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Find video protocols related to scientific articles indexed in Pubmed.
Forces shaping the antibiotic resistome.
Bioessays
PUBLISHED: 09-11-2014
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Antibiotic resistance has become a problem of global scale. Resistance arises through mutation or through the acquisition of resistance gene(s) from other bacteria in a process called horizontal gene transfer (HGT). While HGT is recognized as an important factor in the dissemination of resistance genes in clinical pathogens, its role in the environment has been called into question by a recent study published in Nature. The authors found little evidence of HGT in soil using a culture-independent functional metagenomics approach, which is in contrast to previous work from the same lab showing HGT between the environment and human microbiome. While surprising at face value, these results may be explained by the lack of selective pressure in the environment studied. Importantly, this work suggests the need for careful monitoring of environmental antibiotic pollution and stringent antibiotic stewardship in the fight against resistance.
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Role of PBPD1 in Stimulation of Listeria monocytogenes Biofilm Formation by Subminimal Inhibitory ?-Lactam Concentrations.
Antimicrob. Agents Chemother.
PUBLISHED: 08-18-2014
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Disinfectant-tolerant Listeria monocytogenes biofilms can colonize surfaces that come into contact with food, leading to contamination and, potentially, food-borne illnesses. To better understand the process of L. monocytogenes biofilm formation and dispersal, we screened 1,120 off-patent FDA-approved drugs and identified several that modulate Listeria biofilm development. Among the hits were more than 30 ?-lactam antibiotics, with effects ranging from inhibiting (?50%) to stimulating (?200%) biofilm formation compared to control. Most ?-lactams also dispersed a substantial proportion of established biofilms. This phenotype did not necessarily involve killing, as >50% dispersal could be achieved with concentrations as low as 1/20 of the MIC of some cephalosporins. Penicillin-binding protein (PBP) profiling using a fluorescent penicillin analogue showed similar inhibition patterns for most ?-lactams, except that biofilm-stimulatory drugs did not bind PBPD1, a low-molecular-weight d,d-carboxypeptidase. Compared to the wild type, a pbpD1 mutant had an attenuated biofilm response to stimulatory ?-lactams. The cephalosporin-responsive CesRK two-component regulatory system, whose regulon includes PBPs, was not required for the response. The requirement for PBPD1 activity for ?-lactam stimulation of L. monocytogenes biofilms shows that the specific set of PBPs that are inactivated by a particular drug dictates whether a protective biofilm response is provoked.
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Inhibition of the ANT(2?)-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic ?-hydroxytropolones.
Bioorg. Med. Chem. Lett.
PUBLISHED: 08-09-2014
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Aminoglycoside-2?-O-nucleotidyltransferase ANT(2?)-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic ?-hydroxytropolones against ANT(2?)-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2?)-Ia-expressing bacteria.
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A rifamycin inactivating phosphotransferase family shared by environmental and pathogenic bacteria.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-28-2014
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Many environmental bacteria are multidrug-resistant and represent a reservoir of ancient antibiotic resistance determinants, which have been linked to genes found in pathogens. Exploring the environmental antibiotic resistome, therefore, reveals the diversity and evolution of antibiotic resistance and also provides insight into the vulnerability of clinically used antibiotics. In this study, we describe the identification of a highly conserved regulatory motif, the rifampin (RIF) -associated element (RAE), which is found upstream of genes encoding RIF-inactivating enzymes from a diverse collection of actinomycetes. Using gene expression assays, we confirmed that the RAE is involved in RIF-responsive regulation. By using the RAE as a probe for new RIF-associated genes in several actinomycete genomes, we identified a heretofore unknown RIF resistance gene, RIF phosphotransferase (rph). The RPH enzyme is a RIF-inactivating phosphotransferase and represents a new protein family in antibiotic resistance. RPH orthologs are widespread and found in RIF-sensitive bacteria, including Bacillus cereus and the pathogen Listeria monocytogenes. Heterologous expression and in vitro enzyme assays with purified RPHs from diverse bacterial genera show that these enzymes are capable of conferring high-level resistance to a variety of clinically used rifamycin antibiotics. This work identifies a new antibiotic resistance protein family and reinforces the fact that the study of resistance in environmental organisms can serve to identify resistance elements with relevance to pathogens.
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The antibiotic resistome: what's new?
Curr. Opin. Microbiol.
