Modulation Of CcrAB Expression In Methicillin-resistance Staphylococcus Aureus

Staphylococcus aureus is an important bacterial pathogen in human and animal, and can cause a plenty types of infectious diseases, and antibiotics are the major way in clinical treatment of the infection. However, with the emergence of drug-resistant strains, especially after the worldwide spread of methicillin-resistant Staphylococcus aureus (MRSA), antibiotic treatment has met unprecedented challenges. Due to the acquisition of the gene mecA, which codes for an alternative penicillin-binding protein PBP2a, MRSA strains are resistant to practically the entire class of P-lactam antibiotics. The mecA gene is located on a mobile genetic element SCCmec, and the excision and integration of SCCmec are mediated by the Ccr recombinase encoded by ccrAB or ccrC, which are also located on the mobile element. The expression level of ccrAB or ccrC is greatly related to the stability and horizontal transfer of SCCmec. Previous studies have shown that the ccrAB genes are only expressed in a minority of cells and that the proportion of active cells or their total ccrA expression levels can be affected by certain environmental stimuli such as temperature and antibiotics, suggesting the involvement of a subtle regulatory procedure in ccrAB expression, however, the research on this regulatory mechanism is still blank.This dissertation mainly described the mechanism involved in ccrAB expression through the following two parts of study.1. The regulation of ccrAB transcription and SCCmec excision by alternative sigma factorsAlternative sigma factors play important roles in sensing various environmental stimuli and regulation of bacterial cellular processes. Previous study has shown that SigH is involved in the regulation of the bacteriophage genome excision and integration, implying that alternative sigma factors may also play a role in the excision and integration of SCCmec. In this study, we first confirmed that the genes ccrA and ccrB are in the same operon and their transcriptional levels displayed obviously growth-phase dependence. We overexpressed the three alternative sigma factors SigB, SigH, and SigS in MRSA strain N315 respectively, and the results showed that overexpression of SigB significantly increased the transcriptional level of ccrA. Further study also showed that overexpression of SigB can significantly enhance the SCCmec excision. Through primer extension and 5’RACE (Rapid Amplification of cDNA Ends), we identified a heretofore unrecognized SigB-recognition sequence, which exists in most of the SCCmec type II and IV strains.2. Modulation of ccrAB transcription and SCCmec excision by the inverted repeat sequence and regulatory protein SarSOn analysis of the ccrAB genes promoter sequence in strain N315, we identified an inverted repeat sequence. Further analysis of this sequence in different strains indicated that it exist extensively among different types of SCCmec that carry ccrAB genes, and these sequences are conserved both in base composition and location from the ccrA translation initiation site. Substitution of the related pairing bases can significantly increase the promoter activity of ccrAB, and either substitution of the upstream region or the downstream region of the IR sequence exhibited almost the same effect on ccrAB expression, indicating that the IR sequence affects ccrAB expression through a special secondary structure. Besides, substitution of the IR sequence within the chromosome or in the ccrAB overexpression plasmid can both significantly affect the stability of SCCmec. These results reveal that the IR sequence plays an unignorable role in ccrAB expression and SCCmec stability. Through DNA pull-down assay, we identified the regulatory protein SarS, which can specially bind to the ccrAB promoter. Further EMSA result indicated that SarS can specially bind to the inverted repeat sequence. The qRT-PCR and promoter activity report assay indicated that SarS is involved in the regulation of ccrAB expression at the stationary phase, and overexpression of SarS can significantly increase the transcription of ccrAB and enhance the excision of SCCmec.