The MraY Inhibitor Muraymycin D2 and Its Derivatives Induce Enlarged Cells in Obligate Intracellular Chlamydia and Wolbachia and Break the Persistence Phenotype in Chlamydia.

Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting Chlamydia and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria Chlamydia and Wolbachia, the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with C. trachomatis and Wolbachia showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Chlamydia. Enzymatic inhibition experiments with purified C. pneumoniae MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in Aquifex aeolicus revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing C. trachomatis cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence.

Bacterial Toxin-Triggered Release of Antibiotics from Capsosomes Protects a Fly Model from Lethal Methicillin-Resistant Staphylococcus aureus (MRSA) Infection.

Antibiotic resistance is a severe global health threat and hence demands rapid action to develop novel therapies, including microscale drug delivery systems. Herein, a hierarchical microparticle system is developed to achieve bacteria-activated single- and dual-antibiotic drug delivery for preventing methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections. The designed system is based on a capsosome structure, which consists of a mesoporous silica microparticle coated in alternating layers of oppositely charged polymers and antibiotic-loaded liposomes. The capsosomes are engineered and shown to release their drug payloads in the presence of MRSA toxins controlled by the Agr quorum sensing system. MRSA-activated single drug delivery of vancomycin and synergistic dual delivery of vancomycin together with an antibacterial peptide successfully kills MRSA in vitro. The capability of capsosomes to selectively deliver their cargo in the presence of bacteria, producing a bactericidal effect to protect the host organism, is confirmed in vivo using a Drosophila melanogaster MRSA infection model. Thus, the capsosomes serve as a versatile multidrug, subcompartmentalized microparticle system for preventing antibiotic-resistant bacterial infections, with potential applications to protect wounds or medical device implants from infections.

Assessing the impact of silicon nanowires on bacterial transformation and viability of Escherichia coli.

We investigated the biomaterial interface between the bacteria Escherichia coli DH5α and silicon nanowire patterned surfaces. We optimised the engineering of silicon nanowire coated surfaces using metal-assisted chemical etching. Using a combination of focussed ion beam scanning electron microscopy, and cell viability and transformation assays, we found that with increasing interfacing force, cell viability decreases, as a result of increasing cell rupture. However, despite this aggressive interfacing regime, a proportion of the bacterial cell population remains viable. We found that the silicon nanowires neither resulted in complete loss of cell viability nor partial membrane disruption and corresponding DNA plasmid transformation. Critically, assay choice was observed to be important, as a reduction-based metabolic reagent was found to yield false-positive results on the silicon nanowire substrate. We discuss the implications of these results for the future design and assessment of bacteria-nanostructure interfacing experiments.

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Antibiotics are life-saving medicines, but many bacteria now have the ability to resist their effects. For some infections, all frontline antibiotics are now ineffective. To treat infections caused by these highly resistant bacteria, clinicians must use so-called 'antibiotics of last resort'. These antibiotics include a drug called colistin, which is moderately effective, but often fails to eradicate the infection. One of the challenges to making colistin more effective is that its mechanism is poorly understood. Bacteria have two layers of protection against the outside world: an outer cell membrane and an inner cell membrane. To kill them, colistin must punch holes in both. First, it disrupts the outer membrane by interacting with molecules called lipopolysaccharides. But how it disrupts the inner membrane was unclear. Bacteria have evolved several different mechanisms that make them resistant to the effects of colistin. Sabnis et al. reasoned that understanding how these mechanisms protected bacteria could reveal how the antibiotic works to damage the inner cell membrane. Sabnis et al. examined the effects of colistin on Escherichia coli bacteria with and without resistance to the antibiotic. Exposing these bacteria to colistin revealed that the antibiotic damages both layers of the cell surface in the same way, targeting lipopolysaccharide in the inner membrane as well as the outer membrane. Next, Sabnis et al. used this new information to make colistin work better. They found that the effects of colistin were magnified when it was combined with the experimental antibiotic murepavadin, which caused lipopolysaccharide to build up at the inner membrane. This allowed colistin to punch more holes through the inner membrane, making colistin more effective at killing bacteria. To find out whether this combination of colistin and murepavadin could work as a clinical treatment, Sabnis et al. tested it on mice with Pseudomonas aeruginosa infections in their lungs. Colistin was much better at killing Pseudomonas aeruginosa and treating infections when combined with murepavadin than it was on its own. Pseudomonas aeruginosa bacteria can cause infections in the lungs of people with cystic fibrosis. At the moment, patients receive colistin in an inhaled form to treat these infections, but it is not always successful. The second drug used in this study, murepavadin, is about to enter clinical trials as an inhaled treatment for lung infections too. If the trial is successful, it may be possible to use both drugs in combination to treat lung infections in people with cystic fibrosis.