PUBLISHED: 04-25-2014
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The antibiotic resistome is dynamic and ever expanding, yet its foundations were laid long before the introduction of antibiotics into clinical practice. Here, we revisit our theoretical framework for the resistome concept and consider the many factors that influence the evolution of novel resistance genes, the spread of mobile resistance elements, and the ramifications of these processes for clinical practice. Observing the trends and prevalence of genes within the antibiotic resistome is key to maintaining the efficacy of antibiotics in the clinic.
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Harnessing the Synthetic Capabilities of Glycopeptide Antibiotic Tailoring Enzymes: Characterization of the UK-68,597 Biosynthetic Cluster.
Chembiochem
PUBLISHED: 04-16-2014
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In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.
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An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria.
J. Antimicrob. Chemother.
PUBLISHED: 03-13-2014
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An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli.
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Aspergillomarasmine A overcomes metallo-?-lactamase antibiotic resistance.
Nature
PUBLISHED: 02-26-2014
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The emergence and spread of carbapenem-resistant Gram-negative pathogens is a global public health problem. The acquisition of metallo-?-lactamases (MBLs) such as NDM-1 is a principle contributor to the emergence of carbapenem-resistant Gram-negative pathogens that threatens the use of penicillin, cephalosporin and carbapenem antibiotics to treat infections. To date, a clinical inhibitor of MBLs that could reverse resistance and re-sensitize resistant Gram-negative pathogens to carbapenems has not been found. Here we have identified a fungal natural product, aspergillomarasmine A (AMA), that is a rapid and potent inhibitor of the NDM-1 enzyme and another clinically relevant MBL, VIM-2. AMA also fully restored the activity of meropenem against Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. possessing either VIM or NDM-type alleles. In mice infected with NDM-1-expressing Klebsiella pneumoniae, AMA efficiently restored meropenem activity, demonstrating that a combination of AMA and a carbapenem antibiotic has therapeutic potential to address the clinical challenge of MBL-positive carbapenem-resistant Gram-negative pathogens.
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Draft Genome Sequence of the Sulfonamide Antibiotic-Degrading Microbacterium sp. Strain C448.
Genome Announc
PUBLISHED: 02-15-2014
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We report the draft genome sequence of Microbacterium sp. strain C448, isolated from agricultural soil with a decade of exposure to veterinary antibiotics on the basis of using sulfamethazine and other antibiotics as the sole sources of carbon. The genome sequence revealed that strain C448 harbors several antibiotic resistance genes, including sulI.
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Something old, something new: revisiting natural products in antibiotic drug discovery.
Can. J. Microbiol.
PUBLISHED: 01-22-2014
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Antibiotic discovery is in crisis. Despite a growing need for new drugs resulting from the increasing number of multi-antibiotic-resistant pathogens, there have been only a handful of new antibiotics approved for clinical use in the past 2 decades. Faced with scientific, economic, and regulatory challenges, the pharmaceutical sector seems unable to respond to what has been called an "apocalyptic" threat. Natural products produced by bacteria and fungi are genetically encoded products of natural selection that have been the mainstay sources of the antibiotics in current clinical use. The pharmaceutical industry has largely abandoned these compounds in favor of large libraries of synthetic molecules because of difficulties in identifying new natural product antibiotics scaffolds. Advances in next-generation genome sequencing, bioinformatics, and analytical chemistry are combining to overcome barriers to natural products. Coupled with new strategies in antibiotic discovery, including inhibition of resistance, novel drug combinations, and new targets, natural products are poised for a renaissance to address what is a pressing health care crisis.
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Designing analogs of ticlopidine, a wall teichoic acid inhibitor, to avoid formation of its oxidative metabolites.
Bioorg. Med. Chem. Lett.
