Author response: Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF
2021-12-20

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. Multiple peptide resistance factor (MprF) is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to antimicrobial agents and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding of bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin, and it impaired MRSA survival in human phagocytes. Thus, MprF inhibitors are recommended for new antivirulence approaches against MRSA and other bacterial pathogens. Editor's evaluation This study uses an innovative anti-virulence approach based on monoclonal antibodies that target the Staphylococcus aureus lipid flippase involved in tolerance to cationic peptides. The authors show that this strategy re-sensitizes antibiotic-resistant S. aureus and serves as a proof of principle for anti-virulence approaches to target bacterial infections. https://doi.org/10.7554/eLife.66376.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The continuous increase of antibiotic resistance rates undermines the significance and efficacy of available antibiotics against bacterial infections (Årdal et al., 2020). Several opportunistic antibiotic-resistant bacterial pathogens including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and extended-spectrum beta-lactam or carbapenem-resistant proteobacteria impose a continuously growing pressure on modern healthcare systems (Tacconelli et al., 2018). MRSA is responsible for a large percentage of superficial and severe bacterial infections and the available last-resort antibiotics are much less effective than beta-lactams (Lee et al., 2018). Unfortunately, no new class of antibiotics has entered the clinical phase since the introduction of the lipopeptide antibiotic daptomycin in 2003 (Årdal et al., 2020). Novel anti-infective strategies that would circumvent on the one hand the difficulties in identifying new microbiota-preserving small-molecule antimicrobials and, on the other hand, the enormous selection pressures exerted by broad-spectrum antibiotics, are discussed as potential solutions against a looming postantibiotic era (Dickey et al., 2017). Such strategies could be based for instance on therapeutic antibodies or bacteriophages, which usually have only a narrow activity spectrum. A possible direction could be the inhibition of bacterial targets that are of viable importance only during infection (Lakemeyer et al., 2018). Blocking such targets by so-called antivirulence or antifitness drugs would preserve microbiome integrity and create selection pressure for resistance-conferring mutations only on invading pathogens. Interfering with bacterial virulence factors should ameliorate the course of infection and enable more effective bacterial clearance by the immune system or by antibiotics. Monoclonal antibodies (mABs) directed against antivirulence targets could be interesting alternatives provided the target can be reached by comparatively large antibody molecules. Therapeutic mABs are used in several malignant, inflammatory, and viral diseases (Qu et al., 2018; O’Brien et al., 2021) and have proven efficacy in toxin-mediated bacterial infections such as anthrax or clostridial toxin-mediated diseases (Dickey et al., 2017; Migone et al., 2009; Lowy et al., 2010). Apart from toxin neutralization, however, mABs have hardly been applied in antimicrobial development programs. Moreover, in-depth molecular studies are necessary to devise most promising targets for mABs and elucidate if and how mAB binding could disable pathogens to colonize and infect humans. The multiple peptide resistance factor (MprF), a large integral membrane protein, is crucial for the capacity of bacterial pathogens such as S. aureus to resist cationic antimicrobial peptides (CAMPs) of the innate immune system and CAMP-like antibiotics such as daptomycin (Peschel et al., 2001; Ernst and Peschel, 2011; Slavetinsky et al., 2017). MprF is highly conserved and can be found in various Gram-positive or Gram-negative pathogens (Slavetinsky et al., 2017). MprF proteins proved to be crucial for in vivo virulence of various pathogens in infection models (Peschel et al., 2001; Thedieck et al., 2006; Maloney et al., 