Controlled Dendrimersome Nanoreactor System for Localized Hypochlorite-Induced Killing of Bacteria.

Antibiotic resistance is a serious global health problem necessitating new bactericidal approaches such as nanomedicines. Dendrimersomes (DSs) have recently become a valuable alternative nanocarrier to polymersomes and liposomes due to their molecular definition and synthetic versatility. Despite this, their biomedical application is still in its infancy. Inspired by the localized antimicrobial function of neutrophil phagosomes and the versatility of DSs, a simple three-component DS-based nanoreactor with broad-spectrum bactericidal activity is presented. This was achieved by encapsulation of glucose oxidase (GOX) and myeloperoxidase (MPO) within DSs (GOX-MPO-DSs), self-assembled from an amphiphilic Janus dendrimer, that possesses a semipermeable membrane. By external addition of glucose to GOX-MPO-DS, the production of hypochlorite (-OCl), a highly potent antimicrobial, by the enzymatic cascade was demonstrated. This cascade nanoreactor yielded a potent bactericidal effect against two important multidrug resistant pathogens, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), not observed for H2O2 producing nanoreactors, GOX-DS. The production of highly reactive species such as -OCl represents a harsh bactericidal approach that could also be cytotoxic to mammalian cells. This necessitates the development of strategies for activating -OCl production in a localized manner in response to a bacterial stimulus. One option of locally releasing sufficient amounts of substrate using a bacterial trigger (released toxins) was demonstrated with lipidic glucose-loaded giant unilamellar vesicles (GUVs), envisioning, e.g., implant surface modification with nanoreactors and GUVs for localized production of bactericidal agents in the presence of bacterial growth.

Ca2+-Daptomycin targets cell wall biosynthesis by forming a tripartite complex with undecaprenyl-coupled intermediates and membrane lipids.

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.

Sulfide Protects Staphylococcus aureus from Aminoglycoside Antibiotics but Cannot Be Regarded as a General Defense Mechanism against Antibiotics.

Sulfide production has been proposed to be a universal defense mechanism against antibiotics in bacteria (K. Shatalin, E. Shatalina, A. Mironov, and E. Nudler, Science 334:986-990, 2011, doi:10.1126/science.1209855). To gain insight into the mechanism underlying sulfide protection, we systematically and comparatively addressed the interference of sulfide with antibiotic activity against Staphylococcus aureus, as a model organism. The impact of sulfide and sulfide precursors on the antibiotic susceptibility of S. aureus to the most important classes of antibiotics was analyzed using modified disk diffusion assays, killing kinetic assays, and drug uptake studies. In addition, sulfide production and the impact of exogenously added sulfide on the physiology of S. aureus were analyzed. Sulfide protection was found to be limited to aminoglycoside antibiotics, which are known to be taken up by bacterial cells in an energy-dependent process. The protective mechanism was found to rely on an inhibitory effect of sulfide on the bacterial respiratory chain, leading to reduced drug uptake. S. aureus was found to be incapable of producing substantial amounts of sulfide. We propose that bacterial sulfide production should not be regarded as a general defense mechanism against antibiotics, since (i) it is limited to aminoglycosides and (ii) production levels vary considerably among species and, as for S. aureus, may be too low for protection.

Antimicrobial Dialkylresorcins from Marine-Derived Microorganisms: Insights into Their Mode of Action and Putative Ecological Relevance.

