Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

Rhodomyrtone, purified from Rhodomyrtus tomentosa (Aiton) Hassk, exhibits a high degree of potency against methicillin-resistant Staphylococcus aureus (MRSA). We recently demonstrated that exposure of MRSA to a subinhibitory concentration (0.174 µg/ml) of rhodomyrtone resulted in the alteration of expression of several functional classes of bacterial proteins. To provide further insight into the antibacterial mode of action of this compound, we determined the impact of exposure to rhodomyrtone on the gene transcriptional profile of MRSA using microarray analysis. Exposure of MRSA to subinhibitory concentrations (0.5MIC; 0.5 µg/ml) of rhodomyrtone revealed significant modulation of gene expression, with induction of 64 genes and repression of 35 genes. Prominent changes in response to exposure to rhodomyrtone involved genes encoding proteins essential to metabolic pathways and processes such as amino acid metabolism, membrane function, ATP-binding cassette (ABC) transportation and lipoprotein and nucleotide metabolism. Genes involved in the synthesis of the aspartate family of amino acids, in particular proteins encoded by the dap operon were prominent. The diaminopimelate (DAP) biosynthetic pathway is the precursor of lysine synthesis and is essential for peptidoglycan biosynthesis. However, phenotypic analysis of the peptidoglycan amino acid content of rhodomyrtone-treated MRSA did not differ significantly from that extracted from control cells. Genes involved in the biosynthesis of amino acids and peptidoglycan, and a high affinity ATP-driven K (⁺) transport system, were investigated by quantitative reverse transcription-PCR (qRT-PCR) using EMRSA-16 1, 4, or 18 h after exposure to rhodomyrtone and in general the data concurred with that obtained by microarray, highlighting the relevance of the DAP biosynthetic pathway to the mode of action of rhodomyrtone.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.

Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates.

Microgravity induces physiological deconditioning due to the absence of gravity loading, resulting in bone mineral density loss, atrophy of lower limb skeletal and postural muscles, and lengthening of the spine. SkinSuit is a lightweight compression suit designed to provide head-to-foot (axial) loading to counteract spinal elongation during spaceflight. As synthetic garments may impact negatively on the skin microbiome, we used 16S ribosomal RNA (rRNA) gene amplicon procedures to define bacterial skin communities at sebaceous and moist body sites of five healthy male volunteers undergoing SkinSuit evaluation. Each volunteer displayed a diverse, distinct bacterial population at each skin site. Short (8 h) periods of dry hyper-buoyancy flotation wearing either gym kit or SkinSuit elicited changes in the composition of the skin microbiota at the genus level but had little or no impact on community structure at the phylum level or the richness and diversity of the bacterial population. We also determined the composition of the skin microbiota of an astronaut during pre-flight training, during an 8-day visit to the International Space Station involving two 6–7 h periods of SkinSuit wear, and for 1 month after return. Changes in composition of bacterial skin communities at five body sites were strongly linked to changes in geographical location. A distinct ISS bacterial microbiota signature was found which reversed to a pre-flight profile on return. No changes in microbiome complexity or diversity were noted, with little evidence for colonisation by potentially pathogenic bacteria; we conclude that short periods of SkinSuit wear induce changes to the composition of the skin microbiota but these are unlikely to compromise the healthy skin microbiome.