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IntroductionMicrobial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization–time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRI-TOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria.MethodsWe constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures.ResultsFor validation, we used 58 sequence-confirmed strains and the CLOSTRI-TOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates.DiscussionWe describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS.

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.

Neutrophil granulocytes act as a first line of defense against pathogenic staphylococci. However, Staphylococcus aureus has a remarkable capacity to survive neutrophil killing, which distinguishes it from the less-pathogenic Staphylococcus epidermidis. Both species release phenol-soluble modulin (PSM) toxins, which activate the neutrophil formyl-peptide receptor 2 (FPR2) to promote neutrophil influx and phagocytosis, and which disrupt neutrophils or their phagosomal membranes at high concentrations. We show here that the neutrophil serine proteases (NSPs) neutrophil elastase, cathepsin G and proteinase 3, which are released into the extracellular space or the phagosome upon neutrophil FPR2 stimulation, effectively degrade PSMs thereby preventing their capacity to activate and destroy neutrophils. Notably, S. aureus, but not S. epidermidis, secretes potent NSP-inhibitory proteins, Eap, EapH1, EapH2, which prevented the degradation of PSMs by NSPs. Accordingly, a S. aureus mutant lacking all three NSP inhibitory proteins was less effective in activating and destroying neutrophils and it survived less well in the presence of neutrophils than the parental strain. We show that Eap proteins promote pathology via PSM-mediated FPR2 activation since murine intraperitoneal infection with the S. aureus parental but not with the NSP inhibitors mutant strain, led to a significantly higher bacterial load in the peritoneum and kidneys of mFpr2-/- compared to wild-type mice. These data demonstrate that NSPs can very effectively detoxify some of the most potent staphylococcal toxins and that the prominent human pathogen S. aureus has developed efficient inhibitors to preserve PSM functions. Preventing PSM degradation during infection represents an important survival strategy to ensure FPR2 activation.

ABSTRACT Daptomycin, a calcium-dependent lipopeptide antibiotic whose full mode of action is still not entirely understood, has become a standard-of-care agent for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Daptomycin-resistant (DAP-R) S. aureus mutants emerge during therapy, featuring isolates which in most cases possess point mutations in the mprF gene. MprF is a bifunctional bacterial resistance protein that synthesizes the positively charged lipid lysyl-phosphatidylglycerol (LysPG) and translocates it subsequently from the inner membrane leaflet to the outer membrane leaflet. This process leads to increased positive S. aureus surface charge and reduces susceptibility to cationic antimicrobial peptides and cationic antibiotics. We characterized the most commonly reported MprF mutations in DAP-R S. aureus strains in a defined genetic background and found that only certain mutations, including the frequently reported T345A single nucleotide polymorphism (SNP), can reproducibly cause daptomycin resistance. Surprisingly, T345A did not alter LysPG synthesis, LysPG translocation, or the S. aureus cell surface charge. MprF-mediated DAP-R relied on a functional flippase domain and was restricted to daptomycin and a related cyclic lipopeptide antibiotic, friulimicin B, suggesting that the mutations modulate specific interactions with these two antibiotics. Notably, the T345A mutation led to weakened intramolecular domain interactions of MprF, suggesting that daptomycin and friulimicin resistance-conferring mutations may alter the substrate range of the MprF flippase to directly translocate these lipopeptide antibiotics or other membrane components with crucial roles in the activity of these antimicrobials. Our study points to a new mechanism used by S. aureus to resist calcium-dependent lipopeptide antibiotics and increases our understanding of the bacterial phospholipid flippase MprF. IMPORTANCE Ever since daptomycin was introduced to the clinic, daptomycin-resistant isolates have been reported. In most cases, the resistant isolates harbor point mutations in MprF, which produces and flips the positively charged phospholipid LysPG. This has led to the assumption that the resistance mechanism relies on the overproduction of LysPG, given that increased LysPG production may lead to increased electrostatic repulsion of positively charged antimicrobial compounds, including daptomycin. Here we show that the resistance mechanism is highly specific and relies on a different process that involves a functional MprF flippase, suggesting that the resistance-conferring mutations may enable the flippase to accommodate daptomycin or an unknown component that is crucial for its activity. Our report provides a new perspective on the mechanism of resistance to a major antibiotic.

ABSTRACT Phospholipids are synthesized at the inner leaflet of the bacterial cytoplasmic membrane but have to be translocated to the outer leaflet to maintain membrane lipid bilayer composition and structure. Even though phospholipid flippases have been proposed to exist in bacteria, only one such protein, MprF, has been described. MprF is a large integral membrane protein found in several prokaryotic phyla, whose C terminus modifies phosphatidylglycerol (PG), the most common bacterial phospholipid, with lysine or alanine to modulate the membrane surface charge and, as a consequence, confer resistance to cationic antimicrobial agents such as daptomycin. In addition, MprF is a flippase for the resulting lipids, Lys-PG or Ala-PG. Here we demonstrate that the flippase activity resides in the N-terminal 6 to 8 transmembrane segments of the Staphylococcus aureus MprF and that several conserved, charged amino acids and a proline residue are crucial for flippase function. MprF protects S. aureus against the membrane-active antibiotic daptomycin only when both domains are present, but the two parts do not need to be covalently linked and can function in trans. The Lys-PG synthase and flippase domains were each found to homo-oligomerize and also to interact with each other, which illustrates how the two functional domains may act together. Moreover, full-length MprF proteins formed oligomers, indicating that MprF functions as a dimer or larger oligomer. Together our data reveal how bacterial phospholipid flippases may function in the context of lipid biosynthetic processes. IMPORTANCE Bacterial cytoplasmic membranes are crucial for maintaining and protecting cellular integrity. For instance, they have to cope with membrane-damaging agents such as cationic antimicrobial peptides (CAMPs) produced by competing bacteria (bacteriocins), secreted by eukaryotic host cells (defensins), or used as antimicrobial therapy (daptomycin). The MprF protein is found in many Gram-positive, Gram-negative, and even archaeal commensals or pathogens and confers resistance to CAMPs by modifying anionic phospholipids with amino acids, thereby compromising the membrane interaction of CAMPs. Here we describe how MprF does not only modify phospholipids but uses an additional, distinct domain for translocating the resulting lysinylated phospholipids to the outer leaflet of the membrane. We reveal critical details for the structure and function of MprF, the first dedicated prokaryotic phospholipid flippase, which may pave the way for targeting MprF with new antimicrobials that would not kill bacteria but sensitize them to antibiotics and innate host defense molecules.

Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.