Quantitative Proteomics Analysis Of The Mechaism Of Enterococcus Faecalis With Low-level Linezolid Resistance

Objective: Since the known mechanisms fail to interpret the low-level linezolid-resistant phenomena in E.faecalis,based on our research foundations we deduct that low-level linezolid-resistance may be involved with membrane proteins.This research is mean to understand the role of membrane proteins in low level linezolid-resistant E.faecalis.Method: 1.we performed quantitative proteomics analysis of membrane proteins in an E.faecalis isolate(P10748)with low-level linezolid resistance in comparison with two linezolid-susceptible strains3138 and ATCC 29212,all of which have been previously investigated by whole transcriptome analysis.After bioinformatics analysis,the differentially expressed proteins of linezolid resistant strain compared with two linezolid-susceptible strains were performed to verify their functions.2.Genomic DNA was extracted from 43 E.faecalis strains isolated by the clinical microbiology department of our hospital,and the carrying rate of optrA is 100%.Gene esp and gene sea1 were screened by polymerase chain reaction(PCR)method.The plasmid of strain P10748 was subjected to sequencing analysis.Result: 1.Nano LC-MS/MS analysis of membrane proteins from three E.faecalis isolates P10748,3138 and ATCC 29212 generated a total of 362,473 raw spectra.With the FDR threshold set to 1%,we obtained50,635 qualified spectra including 50,285 unique spectra,which were associated with 8,197 peptides(8,144 unique peptides).These peptides were mapped to 1,170 proteins based on searches against NCBI non-redundant(Nr)and other databases.The most abundant GO terms of all identified proteins included the catalytic activity in the molecular function category,the metabolic process in the biological process category,and the cell,cell part in the cellular component category.The top three abundant categories were carbohydrate transport and metabolism;translation,ribosomal structure,and biogenesis;general function prediction only category in COG analysis.Besides,metabolic and biosynthesis of secondary metabolites,biosynthesis of antibiotics,microbial metabolism in diverse environments,and ABC transporters are the top five abundant KEGG pathways.Based on quantitative spectrum data,significantly differentially expressed proteins between each compared group were identified with a fold change of > 1.2 and a P value < 0.05.Combination of the results from these two comparisons led to identification of 14 significantly up-regulated which including collagen-like protein,AmaP,Sea1,DNA-banding protein(XRE family transcriptional regulator),RepA,lipoprotein,TraB,DUF3329domain-containing protein,Esp,conserved domain protein,optrA and three hypothetical function unknown proteins and 6 significantly down-regulated proteins in the low-level resistant significantly down-regulated proteins in the low-level resistant strain P10748 compared to the two susceptible strains 3138 and ATCC29212.2.There is a significant correlation between the significantly differentially expressed proteins identified in this study and their transcription levels reported previously.Pearson coefficient of protein and mRNA expressions between strains P10748 and ATCC29212 is 0.675,P=0.001.Pearson coefficient of protein and mRNA expressions between strains P10748 and 3138 is 0.698,P=0.001.3.In GO enrichment analysis of those significant expressed proteins,between strain P10748 and ATCC29212,the most enriched GO terms included the primary active transmembrane transporter activity in the molecular function category,mental ion transport in the biology process category,and membrane in the cellular component category.The most enriched GO terms for differentially expressed proteins in the resistant strain P10748 compared to the susceptible strain 3138 included the substrate-specific transmembrane transporter activity in the molecular function category,ion transport in the biology process category,and membrane in the cellular component category.Enriched pathways mainly associated with quorum sensing(QS),phosphotransferase system(PTS),and pantothenate and CoA biosynthesis pathways.4.The transcript of sea1 was not detected in our previous transcriptome analysis,while PCR analysis showed a strong,single band with an expected size was present in strain P10748 but absent in other two strains.Based on sequence analysis of the PCR product,the nucleotide sequence of the sea1 gene in strain P10748 was 91% identical to the E.faecalis plasmid pAD1(GenBank No.X62658.1)and 98% identical to the E.faecalis plasmid pMG2200(GenBank No.AB374546.1).Besides,RT-PCR analysis found that this gene was highly transcribed in strain P10748 while no transcription was detected in strains 3138 or ATCC 29212 strains.5.The rate of gene sea1 and gene esp carrying were 74.4% and 76.7%,respectively,among the 43 E.faecalis strains.Conclusion: 1.Both OptrA an Esp were significantly up-regulated in low-level linezolid resistant strain P10748,which were in generalpositively correlated with transcription levels revealed in previous transcriptome analysis.The results inducted that gene optrA and esp were correspond to linezolid resistance,which provide the reference of drug target.2.Gene optrA might located on sex pheromone plasmid,and proteins like Sea1,RepA,TraB,and XRE family transcriptional regulator may facilitate its horizontal transfer.3.Membrane proteins like Esp may mediate linezolid resistance of E.faecalis through biofilm formation.