Comparative Proteomic Analysis Of The Enterococcus Faecalis V583 And Clinical Isolate V309 Strains Under Vancomycin Treatment

Enterococcus spp is conditioned pathogen. When enterococcus ectopic parasitism, which can cause endocarditis, urinary tract infection, septicemia, wound infection, respiratory tract infection, meningitis, etc, even life-threatening. In the past few years, enterococcus infection is becoming one of the most notable nosocomial pathogens due to antibiotic abuse, the wide application of immunosuppressant and the increase of invasive therapies. Especially the appearance of Vancomycin-Resistant Enterococci (VRE) causes huge problem for clinical therapy. The vancomycin resistance genes in VRE can transmit to other pathogenic bacteria. VRE has attracted more attention now along with the report of Vancomycin-Resistant Staphylococcus aureus (VRSA)caused by VRE.The Enterococcus faecalis (EF) V583 strain and the Clinical Isolates V309 strain were identified in the study and the comparative proteome analysis of above two strains had been done. The mechanism that enterococcus survive under vancomycin pressure were more clear on proteomic level. The specificity and reversibility of vancomycin-resistant genes in V583 and V309 induced by vancomycin were further studied by the first time, and the proteins modification of phosphorylation caused by vancomycin has been analyzed firstly. This study has laid a foundation for controling vancomycin-resistant genes'transmission and guiding clinical medication.By comparing the proteome of vancomycin resistant Enterococcus faecalis under normal and drug induced conditions with Two Dimensional Electrophoresis (2-DE) technology, the differentially expressed proteins has been identified. The resulting 2D gels image were analyzed by ImageMaster, and the differentially expressed proteins between the Enterococcus with control or drug treatment were discovered, the proteins with expression level difference of more than 2.5 times were selected for further Mass spectrometry analysis. There are 57 proteins were identified. Most proteins on the gel showed the same pI and molecular mass with the theological protein but some point exist difference.By semi-quantitative RT-PCR of vancomycin-treated versus untreated samples of both strains, 28 (V583) or 20 (V309) up-regulated proteins, 8 (V583) or 6 (V309) down-regulated proteins, and 1 (V583) or 4 (V309) proteins with mobility changes in 2-D gel analysis were identified. Some of these proteins with known vancomycin resistance functions including VanB and VanXB in V583, VanA and VanX in V309. These proteins increased more than 20-fold and became post-translationally modified after treated with vancomycin. Intriguingly, VanA and VanX in V309 were expressed in cultures either with and without vancomycin, but VanB and VanXB in V583 were expressed only in culture with vancomycin, which suggested that there might be different molecular mechanisms for vancomycin resistance between VanA-type and VanB-type E. faecalis. The rest of these proteins may have unclear vancomycin resistance functions including virulence-related factors (EF2076, EF0577, EF3256), key stress proteins (EF1308, EF174, EF0080), metabolism-related proteins, conjunction-related proteins, proteins related to translation, and some hypothetical proteins. These proteins may play a key role in cell conglutination, antibiotic resistance expression, resistance gene recombination, endoenzyme activation or depression, and so on. The possible function of these proteins were analyzed in this study.Notably, six proteins (Gpm, Ldh, Gap-2, RpsB, EF2076, EF3256) exhibited clear post-translational modifications, especially the EF3256, which is sex pheromone cAD1 precursor lipoprotein, exist clear post-translational modification in both strains treated by vancomycin. In addition, as far as we know, the phosphorylation of EF3256 and the role it play in the progress of signal transmission and release between cells has also not been previously reported. While EF3256-P together with oppA-like protein may play a key role in the regulation of pheromone and transmission of conjugation plasmids.For specificity testing of vancomycin-induced resistance genes in V583 and V309, both strains were cultred 8h, 12h and 16h treated with vancomycin, it's clear that the quantity of these antibiotic-resistant proteins (VanA,VanB,VanX) is increased along with the culture time. This prove these antibiotic -resistance genes is specific to vancomycin. For reversibility testing, both strains were cultred with or without vancomycin for 6 h, at the same the other bacteria cultured with vancomycin for 6 h were split into two cultures. Vancomycin was added to one of the cultures, and both cultures were then incubated further for 6 h. It can be seen that the antibiotic-resistant proteins (VanA,VanB,VanX) increased when cultured with vancomycin were reduced when