Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells

Abstract

Enterococcus faecium is a clinically important pathogen associated with opportunistic infection and multi-drug resistance. E. faecium has been shown to produce membrane vesicles (MVs), but MV production by E. faecium under antibiotic stress conditions and the pathogenic traits thereof have yet to be determined. This study investigated the production of MVs in E. faecium ATCC 700221 cultured with sub-minimum inhibitory concentrations (MICs) of vancomycin or linezolid and determined their pathologic effects on colon epithelial Caco-2 cells. E. faecium ATCC 700221 cultured with 1/2 MIC of vancomycin or linezolid produced 3.0 and 1.5 times more MV proteins than bacteria cultured without antibiotics, respectively. Totals of 438, 461, and 513 proteins were identified in MVs from E. faecium cultured in brain heart infusion broth (MVs/BHI), BHI broth with 1/2 MIC of vancomycin (MVs/VAN), or BHI broth with 1/2 MIC of linezolid (MVs/LIN), respectively. Intact MVs/BHI induced cytotoxicity and the expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells in a dose-dependent manner, but proteinase K-treated MVs significantly suppressed these pro-inflammatory responses. MVs/LIN were more cytotoxic toward Caco-2 cells than MVs/BHI and MVs/VAN, whereas MVs/VAN stimulated more pro-inflammatory cytokine gene expression in Caco-2 cells than MVs/BHI and MVs/LIN. Overall results indicated that antibiotics modulate the biogenesis and proteomes of MVs in E. faecium at subinhibitory concentrations. MVs produced by E. faecium cultured under antibiotic stress conditions induce strong host cell responses that may contribute to the pathogenesis E. faecium.

Keywords: E. faecium; antibiotics; cytotoxicity; inflammatory response; membrane vesicle.

Figure 1

Membrane vesicles (MVs) produced by E. faecium ATCC 700221 cultured with or without antibiotics. (A) Transmission electron micrographs of MVs from E. faecium cultured in BHI broth. (B) SDS-PAGE analysis of proteins obtained from bacterial lysates (lane 1), culture supernatant (lane 2), and MVs (lane 3). Bacteria were cultured in BHI broth to late exponential phase. Lane M, molecular weight marker. (C) Production of MVs from E. faecium cultured with or without antibiotics. MVs were isolated from E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 μg/ml vancomycin (MVs/VAN), or BHI broth with 1 μg/ml linezolid (MVs/LIN). The protein concentration of MVs isolated from 1 L of bacterial culture was measured using a modified BCA assay. Data are presented as the mean ± SD of three independent experiments. ^**P < 0.01 compared to MVs/BHI. (D) SDS-PAGE analysis of MV proteins. Lane M, molecular weight marker; 1, MVs/BHI; 2, MVs/VAN; 3, MVs/LIN. (A,B,D) represent one of three independent experiments.

Figure 2

Proteomic analysis of MVs produced by E. faecium ATCC 700221. MVs were isolated from E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 μg/ml vancomycin (MVs/VAN), or BHI broth with 1 μg/ml linezolid (MVs/LIN). (A,B) A total of 438 proteins in the MVs/BHI were analyzed based on cellular localization (A) and Gene Ontology (B). (C) A Venn diagram of proteins identified in the MVs/BHI, MVs/VAN, and MVs/LIN.

Figure 3

Cytotoxicity of Caco-2 cells treated with MVs from E. faecium ATCC 700221. MVs were isolated from culture supernatants of E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 μg/ml vancomycin (MVs/VAN), or BHI broth with 1 μg/ml linezolid (MVs/LIN). Cells were treated with various concentrations of E. faecium MVs for 24 h. (A) Cell viability was determined using an MTT assay. Data are presented as the mean ± SD of three independent experiments. ⁺P < 0.05, ⁺⁺P < 0.01 compared to untreated control cells. *P < 0.05, **P < 0.01 among the same concentrations of MVs/BHI, MVs/VAN, or MVs/LIN. (B) Flow cytometric analysis of Caco-2 cell death induced by E. faecium MVs. Cells were treated with 20 μg/ml of E. faecium MVs for 24 h. Cells were stained with FITC-Annexin V and propidium iodide, and 10⁴ cells were counted. The figure represents one of three independent experiments that yielded similar results.

Figure 4

Expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells treated with MVs from E. faecium ATCC 700221. MVs were isolated from culture supernatants of E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 μg/ml vancomycin (MVs/VAN), or BHI broth with 1 μg/ml linezolid (MVs/LIN). Cells were treated with various concentrations of E. faecium MVs for 3 h and gene expression was assessed via qPCR. Data are presented as the mean ± SD of three independent experiments. ⁺P < 0.05, ⁺⁺P < 0.01 compared to untreated control cells. *P < 0.05, **P < 0.01 among the same concentrations of MVs/BHI, MVs/VAN, or MVs/LIN.

Figure 5

Host cell responses to proteinase K (PK)-treated E. faecium MVs. (A) Caco-2 cells were incubated with intact or PK-treated MVs/BHI for 24 h and cell viability was determined using an MTT assay. Data are presented as the mean ± SD of three independent experiments. (B) Cells were incubated with intact or PK-treated MVs/BHI for 3 h and gene expression was assessed via qPCR. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 comparing intact and PK-treated MVs.