The Study On The Function Of TRAP And VraX In Staphylococcus Aureus

1. Idetification of the role of TRAP protein involved in the survival of Staphylococcus aureus in long chain unsaturated free fatty acids2. The hypothetical protein VraX is related with natural drug tolerance in Staphylococcus aureusStaphylococcus aureus(S.aureus) is a kind of common Gram-positive pathogens, which can cause many kinds of diseases. The clearance of S.aureus in host is mediated by complement system, phagocytes, unsaturated fatty acids and so on. It has been identified that S. aureus can express a variety of molecules against these clearances.With the the abuse of antibiotics, more and more drug resistant S. aureus comes out. The infection caused by drug resistant S. aureus is a great clinical threaten.This study includes two parts.①Idetification of the role of TRAP protein involved in the survival of S. aureus in long chain unsaturated free fatty acids.②The hypothetical protein VraX is related with natural drug tolerance in S. aureus.Chapter1-Idetification of the role of TRAP protein involved in the survival of S. aureus in long chain unsaturated free fatty acids. Long chain unsaturated free fatty acids (LC-uFFAs) usually contain more than16carbon atoms and one or more double bonds. Some reports show that LC-uFFAs is a component of innate immune system against to S.aureus infection. TRAP is reported to be involved in the pathogenicity of S.aureus. However, the biological function of TRAP is controversial, and its exact function is not yet clear. This Chapter includes the following three parts:1. TRAP mutant (△traP) was sensitive to killing mediated by linoleic acid. Here, it was found that△traP constructed previously became more sensitive to killing mediated by linoleic acid, compared with its parent strain. The results of microarray showed that the mRNA level of the gene of fatty acids metabolism pathway (fadD) was significantly decreased in△traP compared with its parent strain. 2. FadD mutant (△fadD) was sensitive to killing mediated by linoleic acid. We constructed the fadD gene mutant (AfadD). It was found that AfadD was sensitive to killing mediated by linoleic acid, compared with its parent strain. In the further investigation, the level of fadD in△traP was recovered. However, it was found that rescue of FadD in△could not recover the sensitivity to killing mediated by linoleic acid3. The interaction between TRAP and FadD was essential for the killing of S. aureus mediated by linoleic acid. In the following study, the potential binding proteins of TRAP were detemmined as FadB and FadD. The two proteins were expressed and purified. The results of ELISA showed that FadD, but not FadB, can specifically interact with TRAP. Our data suggested the interaction between TRAP and FadD was essential for the killing of S. aureus mediated by linoleic acid.Chapter2-The hypothetical protein VraX was related with natural drug tolerance in S. aureus. A small numbers of drug sensitive bacteria can survive under treantment with antibiotics. It has been reported that the level of some molecules in drug sensitive S. aureus is significantly altered with the treatment with antibiotics. A hypothetical protein VraX level has been identified to be dramatically increased (>20times) incubated with several kinds of antibiotics. Here, we investigated whether VraX was involved in the natural drug tolerance in S. aureus. This Chapter includes the following two parts:1. VraX protein was related with natural drug resistance in S.aureus. The vraX gene deletion mutant (△vraX) was construted by gene homologous recombination method. It was found that AvraX was more sensitive to several antibiotics than its parent strain.2. The level of vraX was regulated by RNaseⅢ. RNA-pulldown assay showed that RNaseⅢ could bind vraX. The mircroarray data showed that the level of vraX was significantly increased in△RNaseⅢ, which was confirmed by RT-PCR and qRT-PCR. The gel-shift results confirmed that RNaseⅢ could specifically bind vra XmRNA. Meanwhile, it was found that RNaseⅢ could degrade vraX mRNA in the presence of Mg2+. All these data suggested that RNaseⅢ could regulate the level of vraX via interaction with vraX.Above all, it was revealed that TRAP could interact with FadD and be involved in the killing of S. aureus mediated by linoleic acid. We also found VraX protein was related with the natural drug tolerance of S. aureus and its level could be regulated by RNaseⅢ. Our study will provide some potential drug targets against the infection caused by S. aureus.