2009) and when exposed to human phagocytes as a result of increased resistance to phagocyte-derived antimicrobial agents such as CAMPs (Slavetinsky et al., 2017; Kristian et al., 2003). Some parts of the protein are located at the outer surface of the cytoplasmic membrane and could in principle be reached by mABs (Ernst et al., 2009; Ernst et al., 2015). MprF forms oligomers and it is a bifunctional enzyme, which can be separated into two distinct domains (Ernst et al., 2015). The C-terminal domain synthesizes positively charged lysyl-phosphatidylglycerol (LysPG) from a negatively charged phosphatidylglycerol (PG) acceptor and a Lys-tRNA donor substrate, while the N-terminal domain translocates newly synthesized LysPG from the inner to the outer leaflet of the cytoplasmic membrane and thus functions as a phospholipid flippase (Ernst et al., 2009; Roy and Ibba, 2009). The exposure of LysPG at the outer surface of the membrane reduces the affinity for CAMPs and other antimicrobials (Ernst et al., 2009). Notably, mprF is a major hot spot for gain-of-function point mutations that lead to daptomycin resistance, acquired during therapy of S. aureus infections (Ernst et al., 2018). In order to assess the suitability of MprF as a target for antivirulence agents we developed mABs targeting several epitopes of potential extracellular loops of its transmembrane part and analyzed their capacity to bind specifically to S. aureus MprF. We identified a collection of mABs, which did not only bind to but also inhibited the LysPG flippase domain of MprF. Our results suggest that a specific loop between two of the transmembrane segments (TMSs) of MprF is exposed at both sides of the membrane suggesting an unusual, potentially flexible topology of this protein part, which may be involved in LysPG translocation. Accordingly, targeting this loop with a specific mAB inhibited the MprF flippase function, rendered S. aureus susceptible to killing by antimicrobial host peptides and daptomycin, and reduced S. aureus survival when challenged by human CAMP-producing polymorphonuclear leukocytes (PMNs). Results Generation of mABs binding to putative extracellular loops of MprF The hydrophobic part of S. aureus MprF appears to include 14 TMS connected by loops with predicted lengths between 2 and 56 amino acids (Ernst et al., 2015). Several of the loops are located at the outer surface of the cytoplasmic membrane, accessible to mABs (Figure 1A). Peptides representing 4 loops with a minimum length of 13 amino acids were synthesized with N- and C-terminal cysteine residues to allow cyclization (Supplementary file 1a). The peptides corresponded to three loops predicted to be at the outer membrane surface (loops 1, 9, and 13) and loop 7, the location of which has remained ambiguous due to conflicting computational and experimental findings (Ernst et al., 2015). The N-terminal amino groups of cyclized peptides were linked to biotin to facilitate their recovery and detection. The antigen peptides were incubated with MorphoSys’s Human Combinatorial Antibody Library (HuCAL), a phage display library expressing human Fab fragments with highly diverse variable regions at the phage surface (Prassler et al., 2011). Antigen-binding phages were enriched in three iterative rounds of panning in solution and antigen-phage complexes were captured with streptavidin-coated beads. The bound phages were extensively washed to remove unspecifically binding phages, eluted, and propagated in Escherichia coli for a subsequent panning round. Washing steps were prolonged and antigen concentrations reduced from round one to round three to increase stringency and discard antibodies with low specificity and affinity. DNA of the eluted, antigen-specific phages was isolated and subcloned in specific E. coli expression vectors to yield His-tagged fragment antigen-binding (Fab) molecules. 