eliminate vancomycin. This prove these antibiotic -resistance genes are reversible to vancomycin. By semi-quantitative RT-PCR, it was approved that the expression level of encoding genes have the same change with it's antibiotic-resistant proteins. Protein post-translation modifications mainly because of the change of charge or molecular weight due to amino acid exchanges or small deletions, shown as differences in intensity or position. We found that post-translational protein modifications may be common in E. faecalis. In the 2-D analysis, 4 proteins: Pgm1(EF0195), Ldh(EF0255), Gap-2(EF1964), RpsB(EF2398) in V309 and 1 protein: Esa (EF2076) in V583 showed clear changes of position, which might result from post-translational proteolytic processing or other modification. There are 3 phosphorylation proteins (Ldh, Gap-2, cAD1) have been identified with Pro-Q and Western Blots. Using PHOSIDA database we confirmed possible phosphorylation sites. This is the first time phosphorylation protein been identified in enterococcus.There were five isoforms in the sex pheromone cAD1 precursor lipoprotein (EF3256). By using Pro-Q Diamond staining of the proteins in 2-D gels, we found five isoforms in both strains exit clear post-translationally modified of phosphorylation in cultures with vancomycin compared to without. Subsequent Western Blot analysis revealed that EF3256 was phosphorylated on both serine and threonine residues. However the sex pheromone cAD1 precursor lipoprotein has neither a phosphorylation motif (PMF) nor a corresponding phosphorpeptide in the PHOSIDA database. It is known that most of antibiotic-resistance genes horizontal transmission due to sex phormone-dependent plasmid transfer, but the function of sex pheromone cAD1 precursor lipoprotein in process of cell signal regulation has never been reported. Therefore, further functional studies will be needed.The determinant for the pheromone in E. faecalis V583 is sex pheromone cAD1 precursor lipoprotein, a 309-amino-acid protein representing an apparent lipoprotein precursor with a typical―lipobox‖associated with a cysteine residue at the signal sequence junction. The signal sequence corresponds to 22 amino acids of lipoprotein, the last 8 of which constitute cAD1. Following the lipoprotein signal sequence for Sec-dependent export are putative signal peptides cleavage sites that indicate that the signal sequence from which the mating pheromones is proteolytically removed from the lipoprotein. Because of another protein oligopeptide pheromone cCF10 transporter (oppA-like protein) which can transport pheromones into the cell was slightly upregulated in E. faecelis V309 cells grown with vancomycin, exogenous cAD1 is believed to bind to OppA-like surface lipoproteins, enhances donor sensitivity and participates in uptake of the peptide via a host-encoded ABC peptide transport system. This implied that vancomycin can increase the potential transmission of conjugation plasmids and improve sensitivity to exogenous bacteria. We postulate that in the E. faecelis V583 and V309 strains, the phosphorylated sex pheromone cAD1 precursor lipoprotein (EF3256) is the active form, and EF3256-P plays a key role in the regulation of pheromone. Therefore, EF3256 phosphorylation may represent a mechanism that enteric bacteria have evolved to interfere with the signaling capabilities of neighboring species of bacteria. By inducing the expression of genes related to virulence, resistance, and conjugation transmission in plasmid, vancomycin can cause E. faecelis to more easily accept or transmit plasmids, especially those carrying drug resistant genes.The intrinsic resistance of E. faecalis to many antibiotics and its acquisition of resistance to other antimicrobial agents, particularly vancomycin is correlated with particular genetic features. Currently, there are probably an unprecedented number of mobile elements contributing to virulence and drug resistance that have accumulated in the E. faecalis genome. Our comparative proteomic analysis of E. faecalis V583 and V309 extends previous studies on their physiological characteristics and supports the hypothesis formulated by Paulsen and colleagues regarding the role of mobile DNA, the transfer of vancomycin resistance and the molecular mechanisms underlying the glycopeptides resistance. More importantly, our data confirmed the expression of some proteins related to their resistance to vancomycin, and we demonstrated that many proteins involved in antibiotic resistance were differentially regulated by vanomycin which also triggered innate signal regulators, adhesion factors, and metabolic gene expression. These responses may enable E. faecelis to adapt, survive and remain pathogenic under pressure of vancomycin treatment. The research results has important significance for us to reveal the molecule mechanism of antibiotic- resistance of E. faecalis and find the strategy to control it.