T7SS proteins are part of the DRM

To identify new FMM-associated protein complex candidates, we quantified proteomes of detergent-sensitive (DSM) and -resistant (DRM) membrane fractions using a biochemical approach designed to purify eukaryotic raft-associated proteins [53,54]. This approach is based on the ability of rafts to resist disaggregation by treatment with non-ionic detergent (Triton X-100, Brij, CHAPS). Differences in lipid composition make rafts more compact than the remainder of the cell membrane and more resistant to detergent disaggregation [53,54]. After detergent treatment, large hydrophobic membrane fragments enriched in FMM can be concentrated in a DRM fraction using a phase separation approach. This allows comparison to a DSM fraction that is sensitive to detergent disaggregation, as it is comprised mainly of phospholipids and does not concentrate to the hydrophobic phase during phase separation [6]. As FloA expression is higher and its activity possibly more important, we used stationary cultures of S. aureus cells to isolate DRM and DSM fractions and quantify the proteins by label-free quantification (LFQ) mass spectrometry [55]. We plotted LFQ intensities of DRM fraction proteins against those of the DSM fraction, which led to separation into four protein populations (I-IV; S1A Fig). The first population (I) consisted of proteins detected exclusively in the DSM fraction; population II and III proteins were found in both DRM and DSM, with greater abundance in the DSM (II) or DRM fraction (III). Population IV was associated exclusively with the DRM fraction (S1A Fig). Most of the proteins we associated with DRM fractions were histidine kinases, lipoproteins, transporters, or components of protein complexes; we identified 12 of 13 known membrane-bound histidine kinases and more than 85% of all identified components of S. aureus membrane transporters (ABC transporters; ion, nutrient and metabolite transporters) in DRM-associated populations (III, IV) (S1B Fig). Proteins involved in division, like FtsA, FtsY or FtsI, were generally detected in DSM populations (S1B and S1D Fig). Nonetheless, sequence analysis of the DRM and DSM proteins categorized by this approach did not lead to the identification of any common features within each protein population (I-IV) (S1C Fig); all proteins identified for each population are listed in S1 Table. These findings are consistent with the hypothesis that FMM, which are very concentrated in the DRM fractions, mainly contain proteins that require protein-protein interaction or form part of protein complexes.

During our data analysis, we detected several proteins of the S. aureus T7SS, significantly enriched in the DRM fraction (Fig 1B); these included membrane proteins EsaA, EssA, EssB, which suggested that S. aureus T7SS is physically and functionally associated to FMM. We used purified DRM and DSM fractions to measure T7SS membrane proteins EsaA, EssA, EssB and EssC by immunodetection, using polyclonal antibodies to EsaA, EssB or EssC [33]. EssA was fused to the codon-optimized RFP variant MARS and detected with polyclonal anti-mCherry antibodies [56]. Western blot analyses confirmed EsaA, EssA and EssB enrichment in the DRM fraction (Fig 1C). Signals attributable to EssB and EsaA were enriched in the DRM fraction (~10- and 2-fold, respectively), and immunodetection of EssA-MARS showed most of the signal associated with this fraction. EssC was also detected in the DRM fraction, although it was not markedly enriched in this fraction compared to the DSM. These data are consistent with the LFQ mass spectrometry findings and correlate T7SS and DRM in S. aureus. Based on these results, we hypothesized that T7SS proteins are associated with the S. aureus FMM, probably in a transient manner, as we did not detect all T7SS membrane-associated proteins exclusively in the DRM fraction.

The FMM are specific membrane regions that probably act as oligomerization platforms. They spatially confine interacting protein partners and promote efficient interaction/oligomerization of multiprotein complexes [23,25,57]. The membrane-bound scaffold protein flotillin localizes preferentially to the FMM. Flotillin probably has an important role in tethering interacting proteins and facilitates oligomerization, similar to its function in eukaryotic lipid rafts [19,22,23]. To determine whether the T7SS-related membrane proteins in the DRM fractions are among the flotillin-tethered protein partners, we used a bacterial two-hybrid assay in a heterologous E. coli system, in which S. aureus flotillin (FloA) and T7SS membrane proteins were tagged with T25 or T18 fragments of an adenylate cyclase. After flotillin interaction with T7SS proteins, the enzyme is reconstituted, produces cAMP and triggers expression of a measurable cAMP-inducible lacZ reporter [58]. We detected strong interaction between FloA and EssB (>2000 Miller Units; a 700 Miller Unit threshold defines positive and negative interaction signals), with no consistent interactions between FloA and EsaA, EssA or EssC (Fig 1D and S2 Fig). Our protein-protein interaction analyses in a heterologous system thus suggest interaction between flotillin and the membrane-bound T7SS protein EssB from S. aureus T7SS.

Flotillin interacts with EssB in S. aureus cells

We extended the FloA-EssB interaction analyses to S. aureus cells and performed pulldown experiments to identify interaction between FloA and EssB, using a FLAG-EssB, FloA-His double-labeled strain. FloA-His is a functional construct used in previous studies [59]. The FLAG-EssB construct was functional when expressed in a ΔessB genetic background, as immunodetection experiments of the EsxC substrate in culture supernatants showed that this complemented strain rescued the ΔessB mutant secretion defect (S3 Fig) [31,33,39]. A purified membrane fraction of this FLAG-EssB, FloA-His double-labeled strain was loaded on a column of Ni-NTA/His-tag resin selective for proteins that bind directly or indirectly to FloA-His. Eluted proteins were resolved by SDS-PAGE and FLAG-EssB was detected by immunoblotting using monoclonal anti-FLAG antibodies. A FLAG-EssB signal was detected in the eluted sample of the double-labeled strain, suggesting that EssB co-eluted with FloA (Fig 2A). In contrast, the elution fraction of FloA-His and FLAG-EssB single-labeled strains showed no signal, which implies that EssB retention on the column was FloA-dependent.

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Fig 2. Flotillin interacts with the T7SS protein EssB.