368 individual colonies per antigen were picked and Fab fragments were expressed and purified. A representative selection of 24 unique Fabs against all 4 peptides were converted to human IgG by cloning in an IgG1 expression vector system and expression in human HKB11 cells and IgGs were purified via protein A chromatography, as recently described (Prassler et al., 2011) (see graphical workflow in Figure 1—figure supplement 1). Figure 1 with 2 supplements see all Download asset Open asset Multiple peptide resistance factor (MprF) topology and binding of monoclonal antibodies (mABs) to MprF-expressing S. aureus cells. (A) MprF membrane topology is given according to our recent study (Ernst et al., 2015) showing synthase and flippase domains in gray and black, respectively. Amino acid (aa) positions of transmembrane segment (TMS) and the C-terminal hydrophilic domain are indicated. TMSs from aa 245–265 and aa 278–298 are shown in two alternative positions as computational and experimental results of transmembrane topology have been contradictory (Ernst et al., 2015). Localizations of MprF’s TMS-connecting loops are numbered starting from the N-terminus, antibody-targeted loops are indicated by red circles and antibody symbols. (B) Specific binding of mABs (100 nM) to S. aureus was analyzed by ELISA using SA113 strains deficient in the IgG-binding protein A (Spa) comparing the SA113 spa mutant (Δspa) and spa mprF double knockout mutant (ΔspaΔmprF). The red line indicates the mean intensity measured at A 405 nm (affinity) of the isotype control mAB L-1 bound to S. aureus SA113Δspa. Means and standard error of the mean (SEM) of at least three biological replicates are shown. Significant differences between SA113Δspa and SA113ΔspaΔmprF were calculated by Student’s paired t-test (ns, not significant; *p < 0.05; ***p < 0.0001). The IgGs were analyzed for binding to the corresponding antigen peptides and also to the three noncognate peptides to assess their selectivity, by ELISA with streptavidin-coated microtiter plates (Figure 1—figure supplement 2). Peptide one bound IgGs developed against different antigen peptides indicating that it may bind antibodies with only low selectivity. Antibodies directed against the four targeted MprF loops were selected based on affinity and analyzed for binding to S. aureus SA113 cells expressing or not expressing MprF. Both, the ‘wild-type’ and mprF deletion mutant strains lacked the gene for protein A (spa), which would otherwise unspecifically bind to IgG (Kim et al., 2012). Bacteria were adsorbed to microtiter plates, blocked with bovine serum albumin, and incubated with IgGs, which were then detected with goat antihuman IgG after extensive washing. Antibodies M-C1, M-C7.1, M-C7.3, M-C9.1, M-C13.1, and M-C13.2 bound significantly stronger to MprF-expressing S. aureus (SA113Δspa) compared to MprF-deficient S. aureus (SA113ΔspaΔmprF), while the humanized isotype control mAB L-1 showed no specific binding (Figure 1B). An additional mAB directed against the S. aureus surface protein EbpS served as positive control, showing equal affinity toward SA113Δspa and SA113ΔspaΔmprF (Figure 1B). These findings are in agreement with the location of the loops 1, 9, and 13 at the outer surface of the cytoplasmic membrane, confirming the overall topology of MprF (Figure 1A). Of note, loop 7 between potential TMS 7 and 8 whose location had previously remained controversial was detected by antibodies M-C7.1 and M-C7.3 (Figure 1B) indicating that loop 7 is accessible from the outside. Antibodies M-C1, M-C7.1, M-C9.1, and M-C13.1 showed the strongest binding to MprF. The MprF epitope bound by M-C7.1 is located at both, outer, and inner surface of the cytoplasmic membrane The MprF loop 7 between TMS 7 and 8 bound by M-C7.1 seemed to have an ambiguous position within the cytoplasmic membrane because the flanking TMS have a comparatively low content of hydrophobic amino acids. Accordingly, only some topology analysis algorithms predict its location at the outer surface of the cytoplasmic membrane and a previous experimental topology investigation using a set of translational fusions with enzymes that are active only at intracellular or extracellular location has revealed a preferential location at the inner cytoplasmic membrane surface in E. coli (Ernst et al., 2015). Since our M-C7.1- and M-C7.3-binding experiments indicated accessibility of loop 7 from the outside, we revisited its location in S. aureus with two experimental strategies. To confirm that M-C7.1 has access to its cognate MprF antigen epitope at the outer surface of the cytoplasmic