PUBLISHED: 01-08-2014
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The thienopyridine antiplatelet agent, ticlopidine and its analog, clopidogrel, have been shown to potentiate the action of ?-lactam antibiotics, reversing the methicillin-resistance phenotype of methicillin-resistant Staphylococcus aureus (MRSA), in vitro. Interestingly, these thienopyridines inhibit the action of TarO, the first enzyme in the synthesis of wall teichoic acid, an important cell wall polymer in Gram-positive bacteria. In the human body, both ticlopidine and clopidogrel undergo a rapid P450-dependent oxidation into their respective antiplatelet-active metabolites, resulting in very low plasma concentrations of intact drug. Herein, a series of analogs of ticlopidine and clopidogrel that would avoid oxidative metabolism were designed, prepared and evaluated as inhibitors of TarO. Specifically, we replaced the P450-labile thiophene ring of ticlopidine and clopidogrel to a more stable phenyl group to generate 2-(2-chlorobenzyl)-1,2,3,4-tetrahydro-isoquinoline) (6) and (2-chloro-phenyl)-(3,4-dihydro-1H-isoquinolin-2-yl)-acetic acid methyl ester (22), respectively. The latter molecules displayed inhibitory activity against TarO and formed the basis of a library of analogs. Most synthesized compounds exhibited comparable efficacy to ticlopidine and clopidogrel. So far, it was introduction of a trifluoromethyl group to compound 6, to generate 2-(2-trifluoromethyl-benzyl)-1,2,3,4-tetrahydro-isoquinoline (13) that exhibited enhanced activity against TarO. Compound 13 represents a novel stable inhibitor of TarO with synergistic impact on ?-lactam antibiotics against MRSA and low potential for P-450 metabolism.
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Antibiotic resistance-the need for global solutions.
Lancet Infect Dis
PUBLISHED: 11-17-2013
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The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.
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Glycopeptide antibiotic biosynthesis.
J. Antibiot.
PUBLISHED: 08-26-2013
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Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.The Journal of Antibiotics advance online publication, 13 November 2013; doi:10.1038/ja.2013.117.
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Metal-Induced Isomerization Yields an Intracellular Chelator that Disrupts Bacterial Iron Homeostasis.
Chem. Biol.
PUBLISHED: 08-15-2013
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The dwindling supply of antibiotics that remain effective against drug-resistant bacterial pathogens has precipitated efforts to identify new compounds that inhibit bacterial growth using untapped mechanisms of action. Here, we report both (1) a high-throughput screening methodology designed to discover chemical perturbants of the essential, yet unexploited, process of bacterial iron homeostasis, and (2) our findings from a small-molecule screen of more than 30,000 diverse small molecules that led to the identification and characterization of two spiro-indoline-thiadiazoles that disrupt iron homeostasis in bacteria. We show that these compounds are intracellular chelators with the capacity to exist in two isomeric states. Notably, these spiroheterocyles undergo a transition to an open merocyanine chelating form with antibacterial activity that is specifically induced in the presence of its transition-metal target.
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Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance in Salmonella Typhimurium.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-02-2013
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The efficacy of antibiotics and host defenses has been linked to the metabolic and redox states of bacteria. In this study we report that a stress-induced export pump belonging to the major facilitator superfamily effluxes citrate and iron from the enteric pathogen Salmonella Typhimurium to arrest growth and ameliorate the effects of antibiotics, hydrogen peroxide, and nitric oxide. The transporter, formerly known as MdtD, is now designated IceT (iron citrate efflux transporter). Iron efflux via an iron-chelating tricarboxylic acid cycle intermediate provides a direct link between aerobic metabolism and bacterial stress responses, representing a unique mechanism of resistance to host defenses and antimicrobial agents of diverse classes.
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A cryptic polyene biosynthetic gene cluster in Streptomyces calvus is expressed upon complementation with a functional bldA gene.
Chem. Biol.
PUBLISHED: 06-18-2013
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Streptomyces calvus is best known as the producer of the fluorinated natural product nucleocidin. This strain of Streptomycetes is also unusual for displaying a "bald" phenotype that is deficient in the formation of aerial mycelium and spores. Genome sequencing of this organism revealed a point mutation in the bldA gene that is predicted to encode a misfolded Leu-tRNA(UUA) molecule. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a polyeneoic acid amide, 4-Z-annimycin, and a minor amount of the isomer, 4-E-annimycin. Bioassays reveal that these compounds inhibit morphological differentiation in other Actinobacteria. The annimycin gene cluster encoding a type 1 polyketide synthase was identified and verified through disruption studies. This study underscores the importance of the bldA gene in regulating the expression of cryptic biosynthetic genes.
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Influence of humans on evolution and mobilization of environmental antibiotic resistome.
Emerging Infect. Dis.