(A) Immunoblot analysis of pulldown assay to show the in vivo EssB and FloA interaction in S. aureus. Lane 1 shows FLAG-EssB FloA-His elution fraction from a Ni-NTA column. Negative controls (lanes 2, 3) are single-labeled strains and lane 4 is an unlabeled strain; lane 5 is the FLAG-EssB FloA-His double-labeled strain membrane fraction as positive control. (B) STED (stimulated emission depletion) microscopy images of a ΔspA strain double-immunolabeled with anti-FloA (Alexa488) and anti-EssB (Alexa546) antibodies. Top panel shows overlay of red and green fluorescent signals, false colored with red and green, respectively. Three individual cells are highlighted and further analyzed in panel C. Bottom panel shows colocalization analysis performed with the ImageJ JACoP plugin. Each pixel containing signal in both red and green fluorescent channel is now represented with a white pixel. Bar, 1 μm. (C) Three representative cells showing EssB and FloA colocalization. Top rows show overlay of false colored red and green fluorescent signals on the left and single red (EssB; Alexa546) and green (FloA, Alexa488) fluorescent channels in the center. Image on the right shows overlay of the white signal of colocalization analysis with the false-colored red and green-fluorescent channels. Bar, 0.2 μm. Each bottom panel shows pixel intensity analysis clockwise around the outline of the cell starting at the top.

https://doi.org/10.1371/journal.ppat.1006728.g002

We used stimulated emission depletion (STED) microscopy to examine EssB and FloA signal colocalization in S. aureus cells. STED microscopy is a super-resolution technique previously used in B. subtilis to demonstrate partial colocalization of FMM-associated proteins with the scaffold protein FloA [24]. Pull-down experiments or BN-PAGE coupled to immunoblotting have shown that flotillin interacts directly or indirectly with these FMM-associated proteins, and that this interaction contributes to FMM-associated complex oligomerization [9,57]. Partial colocalization of FloA with FloA-interacting FMM protein cargo is likely because flotillin does not form part of these oligomeric complexes and is thus not strictly necessary for their activity. The role of flotillin could be transient, to facilitate oligomerization of multimeric complexes that then act in a flotillin-independent manner. This is consistent with the role of FMM as oligomerization platforms, to promote efficient interaction between protein partners. To study FloA and EssB colocalization, we immunodetected these proteins in S. aureus cells, using specific antibodies. Subcellular signal localization in double-labeled samples was examined using STED microscopy (Fig 2B, top panel). Traditional colocalization studies focus on 1:1 ratios of red- and green-labeled proteins, resulting in a merged yellow signal. Proteins abundant at different ratios still colocalize, but are falsely disregarded using this approach. We determined protein colocalization independent of the relative FloA and EssB abundance, and present the results in a white colocalization map (Fig 2B, bottom). The flotillin signal was distributed in 1–6 fluorescent foci per cell, whereas the EssB signal formed 1–4 membrane foci. Using this method, we determined that 27% of the total EssB signal showed an overlap with FloA fluorescent signal. Thus, 73% of the EssB did not overlap with FloA and 68% of FloA did not overlap with EssB (Fig 2C). This partial colocalization indicates biochemical interactions of EssB and FloA and suggests that this EssB and FloA interaction occurs in a transient manner, similar to previous flotillin-associated interactions.

T7SS secretion is low in S. aureus cells lacking flotillin

Having determined that EssB is enriched in the DRM fraction and interacts with FloA, we analyzed FloA influence on T7SS activity; this is an important aspect of S. aureus virulence, since EssB is essential for secretion of T7SS effectors during infection [31,33,39]. We compared the levels of the T7SS substrates EsxA and EsxB and of EsxC in wild type or FloA-defective cells in culture supernatants. EsxA and EsxB were C-terminally labeled with a FLAG-tag and expressed under the control of a constitutive promoter. Culture supernatant proteins were concentrated by trichloroacetic acid (TCA) precipitation, and EsxA- and EsxB-FLAG were immunodetected with monoclonal anti-FLAG antibodies and EsxC with EsxC-specific polyclonal antibodies [33]. The ΔfloA mutation might alter other secretory protein complexes in S. aureus, like the Sec system defect in a B. subtilis flotillin-deficient strain [9]. To normalize total ΔfloA mutant-to-wild type secreted protein in immunoblot analyses, we normalized cell culture optical density and added a defined concentration of an unrelated protein to supernatants. We added purified denatured YtnP lactonase (25 μg/ml) from B. subtilis [60] to supernatants before TCA precipitation, and we used polyclonal anti-YtnP antibodies to trace YtnP concentration and thus ensure comparable concentration of the supernatant. In this system, EsxA-FLAG and EsxB-FLAG showed a marked decrease in supernatants from the ΔfloA mutant compared to wild type (Fig 3); EsxC substrate was also decreased in ΔfloA compared to wild type supernatants, which implied an important flotillin role in T7SS activity. Quantitative differences in T7SS substrates in the supernatant are probably due to reduced T7SS secretion efficiency rather than reduced abundance of its components, as immunodetection indicated similar EsaA, EssA, EssB and EssC protein levels in wild type and ΔfloA mutant whole cell extracts (S4 Fig).

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Fig 3. FloA mutant reduces secretion efficiency of T7SS substrates EsxA, EsxB and EsxC.

Cells were grown to early stationary growth phase. Filtered supernatants (SN) were supplemented with recombinant YtnP as a control of equal loading, then precipitated and immunoblotted and proteins detected with anti-FLAG, -YtnP and -EsxC antibodies. Cell extracts (CE) were probed with anti-FLAG and -EsxC antibodies. Detection of GroEL served as loading control. For FLAG-labeled EsxA and EsxB, an unlabeled wild type strain served as negative control and ΔT7SS strain was used as a secretion-negative strain. For EsxC secretion, a ΔT7SS mutant was used as a negative control and a ΔessB mutant strain as a secretion negative strain.

https://doi.org/10.1371/journal.ppat.1006728.g003

Flotillin mediates T7SS intermolecular interactions

The most direct hypothesis as to how flotillin influences T7SS activity is that its scaffold activity promotes T7SS stability or assembly by tethering interacting proteins [3]. In the absence of FloA, EssB might oligomerize less efficiently and negatively affect correct T7SS organization. We thus used fluorescence microscopy to analyze EssB subcellular distribution in wild type and ΔfloA mutant cells labeled with a GFP-EssB translational fusion. We constructed a GFP-EssB ΔessB complemented strain and confirmed its function as above (S3 Fig). Compared to the punctate GFP-EssB pattern on a wild type background, EssB foci were undetectable in ΔfloA mutants, and the GFP-EssB signal was distributed uniformly over large portions of the membrane (Fig 4A, left) whereas protein abundance was unaffected (Fig 4A, right). This suggests that flotillin determines correct subcellular localization of EssB.

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Fig 4. FloA is important for intermolecular interactions of T7SS membrane proteins.

