Quantitative Proteomics Analysis of Membrane Proteins in Enterococcus faecalis With Low-Level Linezolid-Resistance

Abstract

Despite increasing reports of low-level linezolid-resistant enterococci worldwide, the mechanism of this resistance remains poorly understood. Previous transcriptome studies of low-level linezolid-resistant Enterococcus faecalis isolates have demonstrated a number of significantly up-regulated genes potentially involved in mediation of drug resistance. However, whether the transcriptome faithfully reflects the proteome remains unknown. In this study, 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 strains 3138 and ATCC 29212, all of which have been previously investigated by whole transcriptome analysis. A total of 8,197 peptides associated with 1,170 proteins were identified in all three isolates with false discovery rate (FDR) at 1% and P < 0.05. There were 14 significantly up-regulated and 6 significantly down-regulated proteins in strain P10748 compared to strains 3138 and ATCC 29212, which were in general positively correlated with transcription levels revealed in previous transcriptome studies. Our analysis suggests that the low-level linezolid-resistance in E. faecalis is conferred primarily by the ATP-binding cassette protein OptrA through ribosomal protection and, possibly, also by the enterococcal surface protein (Esp) and other proteins through biofilm formation. The genetic transfer of optrA is potentially regulated by the surface exclusion protein Sea1, conjugal transfer protein TraB, replication protein RepA and XRE family transcription regulator protein. This report represents the first investigation of the mechanisms of linezolid-resistance in E. faecalis by a quantitative proteomics approach.

Keywords: Enterococcus faecalis; linezolid; low-level resistance; membrane proteins; quantitative proteomics.

FIGURE 1

Profiles of membrane proteins identified in three Enterococcus faecalis isolates. (A) Number of unique peptides identified for individual proteins. X axis represents the number of unique peptide. Y axis represents the number of proteins. (B) Distribution of molecular mass among all identified proteins. X axis represents molecular mass (kDa). Y axis represents the percentage of the number of proteins. (C) Coverage of identified proteins. X axis represents coverage, which refers to the proportion of the amino acid sequence of each protein covered by peptides. Y axis represents the number of proteins. (D) Differentially expressed proteins of each compared group. X axis represents each compared group. Y axis represents the number of proteins. Up-regulated proteins are indicated in red and down-regulated proteins are indicated in green. Each isolate was tested in triplicate (indicated as 1, 2, and 3 in parentheses) and compared to other isolate separately.

FIGURE 2

GO function analysis of membrane proteins identified in E. faecalis. Proteins were identified by GO terms based on cellular component, molecular function and biological process. X axis represents the number of proteins. Y axis represents the GO terms.

FIGURE 3

COG function analysis of membrane proteins identified in E. faecalis. X axis represents the number of proteins. Y axis represents the COG terms.

FIGURE 4

Volcano plots of differentially expressed proteins among the linezolid-resistant strain P10748 (resistant) and two linezolid-susceptible strains 3138 (sensitive) and ATCC 29212 (control). These plots show the confidence and fold change of quantified proteins from each compared group (control1, labeled by tag 126; control 2, labeled by tag 127N; control 3, labeled by tag 127C; resistant1, labeled by tag 129N; resistant2, labeled by tag 128N; resistant3, labeled by tag 128C; sensitive1, labeled by tag 129C; sensitive2, labeled by tag 130C; sensitive3, labeled by tag 130N). Spots with color are highly confident (Q-value < 0.05) and differentially expressed proteins. Protein fold changes are expressed as the ratio of all significantly matched peptides. The dashed line represents the applied threshold (P ≤ 0.05, Fold change ≥ 1.2). Up-regulated proteins are highlighted in red and down-regulated proteins are highlighted in green. Each isolate was tested in triplicate (indicated as 1, 2, and 3) and compared to other isolate separately.

FIGURE 5

Clustering and heatmap of significantly differentially expressed proteins among the linezolid-resistant strain P10748 (resistant) and two linezolid-susceptible strains 3138 (sensitive) and ATCC 29212 (control). The heat map was created based on the fold changes of abundance of 20 differentially expressed proteins indicated on the right (from Unigene1072_All to Unigene1531_All). The color-coded z-score on the up right corner indicates the significance level of protein expression, with red indicating up-regulation and green indicating down regulation.