Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF
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Evaluation Summary:
This study is of interest to readers in the field of Microbiology and the control of microbial infectious diseases. The authors address the challenge of antibiotic resistant bacteria with an innovative anti-virulence approach using monoclonal antibodies against a Staphylococcus aureus lipid flippase involved in tolerance to cationic peptides. The work indicates that this approach could re-sensitize antibiotic resistant S. aureus and diminish the severity of infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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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.
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Reviewer #3 (Public Review):
Slavetinsky and colleagues investigated the capability of monoclonal antibodies (mAb) against MprF, a critical protein of S. aureus, to act as re-sensitizing factors towards resistance strains and as supporting factors for S. aureus killing by human polymorphonuclear leukocytes.
They created 8 mAbs against four different loops of MprF and showed that they were able to bind MprF-expressing S. aureus strains. Two of the mAbs led to significant reduction of S. aureus survival upon exposure with nisin (i.e. a cationic antimicrobial against towards which MprF normally confers resistance). The authors focused on the mAb against loop 7 and showed that it reduced survivals also against two other antimicrobials and, most important, it restored Daptomycin killing of a resistant strain. Moreover, although this mAb did …
Reviewer #3 (Public Review):
Slavetinsky and colleagues investigated the capability of monoclonal antibodies (mAb) against MprF, a critical protein of S. aureus, to act as re-sensitizing factors towards resistance strains and as supporting factors for S. aureus killing by human polymorphonuclear leukocytes.
They created 8 mAbs against four different loops of MprF and showed that they were able to bind MprF-expressing S. aureus strains. Two of the mAbs led to significant reduction of S. aureus survival upon exposure with nisin (i.e. a cationic antimicrobial against towards which MprF normally confers resistance). The authors focused on the mAb against loop 7 and showed that it reduced survivals also against two other antimicrobials and, most important, it restored Daptomycin killing of a resistant strain. Moreover, although this mAb did not increase phagocytosis by leukocites, it decreased the survival of the phagocytized S. aureus cells, most likely by rendering them sensitive towards the cationic antimicrobial peptides.
In parallel, the authors used this mAb to revise the ambiguous location of loop 7 of MprF. They employed two different experiment settings and concluded that this loop might have some degree of mobility in the membrane, which also explain the ambiguity of its location in previous studies. By showing that the mAb against loop 7 act by inhibiting the flippase activity of MprF while leaving the synthase activity intact, they speculated that the mobility of loop 7 might play an important role for LysPG translocation process.
The data support the conclusion of the manuscript and show how promising monoclonal antibody are against staphylococcal infections.
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Reviewer #2 (Public Review):
MprF is a lipid flippase involved in determining bacterial tolerance to cationic peptides of the innate immune system and to antibiotics such as daptomycin. Using Staphylococcus aureus as their model organism, the authors assessed the suitability of MprF as a target for anti-virulence treatments. For this purpose, a series of monoclonal antibodies directed against the extracellular loops of MprF were generated. The antibodies were tested for their ability to bind and inhibit the function of MprF, to sensitize S. aureus towards cationic peptides, and to promote phagocyte killing of S. aureus. Moreover, the antibodies were used to investigate the orientation of one specific loop of the MprF protein.
Strenghts:
The manuscript is well-written and the introduction provides a very good overview of the challenges …
Reviewer #2 (Public Review):
MprF is a lipid flippase involved in determining bacterial tolerance to cationic peptides of the innate immune system and to antibiotics such as daptomycin. Using Staphylococcus aureus as their model organism, the authors assessed the suitability of MprF as a target for anti-virulence treatments. For this purpose, a series of monoclonal antibodies directed against the extracellular loops of MprF were generated. The antibodies were tested for their ability to bind and inhibit the function of MprF, to sensitize S. aureus towards cationic peptides, and to promote phagocyte killing of S. aureus. Moreover, the antibodies were used to investigate the orientation of one specific loop of the MprF protein.