membrane in intact bacterial cells, S. aureus SA113ΔspaΔmprF-expressing MprF (pRB-MprF) was grown in the presence of M-C7.1. Cells were then disrupted, membranes were solubilized by treatment with the mild nonionic detergent n-dodecyl-β-D-maltoside (DDM), and proteins were separated in nondenaturing PAGE gels (BN-PAGE). If M-C7.1 bound before cell disruption and remained tightly attached to MprF, it should shift the MprF bands in Western blots of the blue native gels and the MprF–M-C7.1 complex should be detectable after Western blotting with a human IgG-specific secondary antibody. In order to detect MprF independently of M-C7.1, an MprF variant translationally fused to green-fluorescent protein (MprF-GFP, expressed from plasmid pRB-MprF-GFP), which is detectable by a GFP-specific primary antibody (Ernst et al., 2015), was also used and treated in the same way. To detect potentially nonshifted MprF proteins in the native MprF variant (pRB-MprF), M-C7.1 was used as additional primary antibody. S. aureus SA113ΔspaΔmprF bearing the empty vector (pRB) was used as a negative control. In addition to M-C7.1, all three S. aureus strains were also incubated with the control antibody L-1. S. aureus cells expressing either unmodified MprF or MprF-GFP yielded a protein band migrating at a molecular weight of around 900 kDa when preincubated with M-C7.1 but not with L-1 or in cells with the empty-vector control (pRB) (Figure 2A; Figure 2—figure supplement 2). In contrast, the empty-vector control (pRB) strain showed an unspecific band at 300 kDa when preincubated with M-C7.1 or at 150 kDa when preincubated with L-1 but no MprF-specific band (Figure 2A; Figure 2—figure supplement 2). The 900 kDa band of MprF-GFP was detected by both, M-C7.1 and, with a weaker signal, anti-GFP, confirming the identity of MprF (Figure 2A; Figure 2—figure supplement 2). We could recently show that MprF forms oligomers in the staphylococcal membrane, which were migrating at ca. 300 and 600 kDa (Ernst et al., 2015). Bands migrating at similar heights (ca. 250 and 500 kDa) were detected specifically in the MprF-GFP lanes after preincubation with either M-C7.1 or L-1 (Figure 2A; Figure 2—figure supplement 2) suggesting that they represent the oligomerized but not the mAB-complexed MprF proteins (Ernst et al., 2015). Therefore, the 900 kDa bands probably represent a complex formed by MprF and M-C7.1, which confirms that M-C7.1 specifically binds MprF loop seven in live S. aureus cells. The fact, that M-C7.1 used as primary antibody was not able to detect 250 and 500 kDa bands of noncomplexed MprF proteins while the MprF–M-C7.1 complex can directly be detected via antihuman secondary antibody, suggests that M-C7.1 binding only occurs in the living staphylococcal cell. Of note, the MprF–M-C7.1 complex migrating at around 900 kDa indicates that higher-order MprF multimers were shifted by complex formation with M-C7.1. Figure 2 with 2 supplements see all Download asset Open asset Binding of M-C7.1 to multiple peptide resistance factor (MprF) and membrane localization of the M-C7.1-targeted MprF loop 7. (A) Detection of M-C7.1 binding to MprF. Plasmid-encoded native and green fluorescent protein(GFP)-tagged MprF variants were expressed in S. aureus SA113ΔspaΔmprF and living cells were preincubated with M-C7.1 or the isotype control monoclonal antibody (mAB) L-1 (in order to form MprF–mAB complexes). MprF variants complexed with M-C7.1 or control mAB L-1, respectively, were detected by blue native PAGE followed by Western blotting using two different primary (anti-GFP or M-C7.1) and corresponding secondary antibodies. SA113ΔspaΔmprF expressing the empty vector (pRB) served as negative control. Molecular masses in kDa of marker proteins are given on the left of the blot. Arrows mark both the MprF–M-C7.1 complex at 900 kDa and the MprF oligomers at 250 and 500 kDa, which were previously described (Ernst et al., 2015). (B) Cellular localization of the antigen epitope of M-C7.1 using the substituted cysteine accessibility method (SCAM) for specific loops between the MprF transmembrane segments (TMSs). The substituted cysteine T263C is localized in M-C7.1’s target peptide sequence in MprF. Substitution of A99C served as inside