E. faecium Aus0004, S. aureus RN4220, S. aureus ATCC29213, S. pneumoniae R6, Escherichia coli XL1-Blue.

Teicoplanin, vancomycin, oritavancin, and chloramphenicol (Sigma-Aldrich, Germany); dalbavancin (MedChemExpress, Sweden); MA79 (Csávás et al., 2015), ERJ390 (Pintér et al., 2009), and SZZS-12 (Szucs et al., 2017); carbenicillin, gentamicin and erythromycin (Duchefa Biochemie, Netherland); vancomycin BODIPY-FL conjugate (Thermo Fisher Scientific, Germany) and fluorescently labeled teicoplanin (Vimberg et al., 2019).

The vanZ_Tei and vanZ_g genes and their ribosome binding sites were amplified from the plasmid pAT398 (Arthur et al., 1995) and E. faecium Aus0004 chromosomal DNA, respectively, using the primers TecVanZ_SacI_F, TecVanZ_R, gVanZ_SacI_F, and gVanZ_R (Supplementary Table S1). The PCR products were cloned into the pRMC2 shuttle vector under the control of the anhydrotetracycline (AnhTet)-inducible promoter Pxyl/tetO using SacI and EcoRI restriction sites, resulting in the constructs pRMC2:vanZ_Tei and pRMC2:vanZ_g. The constructs were confirmed by sequencing and then electroporated into S. aureus RN4220. Using the same procedure, we prepared the constructs pRMC2:vanZ_Tei-His and pRMC2:vanZ_g-His encoding C-terminal His-tagged VanZ variants. However, primers TecVanZhis_R and gVanZhis_R replaced the reverse primers TecVanZ_R and gVanZ_R.

Strain Sp539 (ΔvanZ) was constructed using a Sweet Janus cassette-based two-step negative selection strategy (Sung et al., 2001; Li et al., 2014). The Sweet Janus cassette contains the kanamycin resistance gene, the recessive rpsL gene and the sacB gene, which confers sucrose sensitivity (Su^S), as counterselectable markers. In the first step, 1000 bp fragments corresponding to the upstream and downstream flanking regions of the vanZ gene (spr0050) were amplified from the wild-type chromosomal DNA with the primer pairs KB60/KB61 and KB62/KB63, respectively. The Sweet Janus cassette (2807 bp) amplified from the Sweet Janus cassette DNA fragment with the primers DP1/DP2 was attached to the regions flanking vanZ by fusion PCR using primers KB60 and KB63. The resulting PCR fragment was used for the transformation of the S. pneumoniae R6 strain, and Kan^R/Su^S transformants (Sp537, vanZ:kan sacB) were selected. The PCR fragments consisting of the upstream and downstream flanking regions of the vanZ gene were amplified by the KB60/KB65 and KB64/KB63 primer pairs, respectively, and fused by overlap extension using primers KB60/KB63. The resulting fragment was transformed into the Sp537 strain to obtain Sp539 (Su^R/Kan^S). To complement the vanZ deletion, we constructed strain Sp635 (Su^R/Kan^S; ΔvanZ:vanZ) by transforming strain Sp537 (Kan^R/Su^S) with the PCR fragment amplified with the primers KB60 and KB63 that contained wild-type loci using R6 chromosomal DNA as a template.

Minimal Inhibitory Concentrations were measured by the broth microdilution method according to ISO standard 20776-1 (EUCAST 2019). S. aureus strains with pRMC2, pRMC2:vanZ_Tei and pRMC2:vanZ_g plasmids were cultured in the presence of 25 μg/ml chloramphenicol and 100 ng/ml AnhTet (Sigma-Aldrich, Germany) to induce vanZ gene expression. All measurements were performed twice in triplicate. S. aureus ATCC29213 was used as a control. MIC values of clinically accepted glycopeptide antibiotics were interpreted according to EUCAST clinical breakpoints (EUCAST, 2019).

Staphylococcus aureus RN4220 strains harboring plasmids pRMC2:vanZ_Tei-His and pRMC2:vanZ_g-His were grown in 2 ml of brain heart infusion medium (Oxoid/Thermo Fisher Scientific, Germany) in the presence of chloramphenicol and AnhTet overnight at 37°C. Cells were harvested and lysed in 1 ml of 1× PBS (phosphate-buffered saline) buffer with 10 μg of lysostaphin (Sigma-Aldrich, Germany) for 15 min at 37°C. Cell debris was removed by centrifugation at 16,000 × g for 30 min. The supernatant was then transferred into fresh tubes and centrifuged at 30,000 × g for 30 min to separate the membrane and cytosolic fractions. Membrane sediment was resuspended in 50 μl of 1 M urea in 1× PBS. Supernatant proteins were precipitated with 10% TCA, washed twice with ice-cold acetone and resuspended in 50 μl of 1 M urea in 1× PBS buffer. Protein concentration was determined using a bicinchoninic acid (BCA)-based protein estimation kit (Thermo Fisher Scientific, Germany). Proteins were further denatured in SDS-loading buffer at 95°C for 10 min, and 20 μl aliquots were loaded on a 12% SDS-acrylamide gel. After separation by SDS-PAGE, proteins were transferred to a PVDF membrane (Immobilon-P, Merck Millipore, United States) at 15 V for 10 min with a BioRad SemiDry blotting system. His-tagged VanZ was detected with monoclonal anti-His antibody (Sigma-Aldrich, Germany) and subsequently with a secondary goat anti-mouse IgG antibody HRP conjugate (Sigma-Aldrich, Germany). Protein abundance was measured using Immobilon Western HRP Substrate (Merck Millipore, United States), and the signal was developed using the ChemiDoc MP Imaging System (Bio-Rad).

