Comparative Proteome Research Of The Vancomycin Resistant Enterococcus Faecalis

Fecal streptococcus is also called Enterococcus faecalis . According to C polysaccharide antigen, Lancefield and serological classification system, streptococcus can be classified into lots of groups. Fecal streptococcus is belong to group D. Group D streptococcus can be sub-classified into Enterococcus and non-enterococci. Enterococcus includes S.faecalis, S. faeciumand and S. durans, non-enterococci includes S. bovis and S.equinus。Enterococcus is a genus of bacteria normally resident in the intestines of human and several other mammalians. When proliferated out of their original residence, Enterococcus can cause septicemia,Urinary tract infection,Respiratory tract infection, endocarditis, meningitis and wound infection, et al. In the past few years, because of the extensive use of the immunosuppressive drugs, invasive treatment, fluoroquinolone and orally administrated Cephalosporins antibiotics, Enterococcus infection is getting more and more prevalence, and becoming the leading cause of the hospital infections. Enterococcus acquired resistant to Streptomycin and Gentamicin due to the antibiotics abuse. The appearance of highly aminoglycosides resistant (HLAR) and Vancomycin resistant Enterococcus (VRE) attracts global attentions. Further more, distribution of the Enterococcus strains also changed a lot. The mechanism of the drug resistant of the Enterococcus is complicated. The drug resistant strains not only have natural resistant to multiple antibiotics, but also easily acquire drug resistance, which makes a huge obstacle of clinical treatment.This thesis is devoted to a compared proteomics research on vancomycin resistant Enterococcus faecalis strains under normal and drug induced conditions.Purpose: By comparing the proteome of Vancomycin resistant Enterococcus faecalis under normal and drug induced conditions, identified the differentially expressed proteins, elucidate the mechanism of the multiple drug resistance of the Enterococcus faecalis, and provide new theological foundations for new treatments.Methods: whole cell protein extracted from Vancomycin resistant Enterococcus Faecalis v583(EF v583)(induced with solvent control or Vancomycin), Vancomycin resistant Enterococcus faecalis isolated by ICU of second affiliated hospital of General hospital of PLA(Enterococcus Faecalisv309,EF309)(induced with solvent control or Vancomycin) were separated with 2D-electrophoresis with pH3-10 and 4-7 stripes respectively. The resulting 2D gels were image analyzed by ImageMaster, and the differentially expressed proteins between the Enterococcus with control or drug treatment were discovered. 147 proteins with expression level difference of more than 2.5 times were selected for further Mass spectrometry analysis by MALDI-TOF/TOF-MS.PMF were acquired and analyzed, and then peptides were selected to be further fragmented to get secondary sequences. PMF and MS/MS sequencing spectrum data were jointly processed by Biotools software and presented to local Mascot and local Enterococcus Faecalis v583 database for protein identification.Results: 64 proteins were identified with scores more than 300, sequence coverage more than 30%, which represents high confidence, with the highest score of 909 and the highest sequence coverage 92%. Most of them have relation with glycolysis, TCA cycle, amino acid metabolism, energy metabolism and nucleotide metabolism.10 hypothetical protein were identified, they were expressed at high level when induced with Vancomycin, indicating that Enterococcus faecalis vancomycin resistance is the combined effect of multiple protein complex mechanism. 3 drug resistance proteins(VanA,VanB,VanX) and 2 transport proteins were identified.The codon preference, protein hydophobics, functional categorization of the identified protein and hypothetical protein were compared by softwares such as CodonW, Expasy ProtParam tool. The results showed that there are significant similarity between identified protein and hypothetical protein. Most proteins on the gel showed the same pI and molecular mass with the theological protein.However, there are also some exceptions, for example, VanA was identified in 10 protein spots in 2 2D gels, VanB was identified in 5 protein spots, average score of the identified VanB protein is as high as 507, average sequence coverage is 57.6%, VanA was identified in 10 protein spots, average score of the identified VanA protein is as high as 618, average sequence coverage is 51.4%.Conclusions:1.We established reference map of EFV583 and Vancomycin-induced EFV583, EF309 and Vancomycin-induced EF309.64 proteins in 147 high pressed protein spots were identified,and the protein database was created in website of NCBA(www.proteomics.cn).2. We use fully automated high-throughput mass spectrometry method in proteins identification process. This method is not only efficient but also reliable.3.The GRAVY value of identified proteins showed that most of the identified proteins are hydrophilic proteins and 8 proteins are hydrophobic proteins.The protein location analysis showed that most of the identified proteins are cytoplasm protein, and there are also two proteins are membrane protein,18 proteins'location are unknown, 1 Extracellular protein was identified.4.Both VanA and VanB were identified in Vancomycin-induced EF309 , which is same as the result of PCR. It may be one of the reasons for the high-resistance.VanA and VanB may play a role in Vancomycin-induced EF309 in common.5. Several ABC transport proteins related to diseases and multiple drug resistance were also identified in this study, suggested a possible function of ABC transport proteins in Enterococcus faecalis Vancomycin resistance, and resistance gene propagation between strains.The results of this research provide important information to elucidate the mechanism of Enterococcus faecalis Vancomycin resistance, which will speed up the discovery of effective regulatory strategies.