control, substitution of T480C served as outside control (see topology model, part C). S. aureus SA113ΔmprF expressing the empty vector (pRB) and an MprF variant lacking all native cysteines (wild-type [WT] (-Cys)) served as additional negative controls. All MprF variants were plasmid-encoded, FLAG tagged at the C-terminus to allow immunoprecipitation and detection, and were expressed in S. aureus SA113ΔmprF. Substituted extracellular cysteine residues were labeled with Na*-(3-maleimidylpropionyl)-biocytin (MPB) (outer leaflet signal, green in overlay), while labeling of substituted internal cysteine with MPB was performed after the blocking of external cysteines with 4-acetamino-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (inner leaflet signal, green in overlay). MprF was detected via antibody staining by an anti-FLAG antibody (red in overlay). (C) MprF topology showing location and amino acid exchanges of artificial cysteine residues for SCAM detection in green. The position of MprF loop 7 was further investigated by inserting a cysteine residue into the loop and analyzing the capacity of the membrane-impermeable agent Na*-(3-maleimidylpropionyl)-biocytin (MPB) to label cysteines covalently, using the substituted cysteine accessibility method (SCAM) (Bogdanov et al., 2005). MPB treatment of intact S. aureus cells should only lead to labeling of extracellular protein portions while blocking cysteines at the outside with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS), followed by cell homogenization and addition of MPB should allow to label only protein parts at the inner surface of the membrane according to a previously established method in E. coli (Bogdanov et al., 2005). An MprF variant lacking all native cysteine residues was generated to exclude background signals. Cysteine residues in MprF were exchanged against serine or alanine residues to minimize structural or S. aureus MprF cysteines of which is conserved in MprF proteins from other suggesting that they not have The mutant protein was found to be because it the of the S. aureus mprF mutant to daptomycin (Figure 2—figure supplement which on intact MprF synthase and flippase (Ernst et al., 2009). the proteins resistance than the protein, because of less protein or were then into MprF loop seven and, as a control, into the intracellular loop and the extracellular loop the localization of which had been by previous computational and experimental (Ernst et al., Figures and Amino acids for were according to a predicted for by substitution with cysteine using et al., The is based on a both information and structural such as predicted secondary and accessibility to the of amino acid exchanges in a protein sequence et al., The SCAM approach further the topology of intracellular loop 2 and extracellular loop 13 (Figure thereby that the can lead to results in S. Notably, MprF loop 7 was found in both at the inner and outer surface of the This the ambiguous position of loop 7 and suggests that this loop may have some of in the membrane, which may the lipid The also M-C7.1 has the capacity to bind MprF loop 7 at the outer surface of the cytoplasmic mABs binding to putative extracellular loops of MprF S. aureus susceptible to CAMPs MprF resistance to cationic antimicrobials such as the by the negative of the membrane outer surface (Peschel et al., 2001; Ernst et al., 2009). If the mABs would not only bind to but also the of MprF, S. aureus should more susceptible to The mABs with specific binding to MprF and the isotype control mAB L-1 were analyzed for their capacity to increase the of S. aureus SA113 to experiments protein A were used to of unspecific IgG binding to protein Bacteria were grown in the presence of one of the mABs and then incubated with at the followed by of viable bacterial cells. of the MprF-specific mABs targeting loops 7 or 13 increased the of S. aureus SA113Δspa to while the other mABs and the isotype control mAB L-1 had no (Figure mAB M-C7.1 the strongest (Figure and it with in a (Figure supplement 1). was selected for further analysis using the highly and methicillin-resistant S. aureus Figure with 1 supplement see all Download asset Open asset and inhibition of S. aureus by antimicrobial peptides