PUBLISHED: 06-15-2013
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The clinical failure of antimicrobial drugs that were previously effective in controlling infectious disease is a tragedy of increasing magnitude that gravely affects human health. This resistance by pathogens is often the endpoint of an evolutionary process that began billions of years ago in non-disease-causing microorganisms. This environmental resistome, its mobilization, and the conditions that facilitate its entry into human pathogens are at the heart of the current public health crisis in antibiotic resistance. Understanding the origins, evolution, and mechanisms of transfer of resistance elements is vital to our ability to adequately address this public health issue.
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Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance.
Biochem. J.
PUBLISHED: 06-14-2013
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Activity of the aminoglycoside phosphotransferase APH(3)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3)-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3)-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.
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The comprehensive antibiotic resistance database.
Antimicrob. Agents Chemother.
PUBLISHED: 05-06-2013
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The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.
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Self resistance to the atypical cationic antimicrobial peptide edeine of Brevibacillus brevis Vm4 by the N-acetyltransferase EdeQ.
Chem. Biol.
PUBLISHED: 03-21-2013
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Edeines are atypical cationic peptides produced by Brevibacillus brevis Vm4 with broad-spectrum antimicrobial activity. These linear nonribosomal peptides bind to the 30S ribosomal subunit and block t-RNA binding to the P-site. To identify the mechanism of high-level self-resistance in the producing organism, the B. brevis Vm4 genome was sequenced and the edeine biosynthetic cluster discovered. A potential edeine-modifying enzyme, EdeQ, showed similarity to spermidine N-acetyltransferases. EdeQ was purified and shown to convert edeine to N-acetyledeine, which is inactive against cells in vivo and against cell-free extracts. Unexpectedly, tandem mass spectroscopy and nuclear magnetic resonance demonstrate that N-acylation occurs on the free amine of the internal diaminopropionic acid rather than the N-terminal spermidine polyamine. Acetylation of edeine by EdeQ abolishes its ability to inhibit translation, thus conferring resistance to the antibiotic in the producing organism.
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Identifying producers of antibacterial compounds by screening for antibiotic resistance.
Nat. Biotechnol.
PUBLISHED: 03-18-2013
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Microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges. We have designed a screen to exploit the self-protection mechanism of antibiotic producers to enrich microbial libraries for producers of selected antibiotic scaffolds. Using resistance as a discriminating criterion we increased the discovery rate of producers of both glycopeptide and ansamycin antibacterial compounds by several orders of magnitude in comparison with historical hit rates. Applying a phylogeny-based screening filter for biosynthetic genes enabled the binning of producers of distinct scaffolds and resulted in the discovery of a glycopeptide antibacterial compound, pekiskomycin, with an unusual peptide scaffold. This strategy provides a means to readily sample the chemical diversity available in microbes and offers an efficient strategy for rapid discovery of microbial natural products and their associated biosynthetic enzymes.
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Intrinsic antibiotic resistance: mechanisms, origins, challenges and solutions.
Int. J. Med. Microbiol.
PUBLISHED: 03-13-2013
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The intrinsic antibiotic resistome is a naturally occurring phenomenon that predates antibiotic chemotherapy and is present in all bacterial species. In addition to the intrinsic resistance mediated by the bacterial outer membrane and active efflux, studies have shown that a surprising number of additional genes and genetic loci also contribute to this phenotype. Antibiotic resistance is rife in both the clinic and the environment; novel therapeutic strategies need to be developed in order to prevent a major global clinical threat. The possibility of inhibiting elements comprising the intrinsic resistome in bacterial pathogens offers the promise for repurposing existing antibiotics against intrinsically resistant bacteria.
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The antibiotic resistance "mobilome": searching for the link between environment and clinic.
Front Microbiol
PUBLISHED: 01-01-2013
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Antibiotic resistance is an ancient problem, owing to the co-evolution of antibiotic-producing and target organisms in the soil and other environments over millennia. The environmental "resistome" is the collection of all genes that directly or indirectly contribute to antibiotic resistance. Many of these resistance determinants originate in antibiotic-producing organisms (where they serve to mediate self-immunity), while others become resistance determinants only when mobilized and over-expressed in non-native hosts (like plasmid-encoded ?-lactamases). The modern environmental resistome is under selective pressure from human activities such as agriculture, which may influence the composition of the local resistome and lead to gene transfer events. Beyond the environment, we are challenged in the clinic by the rise in both frequency and diversity of antibiotic resistant pathogens. We assume that clinical resistance originated in the environment, but few examples of direct gene exchange between the environmental resistome and the clinical resistome have been documented. Strong evidence exists to suggest that clinical aminoglycoside and vancomycin resistance enzymes, the extended-spectrum ?-lactamase CTX-M and the quinolone resistance gene qnr have direct links to the environmental resistome. In this review, we highlight recent advances in our understanding of horizontal gene transfer of antibiotic resistance genes from the environment to the clinic. Improvements in sequencing technologies coupled with functional metagenomic studies have revealed previously underappreciated diversity in the environmental resistome, and also established novel genetic links to the clinic. Understanding mechanisms of gene exchange becomes vital in controlling the future dissemination of antibiotic resistance.
