The Study On The Function Of Hfq In Staphylococcus Aureus

The Gram-positive bacterium Staphylococcus aureus (S.aureus) is an important pathogen which can expressed many kinds of extrotoxins to cause a variety of diseases, such as cutaneous infection, pneumonia, toxic shock syndrome etc. Because most of S.aureus are drug-resistant, many antibiotics are useless.sRNA (small RNA), a kind of non-coding RNA, exists in the eukaryote and prokaryocyte. The microRNA is a kind of sRNA in eukaryote and negatively regulates the target gene by annealing to mRNA. In bacteria, one kind of sRNA similar to microRNA, can regulate the expression of target gene by paring with the 5'UTR of mRNA. In bacteria, one sRNA can inteact with several targets and one gene can be regulated by multiple sRNAs. Hfq (a host factor for RNA phage Qβ), is necessary for sRNA interacting with its targets. Many reports showed that sRNAs involved in the process of metabolism and pathogenesis. Though the studies of sRNA mostly focus on the Escherichia coli (E.coli), some novel sRNAs are identified in S.aureus and Listeria monocytogenes. 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 small RNA, which is identified as a key regulator of toxin expression in S. aureus.The Hfq was 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. Hfq also has ATPase activity. In E.coli, the expression of many proteins is altered in the hfq mutant comparing with wild type and the phenotype of mutant is different with wild type. It is reported that Hfq can influence the expression of exotoxin in some bacteria.There are some proteins interacting with Hfq in E.coli, such as PNP(polynucleotide phosphorylase), PAPI(polyA polymerase I) and RNaseE. These proteins are important for Hfq.In our study, we try to discover the biological function of Hfq in S.aureus from four parts. The first part is the preparation of hfq mutant. In the second part, we try to study the polymer formation and chaperone activity of recombinant Hfq protein. In the third part, the proteins interacting with Hfq are identified. We try to discover biological function of the sRNAs binding to Hfq in the last part. The main results of this study as following:1. The preparation of hfq mutant. The mutant of hfq in S.aureus was obtained through gene recombination. It was found that the mutant was more harmless comparing with wild type in the animal model. The results of gene chip and proteomics showed that the level of many coding genes and intergenic sequences was changed in the mutant. Most sRNAs are transcribed from the intergenic region, so our results suggested that the Hfq protein could influence the expression of mRNA and sRNA in S.aureus.2. The study of the polymer formation and chaperone activity of Hfq. The Hfq protein was recombinant expressed. The recombinant protein had the ATPase activity to accelerate the transcription of mRNA in vitro. The formation of Hfq polymer was tested in the different conditions. It was found that there were different kinds of forms of Hfq protein including monomer, dimmer, tetramer and hexamer. In the further study, we found that the formation mechanism of the different polymers was different. The tyrosine 56 and tyrosine 63 of Hfq protein were indentified as the the key amino acids for the polymer formation by using site mutation method. The result of the monomer of Hfq protein could not bind RNA suggested that the polymer structure was essential to its RNA chaperone function.3. Identification of the interaction proteins of Hfq. First, we found that TRAP (target of RAP) could specifically bind the Hfq protein. TRAP is reported to be involved in the pathogenesis of S.aureus. The polyclonal antibody of TRAP can decrease the level of exotoxins of S.aures. In the further study, we found that TRAP could interact with Hfq though its C terminus and the TRAP protein could stable the polymer of Hfq. The pathogenesis of trap mutant is decreased. The results of gene chip and proteomics indicated that the expression of many genes and exoproteins was altered in the trap mutant. Many of them are also changed in the hfq mutant. These results suggested that the interaction of Hfq and TRAP could regulate some genes expression and influence the pathogenicity of S.aureus.Previously study showed RNaseIII (endoribonuclease III) could rapidly degrades the spa mRNA in the complex of RNAIII/spa mRNA. In our experiment, we found that Hfq could specifically bind RNaseIII and block RNaseIII to degrade the RNA. It suggested that the interaction of Hfq and RNaseIII might involve in the metabolism of RNA in S. aureus.4. Indentification of the function of the sRNAs which could bind to Hfq. We established a novel method named IPL(Immunoprecipitation-Like) to discover the Hfq binding sRNAs and their targets. Using this method, we found that the spa and hla mRNA were the targets of RNAIII, which was accordant to the previous reports. In the further study, we discovered some genes related to S.aureus pathogenesis as the novel targets of RNAIII.ssrA (also called 10Sa and tmRNA) is highly conserved in many kinds of bacteria. In E. coli, ssrA bears properties of both an alanine-tRNA and an mRNA. By a mechanism called trans-translation, ssrA tags incomplete proteins expressed from broken or cleaved mRNA lacking in-frame stop codons The pigment of ssra mutant is up-regulated as same as the hfq mutant. Our results showed that ssrA could act as an antisense RNA to control the expression of CrtM/N and sigB with the aid of Hfq protin.These results indicated that ssrA could also act as a sRNA to regulate some genes expression.We also found some novel sRNA candidates which could specifically bind to Hfq. Two full length novel sRNA were identified by using RACE which named as IP1 and IP3. IP1 and IP3 were up-regulated in the some stress condition. It suggested that these two sRNAs might be important for the stress response of S.aureus. Several genes were indenfied as the potential target of the two sRNAs.In summary, our results showed that Hfq was an important protein in S.aureus. There were some proteins and sRNAs could interact with Hfq. Hfq protein was essential for the complex formation of sRNA and its targets. Many genes were regulated directly or indirectly by Hfq and its interacting molecules. Identification of Hfq function in S.aureus is important for the study of anti-infection in the future..