Strenghts:
The manuscript is well-written and the introduction provides a very good overview of the challenges associated with antibiotic resistance, anti-virulence strategies and the MprF protein. The Figures and the Figure legends are easy to follow. The described approach is innovative, and state of the art methods are used throughout the manuscript.
Weaknesses:
There is a discrepancy between the anti-virulence scope as indicated by the title and the introduction, and the actual content of the result section: here, the anti-virulence strategy is only preliminary addressed, and a lot of effort is instead put into determining the orientation of one specific loop of the MprF protein. This needs to be better aligned, and more compelling data are needed to support that MprF has potential for anti-virulence strategy. The conclusions of this paper are mostly well supported, however, additional controls are needed to fully support that the observed effects of the antibodies are mediated via specific binding to MprF.
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Reviewer #1 (Public Review):
Slavetinsky et al., describe the development of monoclonal antibodies targeting the S. aureus MprF lipid flippase, which is responsible for membrane incorporation of the phospholipid lysyl-phosphatidylglycerol (LysPG). Incorporation renders the cell more positively charged and has been associated with increased virulence and resistance of MRSA to antibiotics and host antimicrobial peptides. MprF is a bifunctional protein; the N-terminal region translocates lipids (flippase), and the C-terminal region synthesizes LysPG. Overall, this is an interesting approach with significant potential.
Strengths:
Several epitopes on MprF (three outer loops) were targeted through the synthesis of peptides, which provided a number of antibodies that inhibit the flippase function. The authors identified one specific antibody …
Reviewer #1 (Public Review):
Slavetinsky et al., describe the development of monoclonal antibodies targeting the S. aureus MprF lipid flippase, which is responsible for membrane incorporation of the phospholipid lysyl-phosphatidylglycerol (LysPG). Incorporation renders the cell more positively charged and has been associated with increased virulence and resistance of MRSA to antibiotics and host antimicrobial peptides. MprF is a bifunctional protein; the N-terminal region translocates lipids (flippase), and the C-terminal region synthesizes LysPG. Overall, this is an interesting approach with significant potential.
Strengths:
Several epitopes on MprF (three outer loops) were targeted through the synthesis of peptides, which provided a number of antibodies that inhibit the flippase function. The authors identified one specific antibody (M-C7.1) that was shown to target a loop whose previous location was debatable; thus, these finding indicate the loop can be accessible from the outside of the cell. Antibody binding sensitized MRSA to host peptides and antibiotics (e.g., daptomycin). The antibody was shown to inhibit flippase function and also decreased bacterial survival in phagocytes. Overall, the antibody could be used as an anti-virulence agent, diminishing the severity of S. aureus-associated disease. The emergence of antibiotic resistance and difficult to treat S. aureus infections requires orthogonal therapeutic approaches; as such, the findings of this study could have significant impact.
Weaknesses:
A major emphasis of the study is that the antibody sensitizes S. aureus to host defenses. This reviewer would like to see dose-responses/titrations of the antibody vs the different CAMPs, using standard susceptibility testing methodology. In addition, during the preliminary ELISAs, have the authors established whether the mprF mutant has lower surface adhesion to maxisorp immuno plates? This would be an important control. When studying M-C7.1 mechanism of action, it is unclear why the data is being normalized to L-1 and why unbound cytochrome C is being quantified. It could be more intuitive to assess bound cytochrome C; can the raw data be included rather than normalized data? A control with delta-mprF alone would also be useful for these experiments. When assessing survival in phagocytes, Figure 5 would benefit from a delta-mprF control to compare M-C7.1 efficacy. This figure also requires statistical analysis. Overall, the conclusions of the study could be further strengthened from additional pre-clinical assessment of the antibody.
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Evaluation Summary:
This study is of interest to readers in the field of Microbiology and the control of microbial infectious diseases. The authors address the challenge of antibiotic resistant bacteria with an innovative anti-virulence approach using monoclonal antibodies against a Staphylococcus aureus lipid flippase involved in tolerance to cationic peptides. The work indicates that this approach could re-sensitize antibiotic resistant S. aureus and diminish the severity of infections.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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