and antibiotics in the presence of M-C7.1. (A) of S. aureus SA113Δspa in the presence of and peptide resistance factor (MprF) antibodies compared to control monoclonal antibody (mAB) L-1. of S. aureus incubated with one of the antibodies and were analyzed after 2 and the negative control mAB was set to (B) of methicillin-resistant S. aureus strain in the presence of and M-C7.1 compared to the isotype control mAB L-1. (C) of in the presence of and M-C7.1 compared to the isotype control mAB L-1. of in the presence of daptomycin and M-C7.1 compared to the isotype control mAB L-1. of strain et al., in the presence of daptomycin and M-C7.1 compared to the isotype control mAB L-1. of SA113 SA113ΔmprF and strain when with compared to control mAB L-1 and to M-C7.1. inhibition of in the presence of 4 and 1 M-C7.1 compared to 1 isotype control mAB L-1. antibodies served as additional negative controls. The standard error of the mean (SEM) of results from at least three biological replicates are shown in of from a representative of three biological for M-C7.1 or other antibodies that were significantly different from for the isotype control mAB L-1 in calculated by Student’s paired t-test are indicated < 0.05; < ***p < 0.0001). all experiments S. aureus strains with intact protein A were used to that the to CAMPs was not by unspecific antibody binding to protein M-C7.1 was found to also increase the of to the human a host defense peptide by and cells and and to daptomycin, a lipopeptide antibiotic in clinical and with CAMPs et al., in addition to (Figure M-C7.1 also increased the antimicrobial activity of daptomycin against the clinical the gain-of-function in MprF et al., Figure Of note, M-C7.1 could daptomycin minimal of both, S. aureus SA113 and the strain (Figure suggesting that M-C7.1 may potentially be able to daptomycin resistance during M-C7.1 but not the isotype control mAB L-1 was able to of in the presence of concentrations of (Figure Thus, mABs specific for extracellular loops of MprF may not only bind to MprF but also its was for several with M-C7.1 at or and with or daptomycin no point mutations in mprF were found suggesting that the MprF segment targeted by M-C7.1 is not to M-C7.1 the flippase of MprF M-C7.1 binds MprF at the between the LysPG synthase and flippase domains (Figure 1). The lipid of SA113 treated with M-C7.1 at concentrations that increased the to or with the isotype control mAB L-1 were compared but showed no differences indicating that the synthase of MprF was not inhibited by M-C7.1 (Figure The flippase activity of MprF the exposure of positively charged LysPG at the outer surface of the cytoplasmic membrane thereby the affinity for the cationic protein (Peschel et al., which binds to negatively charged or for et al., 2009). These proteins have been shown to allow a of in the surface of S. aureus in several previous studies (Ernst et al., 2009; Ernst et al., 2018; Slavetinsky et al., 2012). of SA113 with M-C7.1 at concentrations that increased the to to a increase in the capacity to bind or compared to SA113 treatment or treated with isotype control mAB L-1 (Figure thereby indicating that M-C7.1 the flippase of MprF. is to that the protein of MprF loop 7 and TMSs may a crucial in the process of phospholipid as by the localization of loop 7 on the cytoplasmic and external of the Figure 4 Download asset Open asset M-C7.1 the multiple peptide resistance factor (MprF) lysyl-phosphatidylglycerol (LysPG) flippase but not the LysPG (A) Detection of from S. aureus SA113ΔmprF and treated or not treated with M-C7.1 or the isotype control monoclonal antibody (mAB) L-1. were separated by and with the blue to detect the phosphatidylglycerol (PG) and LysPG of S. aureus and mprF deletion mutant (Slavetinsky et al., 2012). of LysPG in to phospholipid content are given LysPG (B) The of positively charged to MprF LysPG synthase flippase activity while the synthase activity not To assess MprF flippase in the was after with the S. aureus SA113 or with with M-C7.1 or the isotype control mAB L-1 set to SA113ΔmprF with or mAB served as positive control. The standard error of the mean (SEM) of results from three biological replicates are shown. (C) binding to S. aureus SA113 compared to