(A) Subcellular localization of GFP-EssB. Bright field and green fluorescence of a complemented GFP-EssB controlled by its own promoter (P_esxA) in a wild type and a ΔfloA background in stationary growth phase. Bar, 1.5 μm (right). Corresponding immunoblot analysis of P_esxA GFP-EssB strains using polyclonal anti-GFP antibodies. An unlabeled wild type strain served as a negative control (left). (B) BN-PAGE analysis of DSP-crosslinked membrane fractions of S. aureus expressing complemented FLAG-EssB on a wild type or a ΔfloA background using a monoclonal anti-FLAG antibody (left). The right panel shows a pixel intensity analysis of this BN-PAGE. The top arrow indicates higher molecular weight oligomers of EssB and the bottom arrow, low molecular weight oligomers. (C) Pulldown analysis of FLAG-tagged EssB using FLAG-capture beads. The blots show the eluted fractions of wild type, ΔfloA mutant and a ΔT7SS mutant expressing FLAG-EssB; an unlabeled wild type served as negative control. EsaA and EssC were detected using polyclonal antibodies, EssA-MARS was detected using a polyclonal antibody to the mCherry protein. Immunoblot of CoIP elution fractions (Co-immunoprecipitation) (left) and of the input membrane fractions (right). Besides full-length EsaA, several fragments were detected, but only one co-eluted with FLAG-tagged EssB in the pull-down experiment. (D) Bacterial three-hybrid assay to study EssB interaction with EssA, alone or with flotillin on a third plasmid (pSEVA). Quantification of T25-EssB and EssA-T18 interactions were assayed with β-galactosidase activity assay using empty plasmid (pSEVA641), plasmid bearing flotillin (pSEVA641-floA) or no pSEVA plasmid (-).

https://doi.org/10.1371/journal.ppat.1006728.g004

EssB oligomerizes in vitro and forms conditional dimers [39–41], although in vivo homo- or hetero-oligomerization has not been detected [52]. We thus tested whether FloA scaffold activity affects EssB oligomerization directly. FLAG-EssB-labeled S. aureus was cultured to stationary growth phase and membrane protein extracts (0.25% DDM, 4°C) (S5A Fig) were resolved by 3-12% gradient Blue-Native (BN)-PAGE, which allows separation of membrane protein complexes into their natural oligomeric states (30–10,000 kDa) [61–63]. FLAG-EssB was immunodetected using specific anti-FLAG antibodies. We observed a signal attributable to EssB at >250 kDa, which indicates that EssB forms stable oligomers in vivo (S5B Fig). Protein samples crosslinked using DSP (dithiobis(succinimidyl propionate)) prior to cell lysis showed EssB-containing oligomers >1 MDa (S5B Fig), which indicated the existence of large EssB-containing complexes in S. aureus cells. We applied this experimental approach using anti-FLAG epitope antibodies to compare FLAG-EssB-labeled wild type and ΔfloA mutant membrane fractions (Fig 4B). Wild type cells usually showed two signals that corresponded to distinct EssB oligomeric states (>1 MDa), whereas ΔfloA mutant showed one >1 MDa signal that did not coincide with either wild type band (Fig 4B). In addition to these differences, we detected an intensity increase in a low molecular weight signal (~250 kDa) in the ΔfloA mutant compared to the wild type strain (Fig 4B, right, bottom arrow). These results suggest an important role for FloA in formation of EssB-containing high molecular weight complexes in S. aureus.

To ascertain whether these very large protein complexes are EssB homo-oligomers or are constituted by other T7SS proteins, we analyzed FLAG-EssB in a pulldown analysis using FLAG-capture beads. Beads captured FLAG-EssB as well as EssB-associated proteins, which were eluted, resolved in SDS-PAGE, and immunoblotted, which indicated EssA, EssC and an EsaA fragment (Fig 4C). The polyclonal EsaA antibody did not detect full-length EsaA in the pull-down assay, but showed fragmented EsaA, which is also detected in membrane fractions. Similar fragmentation is reported for the B. subtilis EsaA homolog [64]. The wild type and ΔfloA eluted fractions showed EssC and the EsaA fragment in comparable amounts, but no EssA was detected in the ΔfloA fraction (Fig 4C). These data suggest that flotillin mediates the EssB-EssA interaction.

To better understand the flotillin effect on the EssB-EssA interaction, we used a bacterial three-hybrid assay [25,57] to measure EssB-EssA oligomerization efficiency, alone or with FloA (Fig 4D). A bacterial two-hybrid assay in which EssB and EssA were tagged respectively with the T25 and T18 catalytic domains, was complemented with a modular vector [65] that expressed floA. Whereas there was no EssB-EssA interaction in the absence of FloA, the EssB-EssA interaction signal was increased when FloA was present (Fig 4D). FloA did not affect the EssB interaction with other T7SS membrane proteins (EsaA or EssC) (S6 Fig), which implied that FloA scaffold activity is specific to the EssB-EssA interaction. This finding indicates the importance of FloA expression in T7SS protein-protein interactions and supports the hypothesis that flotillin acts as a scaffold to promote EssB-EssA interaction.

To determine whether this FloA activity is sufficient to promote T7SS protein oligomerization, we genetically engineered an orthogonal T7SS system in E. coli, in which the T7SS membrane proteins EsaA, EssA, EssB and EssC were isolated from their native complex oligomerization network, and thus free from interference by potential staphylococcal oligomerization inputs. EsaA, EssA, EssB and EssC proteins were expressed alone (-FloA) or in the presence of FloA (+FloA). Solubilized membrane fractions were purified, proteins extracted and their oligomerization states identified by BN-PAGE and immunoblotting using anti-EssB antibody. In the absence of FloA (-FloA), EssB signals were observed in the 200-400 kDa range (Fig 5A), similar to EssB oligomers detected in uncrosslinked staphylococcal membranes (see Fig 4B and S5B Fig). In the presence of FloA (+FloA), EssB oligomeric species shifted towards higher molecular weight complexes (Fig 5A), indicating that FloA affects EssB oligomerization and potentially other T7SS complex components in the orthogonal biosystem.

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Fig 5. Effect of FloA on reconstituted T7SS in E. coli.