Zobellia galactanivorans has been reported as a seaweed-associated or marine-derived species with largely unknown secondary metabolites. The combination of bioinformatic analysis and MS- and bioactivity guided separation led to the isolation of a new antibiotically active dialkylresorcin from the marine bacterium Z. galactanivorans. The antibiotic profile of the new dialkylresorcin zobelliphol (1: ) was investigated and compared with related and naturally occurring dialkyresorcins (i.e., stemphol (2: ) and 4-butyl-3,5-dihydroxybenzoic acid (3: )) from the marine-derived fungus Stemphylium globuliferum. Bacterial reporter strain assays provided insights into the mode of action of this antibiotic compound class. We identified an interference with bacterial DNA biosynthesis for the dialkylresorcin derivative 1: . In addition, the putative biosynthetic gene cluster corresponding to production of 1: was identified and a biosynthetic hypothesis was deduced.

Deconstructing the Chlamydial Cell Wall.

The evolutionary separated Gram-negative Chlamydiales show a biphasic life cycle and replicate exclusively within eukaryotic host cells. Members of the genus Chlamydia are responsible for many acute and chronic diseases in humans, and Chlamydia-related bacteria are emerging pathogens. We revisit past efforts to detect cell wall material in Chlamydia and Chlamydia-related bacteria in the context of recent breakthroughs in elucidating the underlying cellular and molecular mechanisms of the chlamydial cell wall biosynthesis. In this review, we also discuss the role of cell wall biosynthesis in chlamydial FtsZ-independent cell division and immune modulation. In the past, penicillin susceptibility of an invisible wall was referred to as the "chlamydial anomaly." In light of new mechanistic insights, chlamydiae may now emerge as model systems to understand how a minimal and modified cell wall biosynthetic machine supports bacterial cell division and how cell wall-targeting beta-lactam antibiotics can also act bacteriostatically rather than bactericidal. On the heels of these discussions, we also delve into the effects of other cell wall antibiotics in individual chlamydial lineages.

AmiD Is a Novel Peptidoglycan Amidase in Wolbachia Endosymbionts of Drosophila melanogaster.

Wolbachia endobacteria are obligate intracellular bacteria with a highly reduced genome infecting many arthropod and filarial species, in which they manipulate arthropod reproduction to increase their transmission and are essential for nematode development and survival. The Wolbachia genome encodes all enzymes required for the synthesis of the cell wall building block lipid II, although a peptidoglycan-like structure has not been detected. Despite the ability to synthesize lipid II, Wolbachia from arthropods and nematodes have only a subset of genes encoding enzymes involved in the periplasmic processing of lipid II and peptidoglycan recycling, with arthropods having two more than nematodes. We functionally analyzed the activity of the putative cell wall hydrolase AmiD from the Wolbachia endosymbiont of Drosophila melanogaster, an enzyme not encoded by the nematode endobacteria. Wolbachia AmiD has Zn2+-dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates that are generated during bacterial growth. AmiD may have been maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. This is the first description of a wolbachial lipid II processing enzyme putatively expressed in the periplasm.

Targeting a cell wall biosynthesis hot spot.

Covering: up to 2017History points to the bacterial cell wall biosynthetic network as a very effective target for antibiotic intervention, and numerous natural product inhibitors have been discovered. In addition to the inhibition of enzymes involved in the multistep synthesis of the macromolecular layer, in particular, interference with membrane-bound substrates and intermediates essential for the biosynthetic reactions has proven a valuable antibacterial strategy. A prominent target within the peptidoglycan biosynthetic pathway is lipid II, which represents a particular "Achilles' heel" for antibiotic attack, as it is readily accessible on the outside of the cytoplasmic membrane. Lipid II is a unique non-protein target that is one of the structurally most conserved molecules in bacterial cells. Notably, lipid II is more than just a target molecule, since sequestration of the cell wall precursor may be combined with additional antibiotic activities, such as the disruption of membrane integrity or disintegration of membrane-bound multi-enzyme machineries. Within the membrane bilayer lipid II is likely organized in specific anionic phospholipid patches that form a particular "landing platform" for antibiotics. Nature has invented a variety of different "lipid II binders" of at least 5 chemical classes, and their antibiotic activities can vary substantially depending on the compounds' physicochemical properties, such as amphiphilicity and charge, and thus trigger diverse cellular effects that are decisive for antibiotic activity.

Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly."

AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae.

Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation.

Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae.

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.