The Study On The Function Of Noncoding RNA-L8 And TRAP Protein In Staphylococcus Aureus

Staphylococcus aureus (S.aureus) is an important pathogen capable of causing a variety of diseases in humans through virulence gene expression, ranging from localized infections of skin and soft tissue to life-threatening systemic infections, such as skin infections, toxic shock syndrome, pneumonia, endocarditis and meningitis etc.Non-coding RNAs (ncRNAs) including tRNA, rRNA,miRNA,snRNA etc, exist extensively in the eukaryote and prokaryocyte, and carry out lots of physiological functions. The most well studied ncRNAs is miRNA in the past ten years, which can regulate sepecific gene by complementation with target gene. Recently, it is reported that there are some regulational ncRNAs play the import roles in prokaryocyte, which is named as small RNAs (sRNA). The length of sRNAs in bacteria ranges from about 20 to 500 neucletides. sRNAs perform their function with different mechanisms. About 50-60% of sRNAs play their function through base pairing with target mRNAs. They can regulate the target gene expression by influencing the translation process or the stability of mRNAs. A sRNA can target more than one genes. At the same time, more than one sRNA can regulate the same target. The present study have shown that sRNA play an important role in the bacteria growth metabolism and verulence regulation. There are about one hundred sRNAs have been indentified in Escherichia coli (E.coli). It is also reported that there are some functional sRNAs identified in S.aureus. These sRNAs are predicted to regulate the pathogenesis because they are located in the pathogenicity islands or only expressed in the pathogenic strains. RNAIII is a well studied sRNA, which is identified as a key regulator of toxin expression in S. aureus.The Hfq protein is a RNA chaperone, which is firstly discovered in E.coli. It can form a homohexameric ring to bind RNA and change the stability of RNA or help sRNA pairing with the target mRNA. In E.coli, Hfq is vital for sRNAs functions. At present more than 30% noncoding RNA could bind with Hfq in E.coli. These sRNAs regulate the expession of target mRNA with the aid of Hfq.Chapter I: Identification of a novel functional sRNA L8 in S.aureus. A novel sRNA was identified with prediction of bioinformatics and named as L8. The L8 deletion strain was constructed. The further study suggested that L8 could regulate the expression of arlR and play an important role in the pathogenicicy of S.aureus. This chapter is divided into three parts.1, Identification of the novel small RNA. On the basis of the predication of bioinformatics, the transcript of a novel sRNA was identified. And the full lenth of this sRNA were obtained RACE in S.aureus and named L8. The length of L8 was confirmed by Northern blot. Total RNA under stress condition and cDNA was prepared. The results of qRT-PCR showed that the expression of L8 was significantly altered when S.aureus was grown under some different stress conditions.2, Construction of L8 deletion mutant from S.aureus 8325-4. L8 deletion mutant strain was prepared with homologous recombination. It was found that the pathogenicity of mutant strain decreased significantly. Similarly, whole blood survival assay in vitro showed that the survival rate of L8 mutant strain remarkbly decreased compared with its parent strain. A serial of stress-response experiments results displayed that L8 was involved in growth, biofilm formation, autolysis of S.arueus. These data indicated that L8 play a pivotal role in S.aureus.3, Idenfication of the target genes regulated by L8. The gene arlR was predicted as one target of L8 by bioibformatics. The results of qPCR showed that the expression of arlR was decreased in L8 mutant strain. It was found that L8 could specifically bind arlR with the aid of Hfq. In the further investigation, arlR was indentified as one target of L8 by construction of reporter vector.In summary, a novel functional sRNA-L8 in S.aureus was identified, which could regulate the expression of alrR and play an important role in the pathogenicity of S.arueus.TRAP was thought as an important protein to influence the pathogenicity of S.aureu by regulating the expression of agr, but recent papers showed that TRAP could not influence the expression of agr. The biological function of TRAP is controversial. Our previous report suggested that the antibodies of TRAP could inhibit the infenciton cause by S.aureus. We tried to investigate the biological function of TRAP in the pathogenicity of S.aureus in the following study.Chapter II: Study of the biological function of TRAP in S.aureu. In this chapter, we construced a trap deletion mutant from S.arueus 8325-4. It was investigated whether TRAP could regulate the expression of agr. In the further study, it was demonstrated that the trap deletion mutant was more sensitive to the phagocytosis of neutrophil. This part was divided into two parts.1, Construction of the traP deletion mutant from S.aureus 8325-4. traP deletion mutant strain was prepared by gene reconbination. The agr system was not changed in the mutant, which suggested that TRAP could not influence the expression of agr. While, the infection caused by the mutant strain was decreased significantly, which indicated that TRAP was involved in the pathogenicity of S.aureus. Compared with its parent strain, a serial of phenotypes alternation were observed in the traP deletion strain. These data suggested that TRAP play an important role in S.aureus.2, The TRAP protein was involved in the neutrophil phagocytosis of S.arueus. It was found that traP mutant was sensitive to the neutrophil phagocytosis compared with its parent strain. But the recombinant TRAP protein could not influence this process. In the further study, it was found the level of a protein on the cell wall of S.aureus was increased. This protein was indentified by MS. It was a protein of unknown function and named as SAP. The results of qPCR showed that the expression of sap was higher in the trap mutant strain than that in the wild type. Whether SAP is involved in the neutrophil phagocytosis and killing will be investigated in the future.In a word, The TRAP protein could not influence the expression of agr, but it might play an important role in the pathogenicity of S.arueus by regulationg the interaction between neutrophil and S.aureus.