(A) BN-PAGE and immunoblot analysis of solubilized E. coli membranes expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone or with FloA. EssB was detected using polyclonal antibodies. (B) Size exclusion chromatography on a Superose 6 column with solubilized membrane fractions expressing structural T7SS proteins EsaA, EssA, EssB and EssC alone (- FloA) or with FloA (+FloA). The fractions corresponding to the elution volumes at 8–21 ml were separated in SDS-PAGE and detected by immunoblotting with polyclonal anti-FloA, -EsaA, -EssB or -EssC antibodies.

https://doi.org/10.1371/journal.ppat.1006728.g005

To ascertain whether FloA exclusively affects EssB oligomerization or influences oligomerization of additional T7SS proteins in this system, we purified E. coli membrane fractions, extracted proteins and identified their oligomerization states by size-exclusion chromatography. We used immunoblot to analyze 1 ml fractions of the column volumes in an 8–21 ml range to detect FloA, EsaA, EssB and EssC (Fig 5B). In -FloA fractions, EsaA, EssB and EssC signals were detected in later-eluting fractions of 10–16 ml, corresponding to a 1-0.4 MDa range. In +FloA samples, the signal was detected in early-eluting fractions and concentrated in the 9 ml fraction, which corresponds to high molecular weight complexes >1 MDa (Fig 5B). These results suggest that flotillin affects T7SS membrane protein oligomerization in this orthogonal system, and further highlights the importance of this scaffold protein for S. aureus T7SS.

The T7SS secretion defect of ΔfloA is relevant during an infection

Targeting of flotillin scaffold activity could be an appropriate strategy for fighting bacterial infection by perturbing oligomerization of multiple virulence-related protein complexes such as T7SS. In mice, the T7SS Esx substrates participate in formation of persistent abscesses, probably due to the virulence of EsxA, EsxB, EsxC and EsxD secreted proteins [31,32,37,38,66]. In addition, EsxC is an immunogenic substrate, and kidney abscesses are associated with the generation of anti-EsxC antibodies [32]. In a murine infection model similar to that of Burts et al. [32], we evaluated the link between FloA inactivation and reduced S. aureus virulence mediated by low T7SS activity by measuring EsxC immunoreactivity. Cohorts of 3-week-old BALB/c mice received intravenous injections of sublethal doses of staphylococcal wild type, ΔfloA mutant and ΔT7SS mutant strains. The humoral immune response was boosted by Staphylococcus challenge on days 14 and 28 [67]. After 40 days, mice were sacrificed and serum collected to determine anti-EsxC immunoglobulin titers by indirect ELISA (Fig 6A). We observed significantly lower IgM antibody titers against EsxC in a ΔfloA mutant compared to wild type, consistent with in vitro experiments lowering EsxC secretion in the ΔfloA mutant (Fig 6B). As control, we detected antibodies to the unrelated staphylococcal cell wall protein IsaA [68], which showed that all mice established an S. aureus infection (S7 Fig). These data indicate the importance of FMM integrity in T7SS-mediated virulence phenotypes in an in vivo infection.

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Fig 6. Lack of flotillin lowers EsxC-antibody titers in infected mice.

(A) Scheme of workflow. Mice were challenged three times with sublethal doses of S. aureus (10⁶ CFU) on day 0, 14 and 28. After 40 days, blood samples were collected, serum isolated and used for indirect ELISA. (B) BALB/c mice were challenged as in (A) and IgM titers against EsxC were determined by indirect ELISA. Absorbance corresponds to 1:50 diluted sera. Statistical analysis was carried out using one-way ANOVA (*P<0.05; **P<0.01).

https://doi.org/10.1371/journal.ppat.1006728.g006

Small anti-FMM molecules reduce T7SS activity

To target flotillin activity exogenously and develop alternative strategies against staphylococcal infections, we tested several small molecules known to interfere with FMM organization in S. aureus [59]. The small molecule zaragozic acid (ZA) is a potential inhibitor of flotillin activity [12], as it blocks S. aureus squalene synthase [69], which is needed to produce the polyisoprenoid lipids that stabilize flotillin in the FMM. When bacteria are exposed to micromolar ZA concentrations, flotillin organizes in a smaller number of membrane foci, concomitant with a reduction in its chaperone activity [12]. Similar to ZA, the cholesterol-lowering drug simvastatin used to treat patients with hypercholesterolemia, inhibits the same polyisoprenoid lipid production pathways in S. aureus. Simvastatin is a competitive inhibitor of HMG-CoA reductase, an enzyme upstream of squalene synthase in the constituent lipid biosynthesis pathway [70]. We included the small molecule 5-DSA (5-doxyl-stearic acid), a lipid probe that accumulates in biological membranes and is used to monitor membrane fluidity in lipid raft organization studies in eukaryotic cells [71,72]. In addition, 5-DSA is reported to displace certain membrane lipids and alter the function of various membrane-associated proteins [73,74].

Wild type S. aureus strains were grown in liquid TSB medium with different concentrations of simvastatin, 5-DSA and ZA to define the highest concentrations that did not affect S. aureus growth (20 μM simvastatin, 150 μM 5-DSA, 50 μM ZA; Fig 7A). We used fluorescence microscopy to monitor anti-FMM activity by evaluating FloA subcellular distribution. We quantified the number of fluorescent foci of a FloA-MARS strain grown with simvastatin, 5-DSA or ZA. Most untreated cells showed one, two or three fluorescent foci; occasional cells had no fluorescent foci or had four or more foci. Treatment with 20 μM simvastatin or 50 μM ZA reduced the number of fluorescent FloA foci per cell, and 150 μM 5-DSA treatment showed a drastic reduction in the number of foci per cell (Fig 7B, S8 Fig). The altered subcellular localization of FloA-MARS in the presence of anti-FMM molecules was not a result of decreased flotillin in the cell membrane or its displacement to the cytosol, as shown by immunoblot analysis of fractionated cells after anti-FMM treatment (Fig 7C). We thus suggest that these molecules severely alter FMM organization and probably affect associated processes.

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Fig 7. Anti-FMM molecules affects membrane organization of FloA foci.

(A) Effect on growth of different concentrations of anti-FMM molecules simvastatin (SIM), zaragozic acid (ZA) or 5-doxyl-stearic acid (5-DSA) in TSB medium. (B) Fluorescent foci in FloA-MARS-labeled cells after treatment with 20 μM SIM, 150 μM 5-DSA or 50 μM ZA compared to untreated cells. (C) Immunoblot analysis of membrane and cytosol fractions of wild type cells treated with 20 μM SIM, 150 μM 5-DSA or 50 μM ZA. Untreated cells served as control. Flotillin was detected using polyclonal anti-FloA antibody.

https://doi.org/10.1371/journal.ppat.1006728.g007

To determine whether 5-DSA, simvastatin or ZA also affected T7SS assembly and organization, we repeated BN-PAGE analysis as above. Without crosslinking, the FloA-interacting protein EssB only formed oligomers at ~250 kDa; high molecular weight protein complexes were not detected (Fig 8A, left). DSP treatment before cell lysis allowed stabilization of high molecular weight complexes, and 5-DSA, simvastatin, and ZA treatment led to a decrease in the high molecular weight signal in wild type cells (Fig 8A, center). This was consistent with the increase in low molecular weight species detected in 5-DSA-, simvastatin-, or ZA-treated samples compared to wild type (Fig 8A, right, bottom arrow). BN-PAGE analysis thus suggested that 5-DSA, simvastatin, or ZA treatments interfere with correct T7SS assembly or organization, probably by inhibiting FloA scaffolding activity.