Staphylococcus aureus pRMC2, pRMC2:vanZ_Tei and pRMC2:vanZ_g, S. pneumoniae R6, S. pneumoniae R6, R6ΔvanZ, and R6ΔvanZ:vanZ cells were pregrown in Mueller Hinton medium (Oxoid/Thermo Fisher Scientific, Germany) to A_600nm = 0.4. S. aureus was pregrown in the presence of chloramphenicol and AnhTet. Cells were harvested by centrifugation and resuspended to A_600nm = 1 in 50 mM Tris–HCl buffer (pH = 7.4). Increasing amounts of Bodipy-Vancomycin (Thermo Fisher Scientific, Germany) or Fluorescent Teicoplanin (Vimberg et al., 2019) were added to 1 ml of resuspended cells. Cells were then incubated for 10 min at room temperature with the fluorescent antibiotics, harvested by centrifugation, washed two times with 50 mM Tris–HCl buffer (pH = 7.4), and finally resuspended in 100 μl of the same buffer. The fluorescence of 70 μl of resuspended cells was measured in automatic gain mode at Ex_490nm/Em_520nm in the case of fluorescent vancomycin or Ex_530nm/Em_580nm in the case of fluorescent teicoplanin in 96-well black plates (Thermo Fisher Scientific, Germany) by Tecan Infinite 200Pro. The experiment was repeated three times in duplicate.

The ability of the E. faecium vanZ_Tei and vanZ_g paralogs to confer resistance to glycopeptide antibiotics was tested in S. aureus, which naturally does not encode any proteins of the VanZ superfamily. In particular, we determined the susceptibility of S. aureus RN4220 expressing vanZ_Tei and vanZ_g to the clinically used glycopeptide antibiotic vancomycin (VAN); the lipoglycopeptide antibiotics teicoplanin (TEI), oritavancin (ORI), and dalbavancin (DALB); and three experimental lipoglycopeptide antibiotics derived from teicoplanin pseudoaglycone: MA79 (Csávás et al., 2015), ERJ390 (Pintér et al., 2009) and SZZS-12 (Szucs et al., 2017; Figure 1). In addition, the non-glycopeptide antibiotics carbenicillin (CARB, cell wall-targeting) gentamicin (GEN, 30S ribosome-targeting) and erythromycin (ERY, 50S ribosome-targeting) were used as controls.

As shown in Table 1, the expression of vanZ_g decreased the susceptibility of S. aureus to TEI and ERJ390 16-fold, to DALB four-fold, and to ORI and MA79 two times, and the expression of vanZ_g had no effect on the susceptibility of S. aureus to VAN, SZZS-12 or the control drugs. Similar to vanZ_g, the expression of vanZ_Tei decreased the susceptibility of S. aureus to ERJ390 16-fold but had less or no activity against TEI, ORI and DALB. However, at the same time, cells expressing vanZ_Tei were more active against MA79 and SZZS-12 (Table 1). To test whether different levels of protein expression cause different activities of VanZg and VanZTei, we performed Western blot analysis of the strains expressing C-terminal His-tagged versions of VanZ proteins. However, the analysis showed that both proteins were expressed at similar levels and that they were localized exclusively in the cell membrane (Supplementary Figure S1).

To further explore the effect of VanZ proteins on the resistance to glycopeptide antibiotics in the natural genetic background, we employed S. pneumoniae R6, which encodes the VanZ ortholog encoded by genome locus spr0050. We constructed a clean knockout of vanZ (ΔvanZ) and complemented vanZ in the S. pneumoniae R6 genome and tested the susceptibility of the strains to antibiotics. According to MIC measurements, the ΔvanZ mutant was up to four-fold more sensitive to lipoglycopeptide antibiotics, but not to VAN or non-glycopeptide antibiotics, than the wild-type strain (Table 1). Altogether, these data indicate that the transmembrane proteins VanZ_g and VanZ_Tei, as well as VanZ from S. pneumoniae, decrease susceptibility to TEI and its derivatives, while they have no or a minor effect on VAN and its derivative ORI.

To determine whether the expression of VanZ might interfere with the binding of lipoglycopeptide antibiotics to the bacterial surface, we followed the binding of fluorescently labeled VAN and TEI (FL-VAN and FL-TEI) to S. aureus expressing vanZ_Tei and vanZ_g, as well as to S. pneumoniae R6 wild-type, ΔvanZ and reverted strains. The titration curves of FL-VAN binding clearly showed that the presence of VanZ did not affected FL-VAN binding to S. aureus or S. pneumoniae cells (Figures 2A,B). On the other hand, FL-TEI bound less efficiently to S. aureus with VanZ_g or VanZ_Tei than to cells without VanZ (Figure 2C). Similarly, S. pneumoniae R6 ΔvanZ was saturated with a lower amount of FL-TEI than the wild type (Figure 2D). Altogether, this experiment demonstrates that VanZ proteins might indeed affect the binding of lipoglycopeptide antibiotics to cells.