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Small-molecule modulators of Listeria monocytogenes biofilm development.
Appl. Environ. Microbiol.
PUBLISHED: 12-22-2011
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Listeria monocytogenes is an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay for L. monocytogenes and, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains of Listeria at micromolar concentrations, as well as molecules that decreased (? 50% of vehicle control) or increased (? 200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules-including the protein kinase C antagonist sphingosine-had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that while Listeria biofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 ?M, d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C(9:0) and C(14:0) were Listeria biofilm inhibitors, whereas fatty acids of C(16:0) or longer were stimulators, showing chain length specificity. De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes of L. monocytogenes biology and promising leads for the further development of antibiofilm strategies.
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Inactivation of the lipopeptide antibiotic daptomycin by hydrolytic mechanisms.
Antimicrob. Agents Chemother.
PUBLISHED: 11-14-2011
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The lipopeptide daptomycin is a member of the newest FDA-approved antimicrobial class, exhibiting potency against a broad range of Gram-positive pathogens with only rare incidences of clinical resistance. Environmental bacteria harbor an abundance of resistance determinants orthologous to those in pathogens and thus may serve as an early-warning system for future clinical emergence. A collection of morphologically diverse environmental actinomycetes demonstrating unprecedented frequencies of daptomycin resistance and high levels of resistance by antibiotic inactivation was characterized to elucidate modes of drug inactivation. In vivo studies revealed that hydrolysis plays a key role, resulting in one or both of the following structural modifications: ring hydrolysis resulting in linearization (in 44% of inactivating isolates) or deacylation of the lipid tail (29%). Characterization of the mechanism in actinomycete WAC4713 (a Streptomyces sp. with an MIC of 512 ?g/ml) demonstrated a constitutive resistance phenotype and established daptomycins circularizing ester linkage to be the site of hydrolysis. Characterization of the hydrolase responsible revealed it to be likely a serine protease. These studies suggested that daptomycin is susceptible to general proteolytic hydrolysis, which was further supported by studies using proteases of diverse origin. These findings represent the first comprehensive characterization of daptomycin inactivation in any bacterial class and may not only presage a future mechanism of clinical resistance but also suggest strategies for the development of new lipopeptides.
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A small molecule discrimination map of the antibiotic resistance kinome.
Chem. Biol.
PUBLISHED: 09-19-2011
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Kinase-mediated resistance to antibiotics is a significant clinical challenge. These enzymes share a common protein fold characteristic of Ser/Thr/Tyr protein kinases. We screened 14 antibiotic resistance kinases against 80 chemically diverse protein kinase inhibitors to map resistance kinase chemical space. The screens identified molecules with both broad and narrow inhibition profiles, proving that protein kinase inhibitors offer privileged chemical matter with the potential to block antibiotic resistance. One example is the flavonol quercetin, which inhibited a number of resistance kinases in vitro and in vivo. This activity was rationalized by determination of the crystal structure of the aminoglycoside kinase APH(2?)-IVa in complex with quercetin and its antibiotic substrate kanamycin. Our data demonstrate that protein kinase inhibitors offer chemical scaffolds that can block antibiotic resistance, providing leads for co-drug design.
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Palmitoyl-DL-carnitine is a multitarget inhibitor of Pseudomonas aeruginosa biofilm development.