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Fig 8. Anti-FMM molecules interfere with T7SS complex formation and can inhibit secretion of T7SS substrates in vitro and in vivo.

(A) Effect of anti-FMM molecules on T7SS complex formation. BN-PAGE analysis of untreated vs. DSP-crosslinked membrane fractions of S. aureus cells grown to stationary phase in the presence of 20 μM SIM, 150 μM 5-DSA or 50 μM ZA. Pixel intensity analysis of DSP-crosslinked samples are shown (right). (B) Effect of anti-FMM molecules on secretion of T7SS substrates. Cells were grown to the end of exponential growth phase in the presence of 20 μM SIM, 150 μM 5-DSA or 50 μM ZA. Precipitated supernatant (SN) and corresponding cell extracts (CE) were separated in SDS-PAGE and tested in immunoblot with anti-FLAG or -EsxC antibodies. Recombinant YtnP was added to supernatants as loading control and probed with anti-YtnP antibodies; for cell extracts anti-GroEL was used as a loading control. (C) Indirect ELISA to study the ZA effect on antibody response to EsxA-D. Zaragozic acid was administered to BALB/c mice by intraperitoneal injection, followed by challenge with a sublethal dose of staphylococci. The procedure was repeated on days 14 and 28 to boost the antibody response. Graphs show IgM antibody titers to EsxA-D in untreated vs. treated mice. Statistical analysis was performed using an unpaired t-test (*P<0.05; **P<0.01).

https://doi.org/10.1371/journal.ppat.1006728.g008

We tested whether 5-DSA, simvastatin or ZA treatments also inhibit T7SS secretion in vitro. To define this, we monitored secretion of the T7SS substrates EsxA, EsxB and EsxC in stationary phase cultures. Concentrated proteins from supernatants of untreated and simvastatin-, 5-DSA- and ZA-treated cultures were resolved by SDS-PAGE and analyzed in immunoblot. Simvastatin-, 5-DSA- and ZA-treated cultures showed a notable reduction in secreted EsxA, EsxB and EsxC, particularly marked in ZA-treated cells, for which no signals were detected (Fig 8B). To determine the localization of the T7SS substrates, we analyzed cell extracts by immunoblot, which showed EsxA, EsxB and EsxC signals in the cytoplasmic fraction of treated cultures. Simvastatin, 5-DSA and ZA in S. aureus cultures thus compromised T7SS activity and reduced T7SS substrate secretion (Fig 8B). While treatment of S. aureus cultures probably affects T7SS organization and assembly, simvastatin, 5-DSA or ZA addition did not alter EsaA, EssB or EssC concentrations in cell extracts (S9 Fig).

The ZA inhibitory effect on T7SS activity via FMM perturbation led us to test ZA-mediated T7SS inhibition in vivo in the murine infection model. We administered 20 mg/kg ZA to a cohort of BALB/c mice via intraperitoneal injection, followed by challenge with sublethal doses of S. aureus. This procedure was repeated twice (days 14 and 28); after 40 days mice were sacrificed and serum was collected for ELISA determination of immunoglobulin titers to EsxA, EsxB, EsxC and EsxD substrates. IgM antibody titers against all T7SS Esx substrates decreased for all T7SS Esx substrates, which was statistically significant for EsxB, EsxC and EsxD compared to levels in infected untreated mice (Fig 8C). These results indicate that ZA inhibits secretion of T7SS-related substrates in vivo, probably by reducing secretion efficiency. This observation makes ZA an attractive molecule for development as an alternative antimicrobial therapy in S. aureus infections.

Strains, media and culture conditions

Strains used in this study were Staphylococcus aureus RN4220 [29] and S. aureus USA300_TCH1516 [83]. Escherichia coli DH5α and XL-1 Blue were used for cloning, and BL-21 Gold for recombinant protein expression. E. coli strains and S. aureus were propagated on LB and TSB media, respectively. Selective LB plates for E. coli were prepared using ampicillin (100 μg/ml), chloramphenicol (25 μg/ml), kanamycin (50 μg/ml) and gentamycin (2 μg/ml). S. aureus was selected on TSB plates containing spectinomycin (600 μg/ml) or erythromycin (2 μg/ml for RN4220 isolates and 100 μg/ml for the USA300_TCH1516 isolate, which bears an erythromycin resistance gene). For blue/white screenings, X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was added to the plates (final concentration 50 μg/ml). If proteins were controlled by inducible promoters and induction was required, IPTG (isopropyl-β-thiogalactopyranoside) or xylose was added at 1 mM or 1% (v/v), respectively. The anti-FMM molecules 5-doxyl-stearic acid and simvastatin were purchased from Sigma-Aldrich and zaragozic acid from Santa Cruz. Simvastatin and zaragozic acid were dissolved in DMSO to a 10 mg/ml stock solution and 5-doxyl-stearic acid was dissolved in methanol to 75 mM stock solution. Compounds were added to cultures at specified concentrations.

Strain construction

All strains and plasmids were generated according to standard molecular biology techniques [84]. A complete list of strains, plasmids and primers used can be found in S2 and S3 Tables. For protein tagging (with GFPmut2, RFPMars or FLAG-Tag), the CDS of the protein of interest was fused to the respective tag via LFH (long flanking homology)-PCR [85]; the PCR fragment was subsequently cloned into target plasmids using suitable restriction enzymes. If the native promoter was required, a 500 bp fragment upstream of the corresponding operon was fused upstream to the CDS by LFH-PCR. For gene deletion and ectopic gene expression in S. aureus, we used the pMAD plasmid and its derivatives pAmy and pLac [86,87]. pMAD plasmids were constructed and maintained in E. coli and then transformed in S. aureus RN4220 via electroporation. After successful integration of the entire plasmid into the genome (first recombination), constructs were shuttled to USA300 via ϕ11-phage transduction and selected for blue color and erythromycin resistance. To eliminate the pMAD plasmid backbone (containing the erythromycin resistance gene and β-galactosidase), strains were grown at 42°C, plated on TSB X-Gal and white colonies were screened for loss of plasmid and presence of target construct by colony PCR (second recombination).

