D272, R331, I379, K391, E490) as potential sites for binding QEN, which would indirectly influence the changes in the functional sites N234, D270, Y273, F328, inhibiting the formation of biofilm. Meanwhile, 128 µg/ml of QEN could significantly inhibit the S. aureus biofilm formation. This manuscript serves as a molecular foundation for QEN as an antibacterial drug providing a new perspective for developing antibacterial drugs.

DNA methylation data-based molecular subtype classification and prediction in patients with gastric cancer.

BACKGROUND: Genetic and epigenetic alterations have been indicated to be closely correlated with the carcinogenesis, DNA methylation is one of most frequently occurring molecular behavior that take place early during this complicated process in gastric cancer (GC). METHODS: In this study, 398 samples were collected from the cancer genome atlas (TCGA) database and were analyzed, so as to mine the specific DNA methylation sites that affected the prognosis for GC patients. Moreover, the 23,588 selected CpGs that were markedly correlated with patient prognosis were used for consistent clustering of the samples into 6 subgroups, and samples in each subgroup varied in terms of M, Stage, Grade, and Age. In addition, the levels of methylation sites in each subgroup were calculated, and 347 methylation sites (corresponding to 271 genes) were screened as the intrasubgroup specific methylation sites. Meanwhile, genes in the corresponding promoter regions that the above specific methylation sites were located were performed signaling pathway enrichment analysis. RESULTS: The specific genes were enriched to the biological pathways that were reported to be closely correlated with GC; moreover, the subsequent transcription factor enrichment analysis discovered that, these genes were mainly enriched into the cell response to transcription factor B, regulation of MAPK signaling pathways, and regulation of cell proliferation and metastasis. Eventually, the prognosis prediction model for GC patients was constructed using the Random Forest Classifier model, and the training set and test set data were carried out independent verification and test. CONCLUSIONS: Such specific classification based on specific DNA methylation sites can well reflect the heterogeneity of GC tissues, which contributes to developing the individualized treatment and accurately predicting patient prognosis.