Chembiochem
PUBLISHED: 08-06-2011
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Bacteria growing in biofilms are often in metabolic and physiological states that do not respond well to antibiotics, and thus, are major contributors to chronic diseases. Biofilm inhibitors, therefore, have the potential to be used alone or as adjuvants to conventional antibiotic therapies. Here, we screened a chemically diverse collection of protein kinase inhibitors for molecules that perturb biofilm development. Among the inhibitory molecules identified, palmitoyl-DL-carnitine (pDLC) impaired Pseudomonas aeruginosa and Escherichia coli biofilm formation in a dose-dependent manner. The pDLC affected multiple pathways implicated in P. aeruginosa biofilm development; it stimulated motility, inhibited activity of the Las quorum sensing system, and overrode the biofilm-promoting effects of subminimal inhibitory concentrations of aminoglycosides and high levels of the second messenger, cyclic-di-GMP. Palmitic acid, but not carnitine, inhibited biofilm formation but did not stimulate motility, suggesting that pDLC works through unique mechanisms. The ability to target multiple pathways involved in biofilm formation is desirable in an inhibitor, which makes pDLC an interesting lead for antibiofilm therapies.
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?-Lactone natural products and derivatives inactivate homoserine transacetylase, a target for antimicrobial agents.
J. Antibiot.
PUBLISHED: 04-27-2011
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Homoserine transacetylase (HTA) catalyzes the transfer of an acetyl group from acetyl-CoA to the hydroxyl group of homoserine. This is the first committed step in the biosynthesis of methionine (Met) from aspartic acid in many fungi, Gram-positive and some Gram-negative bacteria. The enzyme is absent in higher eukaryotes and is important for microorganism growth in Met-poor environments, such as blood serum, making HTA an attractive target for new antimicrobial agents. HTA catalyzes acetyl transfer via a double displacement mechanism facilitated by a classic Ser-His-Asp catalytic triad located at the bottom of a narrow actives site tunnel. We explored the inhibitory activity of several ?-lactones to block the activity of HTA. In particular, the natural product ebelactone A, a ?-lactone with a hydrophobic tail was found to be a potent inactivator of HTA from Haemophilus influenzae. Synthetic analogs of ebelactone A demonstrated improved inactivation characteristics. Covalent modification of HTA was confirmed by mass spectrometry, and peptide mapping identified Ser143 as the modified residue, consistent with the known structure and mechanism of the enzyme. These results demonstrate that ?-lactone inhibitors are excellent biochemical probes of HTA and potential leads for new antimicrobial agents.
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Antibiotic resistance is ancient.
Nature
PUBLISHED: 03-28-2011
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The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to ?-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.
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Structural basis for a new tetracycline resistance mechanism relying on the TetX monooxygenase.
FEBS Lett.
PUBLISHED: 02-28-2011
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The flavin-dependent monooxygenase TetX confers resistance to all clinically relevant tetracyclines, including the recently approved, broad-spectrum antibiotic tigecycline (Tygacil®) which is a critical last-ditch defense against multidrug-resistant pathogens. TetX represents the first resistance mechanism against tigecycline, which circumvents both the tet-gene encoded resistances, relying on active efflux of tetracyclines, and ribosomal protection proteins. The alternative enzyme-based mechanism of TetX depends on regioselective hydroxylation of tetracycline antibiotics to 11a-hydroxy-tetracyclines. Here, we report the X-ray crystallographic structure determinations at 2.1Å resolution of native TetX from Bacteroides thetaiotaomicron and its complexes with tetracyclines. Our crystal structures explain the extremely versatile substrate diversity of the enzyme and provide a first step towards the rational design of novel tetracycline derivatives to counter TetX-based resistance prior to emerging clinical observations.
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Receptor domains of two-component signal transduction systems.
Mol Biosyst
PUBLISHED: 02-24-2011
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Two-component signal transduction systems are found ubiquitously in prokaryotes, and in archaea, fungi, yeast and some plants, where they regulate physiologic and molecular processes at both transcriptional and post-transcriptional levels. Two-component systems sense changes in environmental conditions when a specific ligand binds to the receptor domain of the histidine kinase sensory component. The structures of many histidine kinase receptors are known, including those which sense extracellular and cytoplasmic signals. In this review, we discuss the basic architecture of two-component signalling circuits, including known system ligands, structure and function of both receptor and signalling domains, the chemistry of phosphotransfer, and cross-talk between different two-component pathways. Given the importance of these systems in regulating cellular responses, many biochemical techniques have been developed for their study and analysis. We therefore also review current methods used to study two-component signalling, including a new affinity-based proteomics approach used to study inducible resistance to the antibiotic vancomycin through the VanSR two-component signal transduction system.
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Antibiotic adjuvants: multicomponent anti-infective strategies.