The flotillin deletion mutant (ΔfloA::spc) was constructed previously [59] and transferred into the USA300_TCH1516 isolate by ϕ11-phage transduction. The MARS-fluorescent protein fusion to FloA was generated by replacing the CDS of floA-yfp with floA-mars in a pCel plasmid created previously [59].

For analysis of protein-protein interaction via bacterial two- and three-hybrid systems [25,57,58], the coding sequences of the corresponding gene without the stop codon were cloned in-frame into each of the bacterial two-hybrid vectors (pKT25, pKNT25, pUT18, pUT18C). For bacterial three-hybrid analysis, the CDS of flotillin controlled by an IPTG-inducible P_lac promoter was introduced into plasmid pSEVA631 of the SEVA system [59,65].

For recombinant expression of EsxA, EsxB, EsxC and EsxD, we used the plasmid pET20b(+), which allows tagging with a hexahistidine tag for purification. The plasmid also contains a periplasm-targeting sequence that was removed by cloning the inserts in-frame with NdeI and XhoI. For recombinant expression of FloA and EssB, the CDS were cloned into pASK-IBA3 using reverse PCR to linearize the vector bearing a C-terminal StrepII-tag. The construct to express recombinant T7SS structural genes EsaA, EssA, EssB and EssC was from GenScript. The genes are codon-optimized for E. coli and contain a ribosome-binding site between each gene to ensure translation of all components. The esaAessABC-CDS was cloned into the pBAD-HisB vector by in-fusion PCR, excluding the hexahistidine-tag encoded in the vector.

EssB antibody production

For antibody production, pASK-IBA3C-essB was transformed in E. coli TOP10 (Life Technologies) and a single colony used to inoculate an overnight pre-culture. The pre-culture was diluted 1:100 and grown at 37°C to an optical density of OD₆₀₀ = 0.4–0.6. EssB was expressed by adding 2.5 mM anhydrotetracycline (24 h, 18°C). Cells were harvested by centrifugation (6000 xg, 20 min, 4°C) and resuspended in 300 mM NaCl, 50 mM Tris pH 8.0. Cells were lysed by two rounds of French press-mediated lysis (10,000 psi) and the membrane fraction of cleared lysate was collected by ultracentrifugation (185,000 xg, 1 h, 4°C). Membrane proteins were then extracted in 300 mM NaCl, 50 mM Tris pH 8.0, 1% DDM (1 h, 4°C). Insoluble proteins were removed by ultracentrifugation (185,000 xg, 1 h, 4°C). Membrane proteins were loaded on a pre-equilibrated 2x 1 ml StrepTrap HP column (GE Healthcare) and eluted with 2.5 mM desthiobiotin. Peak fractions were concentrated with a 10 kDa concentrator and further purified on an S200 size exclusion chromatography column. For antibody generation, protein was diluted in 1x PBS + 0.05% DDM (final concentration 0.3 mg/ml). Rabbit immunization and antibody production were performed by ImmunoGlobe GmbH (Himmelstadt, Germany).

LFQ protein quantification by mass spectrometry

For label-free quantification, protein samples were reduced in 1x Laemmli buffer (BioRad) containing 50 mM dithiothreitol (DTT, Thermo Scientific; 5 min, 95°C). Proteins were alkylated with 120 mM iodoacetamide (20 min, room temperature, light-protected) then precipitated and washed three times in ice-cold acetone. Protein pellets were dissolved in 100 mM ammonium bicarbonate containing 0.5% (w/v) sodium deoxycholate (Sigma-Aldrich). Digestions were performed with the lysyl endopeptidase LysC (Wako; 1 h, 30°C), followed by treatment with trypsin (Promega; overnight, 37°C). Sodium deoxycholate was removed by ethylacetate extraction [88] and samples were dried using a vacuum concentrator. Peptides were desalted using C18 stage tips with five C18 Empore SPE disks (3M) and eluted with 60% (v/v) acetonitrile/0.1% (v/v) formic acid. NanoLC-MS/MS analyses were performed on an Orbitrap Fusion instrument equipped with an EASY-Spray Ion Source coupled to an EASY-nLC 1000 (Thermo Scientific). Peptides were loaded on a trapping column (2 cm x 75 μm ID, PepMap C18, 3 μm particles, 100 Å pore size) and separated on an EASY-Spray column (25 cm x 75 μm ID, PepMap C18, 2 μm particles, 100 Å pore size) with a 120 min linear gradient from 3 to 32% acetonitrile and 0.1% formic acid. MS scans were acquired in the Orbitrap analyzer at 120,000 resolution at m/z 200. Data-dependent MS/MS scans were measured by a Top Speed method (cycle time 3 s in the ion trap analyzer, with rapid scan rate and HCD fragmentation with 35% normalized collision). Dynamic exclusion was applied for 60 s; isotopes, singly charged precursors and charge states >7 were excluded from selection. Minimum signal threshold for precursor selection was 1 x 10⁴. Predictive AGC was used with a target value of 2 x 10⁵ for MS scans. For MS/MS scans, recommended universal method settings were applied (AGC target 3 x 10³, max. injection time 0.25 s, injection of ions for all available parallelizable time). EASY-IC was used for internal calibration. Data analysis was performed using MaxQuant v1.5.3.30.

Bacterial two/three-hybrid assays

Bacterial two- and three-hybrid assays were performed using a kit (Euromedex) and screened as reported [25,57]. Briefly, plasmids were transformed in the BTH101 strain and selected on LB plates containing ampicillin and kanamycin. Single colonies were picked and grown at 30°C in liquid LB medium with antibiotics. After overnight growth, 2 μl were spotted on LB plates with ampicillin, kanamycin, 0.5 mM IPTG and 40 μg/ml X-Gal and incubated (48 h, 30°C). For quantification of interactions, single transformants were picked and grown in 1 ml LB medium (48 h, 30°C) with antibiotics and inducer (0.5 mM IPTG), and β-galactosidase activity was determined in Miller Units, as described [89].

