Biology subjects

Ledger, E. V. K.

Publications and source records attributed to Ledger, E. V. K..

6 recordsLinked to original sources

bioRxiv2025

Small molecule inhibitors of the NorA multidrug efflux pump potentiate antibiotic activity by binding the outward-open conformation

Antibiotic resistance is among the greatest threats of the modern era. Multidrug efflux pumps expel antibiotics from bacterial cells and present a particular challenge by conferring resistance to a broad range of antibiotic classes; however, there is currently a lack of potent and selective inhibitors. Here, we report the discovery of IMP-2380, a drug-like chemical probe for the multidrug efflux pump NorA that delivers low-nanomolar potentiation of ciprofloxacin activity in vitro and activity in an in vivo S. aureus infection model. A phenotypic high-throughput screen for inhibitors of the ciprofloxacin-activated SOS DNA repair pathway in methicillin-resistant Staphylococcus aureus (MRSA) identified hit compounds targeting NorA, and subsequent optimization established IMP-2380 as the most potent NorA inhibitor discovered to date. The structure of NorA bound to IMP-2380 was solved by cryo-electron microscopy at 2.52 [A] resolution, revealing that the small molecule locks the pump in the outward-open conformation. This closes the inner face and prevents antibiotics binding from the cytosol, providing an explanation for the exceptional potency of IMP-2380 and structure-activity relationship across the series. IMP-2380 represents an in vivo active NorA inhibitor, functioning via a structurally defined outward-open binding mode, and will enable future exploration of NorA as a druggable target to combat antibiotic resistance.

biochemistry↗

bioRxiv2024

Acquisition of daptomycin resistance in patients results in decreased virulence in Drosophila

Staphylococcus aureus can acquire antimicrobial resistance which in turn may affect its pathogenic potential. Using a panel of paired clinical isolates collected before and after daptomycin resistance acquisition, most frequently through single mprF mutation, we show a relationship between increasing daptomycin minimum inhibitory concentration and reduced virulence in a Drosophila systemic infection model. Analysing toxin production, in vitro bacterial growth characteristics, and cell surface properties, we failed to link daptomycin resistance-related attenuated virulence to either reduced virulence factor production, reduced fitness or to any of the cell surface characteristics investigated. Competition assays in Drosophila also did not support any altered ability in immune evasion. Instead, using a panel of mutant flies defective for various immune components, we show that this daptomycin resistance-related attenuated virulence is mostly explained by greater susceptibility to activity of Drosophila prophenoloxidase, a tyrosinase involved in melanization, but not to antimicrobial peptides or to Bomanin antimicrobial effectors. Further investigation could not link daptomycin resistance-related attenuation of virulence to a differential susceptibility to reactive oxygen species or to quinones prominently associated with phenoloxidase bacterial-killing activity. Taken together, it appears that daptomycin resistance attenuates Staphylococcus aureus virulence through an enhanced sensitivity to phenoloxidase based on a complex mechanism. Our study provides new insights in the understanding of the crosstalk between antimicrobial resistance, escape from immune killing, and virulence. Author summaryAcquiring antimicrobial resistance can increase or decrease bacterial virulence. However, the mechanisms causing these resistance-virulence linked effects are unclear. Here, we bring new insights on the crosstalk between antimicrobial resistance and virulence. We characterized a panel of Staphylococcus aureus strains isolated from patients before and after resistance acquisition to the antibiotic daptomycin. Relative to the parental strain, resistant isolates most often varied by one single mutation, in a gene involved in the composition of the bacterial membrane, and these strains were much less virulent when fruit-flies were infected. Our results indicate that the difference of virulence is unrelated to changes in bacterial toxin production, bacterial growth, immune evasion or cell surface properties. Instead, resistant strains were more vulnerable to a host proenzyme involved in the antibacterial melanization response, an important response deployed throughout the arthropods. We predict that daptomycin resistance forces staphylococci to alter the composition of their cell surface. This alteration causes the bacteria to become more vulnerable to killing by melanization. Our results contribute to our understanding of the link between antimicrobial resistance and pathogenicity.

microbiology↗

bioRxiv2024

PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesising a thick peptidoglycan cell wall which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin binding protein PBP4 is crucial for serum-induced cell wall thickening. Firstly, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant towards the last resort antibiotic daptomycin relative to wildtype cells. Secondly, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteraemia isolates, except for one strain with a naturally-occurring mutation that confers a S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and in clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.

microbiology↗

bioRxiv2023

Staphylococcal host adaptation compromises neutrophil killing by concealing bound opsonins with peptidoglycan

The bacterial pathogen Staphylococcus aureus adapts to the host environment by increasing the thickness of its cell wall. However, the impact of cell wall thickening on susceptibility to host defences is unclear. Here, we show that as bacteria adapted to serum, the resulting increase in cell wall thickness led to a reduction in the exposure of bound antibody and complement and a corresponding reduction in phagocytosis and killing by neutrophils. The exposure of opsonins bound to protein antigens or LTA were most significantly reduced, whilst opsonisation by IgG against wall teichoic acid or peptidoglycan was largely unaffected. Partial digestion of accumulated cell wall in host adapted cells using the enzyme lysostaphin restored opsonin exposure and promoted phagocytosis and killing. Concordantly, the antibiotic fosfomycin inhibited cell wall remodelling and maintained full susceptibility of S. aureus to opsonophagocytic killing by neutrophils. These findings reveal that the adaptation of S. aureus to the host environment reduces the ability of the immune system to detect and kill this pathogen through reduced exposure of protein- and LTA-bound opsonins via cell wall remodelling.

microbiology↗

bioRxiv2022

Growth arrest of Staphylococcus aureus induces daptomycin tolerance via cell wall remodelling

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill non-dividing bacteria. However, these studies employed only brief periods of growth arrest. We found that a bacteriostatic concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with ~100,000-fold increase in survival after 16 h growth arrest relative to exponential phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, whilst accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whilst tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling.

microbiology↗

bioRxiv2017

Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids

Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We have shown previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, Group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipid in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

microbiology↗