| Staphylococcus aureus is the leading cause of hospital- and community-acquired infections worldwide which can cause infections at many anatomical sites, and the most common infections include cellulitis, impetigo and soft tissue abscesses. The pathogenic success of S. aureus is, at least partially, attributed to numerous virulence factors, including hemolysins, proteases, toxins, and immune modulators. This thesis focuses on the sensory regulation system and material transportation and metabolism of S. aureus, and the content is divided into two parts below:1. Study of two-component system （TCS） in S. aureusTCS is a kind of signal cascade transduction system which has been extensively found in archaea, eubacteria, and a small part of fungus and plants. TCS assists organisms to respondquickly to various stimuli from surrounding milieu. Acting as a kind of virulence regulator, TCS plays a detrimental role in maintaining cell survival, invading into host tissue and resisting to antibiotics in S. aureus. It has been reported that S. aureus habours 16 pairs of TCSs on the chromosome, and most of them have been characterized. However, there are still TCSs that have not been well studied in S. aureus. Based on this, we chose the less reported TCS HptRSA and SAOUHSC01313-1314 in S. aureus as our object.Through sequence blast we discovered thatHptRSA has homology to hexose phosphate uptake regularoy system UhpABC in Eschrichia coli. Virtually HptRSA is a three-component signal transduction system. We found that the HptRSA mutant strain could not grow well in the medium where glucose-6-phosphateis the only carbon source. Rreal time RT-PCR result confirmed that HptRSA could facilitate the uptake of extracellular glucose-6-phosphate by modulating expression of the hexose transporter UhpT in S. aureus. We further found that UhpTexhibits high substrate specificity in S. aureus since it could only be activated by glucose-6-phospate but not other sugar phosphates we tested. In addition, we discovered that HptA has the ability to bind to glucose-6-phosphate, suggesting that it may be the major glucose-6-phosphate sensor of HptRSA system. Furthermore, we proved that the response regulator HptR could directly bind to uhpT promoter, and a length of 30-bp nucleotide sequence of uhpT promoter was essential for HptR binding.The functional study of TCS SAOUHSC01313-1314 is still in the primary stage.Mutation of this TCS had no significant change in bacterial growth, split, bio film formation, etc. More effort is needed to uncover the responding signal and target genes of this TCS, and the main strategy is to explore differential expression of genes in SAOUHSC01313-1314 mutant strain under different stresses.2. Research on the ATP-binding cassette （ABC） transporter WmrAB in S. aureusABC transporter is a kind of membrane transporter which usesthe binding and hydrolysis of ATP to power the translocation of vrious substances across membranes. The functional report of ABC transporters in S. aureus usually concentrates on several known multidrug transporters, while the majority is still less investigated.We had an elementary exploration on the function unknown ABC transporter Wmr （cell wall metabolism relating transporter） AB in S. aureus,and found that the WmrAB mutant strain displayed significantly decreased autolysis rate and reduced susceptibility to glycopeptide antibiotics （vancomycin and tericoplanin）. Further real time RT-PCR results indicated that the expression level of some genes and regulators involved in cell wall metabolism （such as aaa, lytRS, ddl, walKR） changed significantly, eventually leading to the disruption of cell wall metabolism balance.Although WmrAB coding genes are located closely to SAOUHSC01313-1314, wmrAB transcription level has no obvious change in SAOUHSC01313-1314 mutant strain. What’s more, the response regulator SAOUHSC01314 could not bind directlyto wmrAB promoter in electrophoresis mobility shift assay. All above could basically eliminate the regulation of SAOUHSC01313-1314 on WmrAB.Through the above studies, we have gained a deeper understanding of the regulatory mechanism of two-component system and multiple physiological functions of ABC transporters in S. aureus. In the meantime, we have complemented the previous study and further completed the acquaintance of global regulatory network and transport metabolism in S. aKreusWehopethat our work may provide some new thoughts on the development of novel antibiotics and treatment of infectious diseases caused by S. aureus. Nevertheless, there are still some remaining questions which need more efforts to solve. For example, the potential stimulus SAOUHSC01313-1314senses, and the functional interaction bewteen WmrAB and other regulators. |