Recombinant protein expression and size exclusion chromatography

For recombinant expression of Esx-proteins (EsxA-EsxD) and YtnP, the pET20b(+) vector containing the respective His-tagged protein was transformed in E. coli BL-21 DE3 Gold (Stratagene). Cells were grown to an optical density of OD₆₀₀ = ~0.6 and expression of recombinant proteins was induced with 1 mM IPTG (4–5 h, 37°C). Cell pellets were resuspended in 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol, 1% (v/v) Tween-20 and 0.2 μg/ml lysozyme (Sigma-Aldrich; 10 min, 37°C), then lysed mechanically in a fast-prep shaker (two times, 45 s each, 6.5 m/s) and lysate was cleared by centrifugation (10,000 xg, 10 min, 4°C). Lysates were mixed with pre-equilibrated Ni-NTA resin (Qiagen) and incubated (30 min, 4°C with mild agitation). The resin was washed twice in 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% (v/v) glycerol and 1 mM PMSF (phenylmethylsulfonylfluoride; Sigma-Aldrich) with increasing imidazole concentrations from 20 to 50 mM. His-tagged proteins were eluted from the resin with 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 M imidazole, 10% (v/v) glycerol and 1 mM PMSF. Imidazole was removed using PD-10 desalting columns (GE Healthcare). Proteins were supplemented with 20% (v/v) glycerol and stored at -80°C.

pBAD-esaAessABC was transformed in E. coli One Shot TOP10 expression strain (Life-Technologies) and if necessary, pASK-IBA3C-floA was co-transformed. Cells were grown at 37°C in liquid LB medium to an optical density of OD₆₀₀ = ~0.8 and expression induced with 0.2% (w/v) L-arabinose (for pBAD plasmid) and 0.2 μg/ml anhydrotetracycline (for pASK-IBA3C) (24 h, 18°C). Cell pellets were resuspended in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM MgCl₂ and 1 mM DTT and lysed in a French-press (3 cycles at 10,000 psi). The lysate was clarified by centrifugation (10,000 xg, 20 min, 4°C) and membrane fractions collected by ultracentrifugation (185,000 xg, 1 h, 4°C). Membranes were homogenized and solubilized in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.25% (w/v) n-tetradecyl β-D-maltoside (TeDM) (Anatrace). Membrane proteins were clarified by ultracentrifugation (100,000 xg 1 h, 4°C). The supernatant was loaded onto a Superose 6 10/300 column (GE Healthcare) equilibrated with 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM MgCl₂ and 0.00002% (w/v) TeDM. Eluted fractions were collected and analyzed by western blot.

Sample preparation for SDS-PAGE and immunoblot analysis

For analysis of whole cell extracts by immunoblot, cells were grown to the stated growth phase. Cells equivalent to 1 ml at optical density OD₆₀₀ = 1.5 were collected by centrifugation (9000 xg, 3 min) and suspended in 50 μl lysis buffer (10 mM EDTA, 50 mM Tris-HCl pH 7.5) supplemented with 50 μg/ml lysostaphin (Ambi Products) and 1 mM PMSF. After incubation (37°C, 30 min), 50 μl 2x Laemmli buffer were added and samples boiled (10 min, 95°C). Depending on protein, 5–20 μl were loaded on an SDS-PAGE gel for subsequent immunoblot analysis.

Culture supernatants were filter-sterilized with 0.25 μm syringe filters and precipitated with 5% trichloroacetic acid (overnight, 4°C), washed with ice-cold acetone, and resuspended in 1x Laemmli buffer. Samples were boiled (10 min, 95°C) and proteins equivalent to 0.4 ml (for YtnP detection), 0.6 ml (for EsxC) or 1.6 ml culture supernatant (for FLAG) were resolved on 18% SDS-PAGE gels with a Tris-Tricine buffer system.

Western blot was performed using standard protocols for semi-dry and wet-blot methods. Proteins were transferred to a PVDF membrane, blocked with 5% (w/v) non-fat dried milk powder in TBS-T, incubated with primary antibodies overnight, followed by 1 h incubation with secondary antibodies. Antibodies were used as follows: anti-mCherry (1:5000; BioVision), -GFP (1:5000; Takara), -FLAG (1:1000; Sigma-Aldrich), -EsxC (1:2000; [33]), -EssC (1:10,000; [33]), -EsaA (1:10,000; [33]), -EssB (1:4000), -YtnP (1:1000; [60]), -GroEL (1:5000, Sigma-Aldrich), -FloA (1:10000; [55]), chicken IgY-HRP (1:2,500; Life Technologies), -mouse IgG-HRP (1:10,000; Life Technologies) and -rabbit IgG-HRP (1:20,000; Bio-Rad). Polyclonal antibodies to EsxC, EsaA and EssC were a kind gift of Tracy Palmer (School of Life Science, University of Dundee, Scotland). Image processing was performed using ImageJ software [90]. Signals were quantified with the ImageJ Gel-Analyzing tool.

Isolation of crude membrane fraction of S. aureus

For isolation of the crude membrane fraction, cells were grown in 50 ml liquid TSB medium overnight with vigorous agitation. Cells were collected by centrifugation (4000 xg, 15 min) and washed in PBS. If required, cells were chemically crosslinked using amine-reactive crosslinker DSP (dithiobis(succinimidyl propionate); Thermo Scientific) prior to lysis. For crosslinking, cells were resuspended in 10 ml PBS supplemented with 1 mM DSP and incubated on ice for 2 h. Crosslinking reaction was quenched by adding Tris-HCl pH 7.5 to a final concentration of 20 mM (on ice, 15 min). Cells were then collected and resuspended in 10 ml PBS lysis buffer (PBS, 250 mM sucrose, 1 mM EDTA) supplemented with 50 μg/ml lysostaphin, 1 mM PMSF followed by incubation (37°C, 10 min). Cells were lysed mechanically with glass beads in a fast-prep shaker (twice 45 s each with 6.5 m/s); debris and unbroken cells were removed by centrifugation (11,000 xg, 10 min, 4°C). The supernatant containing cytosolic and membrane proteins was ultracentrifuged (100,000 xg, 1 h, 4°C). Pelleted membrane proteins were resuspended in PBS + 250 mM sucrose and 10% (v/v) glycerol, flash-frozen in liquid nitrogen and stored at -80°C.

DRM-DSM isolation

To isolate detergent-resistant and -sensitive membrane (DRM and DSM) fractions, we used the Cellytic MEM protein extraction kit (Sigma-Aldrich) according to manufacturer’s protocols with minor modifications. Briefly, the crude membrane fraction was isolated as above and 1 μg total protein was mixed with kit lysis and separation buffer. After separating DRM from DSM proteins, DRM was washed three times and fractions precipitated with acetone, air-dried (5 min), suspended in 200 μl Laemmli buffer, and 10-20 μl loaded on an SDS-PAGE gel.

