Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediates and van Operon mRNA Levels in VanA-Type Vancomycin-Resistant Enterococcus faecium

Abstract

Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (vanRSHAXYZ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Ala-D-Ala is replaced by D-Ala-d-lactate (Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in vanA operon mRNA levels measured by real-time quantitative PCR (RT-qPCR). Normal pathway intermediates predominated in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18-h) vancomycin exposure resulted in a mixture of the terminal normal (UDP-N-acetylmuramic acid [NAM]-l-Ala-D-Glu-l-Lys-D-Ala-D-Ala [UDP-Penta]) and alternative (UDP-NAM-l-Ala-γ-D-Glu-l-Lys-D-Ala-D-Lac [UDP-Pentadepsi]) pathway intermediates (2:3 ratio). Time course analyses revealed normal pathway intermediates responding rapidly (peaking in 3 to 10 min) and alternative pathway intermediates responding more slowly (peaking in 15 to 45 min). RT-qPCR demonstrated that vanA operon mRNA transcript levels increased rapidly after exposure, reaching maximal levels in 15 min. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin-sensitive Enterococcus faecium, and methicillin-resistant Staphylococcus aureus after vancomycin exposure demonstrated substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistant to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode enzymes that provide for an alternative PG biosynthesis pathway. In VanA-type resistance, these alternative pathway enzymes replace the D-Ala-D-Ala terminus of normal PG intermediates with D-Ala-D-Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, vanA gene induction, and changes in the normal and alternative pathway intermediate levels have not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

Keywords: Enterococcus faecium; LC-MS/MS; VRE; VanA; antibiotic resistance; cell wall; mass spectrometry; metabolism; metabolomics; peptidoglycan; vancomycin.

FIG 1

(A) Generic PG biosynthesis process in Gram-positive bacteria. Note that in E. faecium a d-iAsp “bridging” residue is added to the ε-amino group of l-Lys (25), which is not included in this figure. (B) VanA-type resistance gene cluster (adapted from reference 15). (C) Alternative CWB pathway in VanA-type resistance. GlcN, glucosamine; GlcNAc, N-acetylglucosamine (NAG); GlcNAc-1P, N-acetylglucosamine-1-phosphate; PEP, phosphoenolpyruvate; AEK, L-Ala–γ-D-Glu–L-Lys.

FIG 2

Time course of VREfm PG intermediates in response to added vancomycin (16 μg/ml added at a culture OD₆₀₀ of 0.5), plotted as fold changes versus the time zero values (shown in Table 1). UDP-Sum is the sum of all UDP-linked intermediates. DADA, D-Ala–D-Ala; DADL, D-Ala–D-Lac.

FIG 3

Time course of the effect of vancomycin (16 μg/ml) on RT-qPCR-determined mRNA levels in VREfm. Data are presented as mean ± SE (n = 3).

FIG 4

(Top) Fold changes (relative to no-vancomycin control) in key VREfm PG intermediate levels after 15-min exposure to different vancomycin concentrations (n = 4), shown with a semilogarithmic y axis. (Bottom) Corresponding fold changes in mRNA levels (n = 4).

FIG 5

Fold changes (relative to no-vancomycin control) in key VREfm PG intermediate levels after 15-min exposure to vancomycin (Vm) and/or linezolid (n = 3), shown with a semilogarithmic y axis.