Co-immunoprecipitation (Ni-NTA, FLAG capture)

His- or FLAG-tagged proteins were co-immunoprecipitated with Ni-NTA resin (Qiagen) or M2 FLAG-capture beads (Sigma-Aldrich), respectively. For immunoprecipitation with His-tagged FloA, crude membrane fractions were isolated as above, solubilized in His-binding buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol, 1% (v/v) Tween-20) containing 0.25% DDM. Insoluble material was removed by centrifugation (100,000 xg, 1 h, 4°C). Solubilized protein (1 mg) was incubated with 150 μl Ni-NTA resin and rotated (2 h, 4°C), washed twice in buffer W1 (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol), twice in buffer W2 (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 50 mM imidazole, 10% (v/v) glycerol), and eluted with 200 μl elution buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 M imidazole, 10% (v/v) glycerol). The eluted fraction was concentrated by TCA precipitation before SDS-PAGE and immunoblot.

To capture FLAG-tagged proteins, crude membrane fractions were solubilized in 50 mM Tris-HCl pH 7.5, 50 mM NaCl supplemented with 0.25% DDM. After removal of insoluble material (100,000 xg, 1 h, 4°C), a total of 4 mg protein was mixed with 20 μl pre-equilibrated beads. After rotation (2 h, 4°C), beads were washed four times in 50 mM Tris-HCl pH 7.5, 50 mM NaCl with decreasing amounts of detergent (two washes with 0.125% DDM, two washes with 0.02% DDM). Captured FLAG-tagged proteins were eluted by boiling beads in 50 μl Laemmli buffer, followed by SDS-PAGE and immunoblot.

BN-PAGE

Blue Native PAGE (BN-PAGE) was performed with the Novex NativePAGE Bis-Tris system (Life Technologies), used according to manufacturer’s protocols with minor modification. Briefly, crude membrane fractions were isolated as described above and solubilized (overnight, 4°C) in 1x Native PAGE sample buffer with 0.25% DDM. Insoluble material was removed by centrifugation (20,000 xg, 30 min, 4°C) and 150 μg solubilized membranes were loaded on 3–12% gradient gels. After electrophoresis and blotting, PVDF membranes were fixed (15 min, 8% acetic acid), air-dried and rewetted in methanol before blocking and antibody incubation.

Microscopy

For microscopy analysis, cells were harvested from liquid TSB culture, washed twice with PBS and fixed with 4% paraformaldehyde (5 min). After two additional washes in PBS, cells were spotted on an agarose pad (0.8% agarose in PBS) to immobilize them for image acquisition. Epifluorescence microscopy was performed using a Leica DMI6000B microscope, equipped with a Leica CRT 60000 illumination system and DFC630FX camera. Raw images were processed and deconvoluted using image processing software Leica LAS AF v3.7.

For super-resolution stimulated emission depletion (STED) microscopy, immunofluorescent staining of the samples was guided by [91]. Δspa mutant cells were grown to mid-exponential phase and fixed with 4% paraformaldehyde for 30 min. After washing with PBS, cells were resuspended in GTE buffer (50 mM glucose, 20 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8) and mounted on 0.1% poly-L-lysine-coated coverslips. Samples were treated with 10 μg/ml lysostaphin in GTE for 5 min, washed with PBS and incubated in 70% EtOH for 10 min. Coverslips were air dried and blocked with 5% BSA for 30 min. Blocking solution was removed and the primary antibody (anti-EssB 1:25 and anti-FloA 1:100) was incubated overnight in 0.5% BSA at 4°C. For each experiment a negative control was incubated without the primary antibody. Coverslips were washed with PBS and the Alexa conjugated secondary antibody was incubated for 2 h in the dark at room temperature (anti-rabbit-Alexa546 1:250, anti-chicken-Alexa488 1:500, ThermoFisher). After washing with PBS, coverslips were air dried and mounted on microscope slides with 3 μl of Prolong Gold Antifade Mountant (ThermoFisher). Slides were incubated in the dark overnight and images taken with a Leica SP8 TCS STED 3X system. Dual channel images were obtained exciting Alexa 546 and Alexa 488 at 561 nm and 488 nm respectively, with a pulsed white light laser depleting at 660 nm and 592 nm, respectively. Deconvolution of z-stacks was obtained with the software Huygens Professional and colocalization analysis was performed with the Costes’ threshold function of the JACoP Plugin for ImageJ [78,92].

Animal experiments and ELISA

Cohorts of 3-week-old BALB/c mice (n = 6) were infected intravenously with 100 μl of suspension containing 10⁶ CFU staphylococci in PBS. For anti-FMM treatment, 20 mg/kg zaragozic acid (in PBS) was administered intraperitoneally 30 min prior to challenge with staphylococci. The procedure was repeated twice (days 14 and 28). Mice were sacrificed after 40 days and blood samples collected by cardiac puncture. Serum EsxA-D and IsaA antibody titers were examined by ELISA. Briefly, recombinant His-tagged Esx proteins and IsaA were purified as above, diluted in PBS to 10 μg/ml final concentration and coated on 96-well plates (Nunc, MaxiSorp). Plates were incubated (overnight, 4°C) and blocked with 5% BSA (bovine serum albumin) in PBS. Serum samples were diluted (1:50 in PBS), incubated on plates (1 h, 37°C); plates were washed with PBS-T (PBS + 0.05% Tween) and incubated with anti-mouse IgM antibodies (1:5000; Life Technologies; 1 h, 37°C). After a final wash with PBS-T, 100 μl TMB (3,3’,5,5’-tetramethylbenzidine; Life Technologies) was used to develop the reaction, which was terminated with 100 μl 1 N NaOH and absorbance measured at 450 nm.

Ethics statement

All experimental animal studies were approved by the Committee on the Ethics of Animal Experiments of the government of Lower Franconia (55.2-2532-2-57) and were in strict accordance with the guidelines for animal care and experimentation of German Animal Protection Law and EU Directive 2010/63/EU. Mice were housed in cages in standardized lighting conditions and had ad libitum access to food and water. All efforts were made to minimize suffering and animals were sacrificed at the end of the experiment by CO₂ inhalation.

Statistical methods

Statistical analysis was performed with GraphPad Prism (GraphPad software, version 7) using appropriate statistical methods as indicated in the figure legends. P-values ≤0.05 were considered significant. Pairwise comparisons were assessed using unpaired Student’s t-test. Analysis of variance (ANOVA) was performed to determine whether a group of means was significantly different from each other.