Expert Rev Mol Med
PUBLISHED: 02-24-2011
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The unremitting emergence of multidrug-resistant bacterial pathogens highlights a matching need for new therapeutic options. For example, new carbapenemases such as KPC (class A Klebsiella pneumoniae) and NDM-1 (New Delhi metallo-?-lactamase 1) are surfacing, resulting in almost total resistance to ?-lactam antibiotics. Furthermore, resistance is quickly disseminated, not only in the healthcare sector, but also within the community at large, because many resistance determinants are carried on mobile genetic elements readily shared among pathogens. The absence of new antibiotics has led to a growing reliance on older, more toxic drugs such as colistin, but resistance to these is already arising. One approach to combat this growing problem is the use of combination drug antibiotic adjuvant therapy, which potentiates the activity of antibiotics. Here, we review the current situation and discuss potential drug combinations that may increase the potency of antibiotics in the future. Adjuvant therapies include antibiotic combinations, synergy between antibiotics and nonantibiotics, inhibition of resistance and molecules that alter the physiology of antibiotic-insensitive cells, such as those in biofilms. We provide a rationale for these multicomponent strategies, highlighting current research and important considerations for their clinical use and pharmacological properties.
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An ecological perspective of microbial secondary metabolism.
Curr. Opin. Biotechnol.
PUBLISHED: 02-16-2011
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Bacteria and fungi produce a remarkable array of bioactive small molecules. Many of these have found use in medicine as chemotherapies to treat diseases ranging from infection and cancer to hyperlipidemia and autoimmune disorders. The applications may or may not reflect the actual targets for these compounds. Through careful studies of microbes, their associated molecules and their targets, a growing understanding of the ecology of microbial secondary metabolism is emerging that exposes the central role of secondary metabolites in many complex biological systems.
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Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy.
Nat. Chem. Biol.
PUBLISHED: 02-10-2011
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Combinations of antibiotics are commonly used in medicine to broaden antimicrobial spectrum and generate synergistic effects. Alternatively, combination of nonantibiotic drugs with antibiotics offers an opportunity to sample a previously untapped expanse of bioactive chemical space. We screened a collection of drugs to identify compounds that augment the activity of the antibiotic minocycline. Unexpected synergistic drug combinations exhibited in vitro and in vivo activity against bacterial pathogens, including multidrug-resistant isolates.
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Molecular mechanisms of antibiotic resistance.
Chem. Commun. (Camb.)
PUBLISHED: 02-01-2011
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Over the past decade, resistance to antibiotics has emerged as a crisis of global proportion. Microbes resistant to many and even all clinically approved antibiotics are increasingly common and easily spread across continents. At the same time there are fewer new antibiotic drugs coming to market. We are reaching a point where we are no longer able to confidently treat a growing number of bacterial infections. The molecular mechanisms of drug resistance provide the essential knowledge on new drug development and clinical use. These mechanisms include enzyme catalyzed antibiotic modifications, bypass of antibiotic targets and active efflux of drugs from the cell. Understanding the chemical rationale and underpinnings of resistance is an essential component of our response to this clinical challenge.
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Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole.
Mol. Syst. Biol.
PUBLISHED: 01-11-2011
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Resistance to widely used fungistatic drugs, particularly to the ergosterol biosynthesis inhibitor fluconazole, threatens millions of immunocompromised patients susceptible to invasive fungal infections. The dense network structure of synthetic lethal genetic interactions in yeast suggests that combinatorial network inhibition may afford increased drug efficacy and specificity. We carried out systematic screens with a bioactive library enriched for off-patent drugs to identify compounds that potentiate fluconazole action in pathogenic Candida and Cryptococcus strains and the model yeast Saccharomyces. Many compounds exhibited species- or genus-specific synergism, and often improved fluconazole from fungistatic to fungicidal activity. Mode of action studies revealed two classes of synergistic compound, which either perturbed membrane permeability or inhibited sphingolipid biosynthesis. Synergistic drug interactions were rationalized by global genetic interaction networks and, notably, higher order drug combinations further potentiated the activity of fluconazole. Synergistic combinations were active against fluconazole-resistant clinical isolates and an in vivo model of Cryptococcus infection. The systematic repurposing of approved drugs against a spectrum of pathogens thus identifies network vulnerabilities that may be exploited to increase the activity and repertoire of antifungal agents.
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Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
J. Biol. Chem.
PUBLISHED: 11-17-2010
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The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 ? resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2?) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.
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The genomic enzymology of antibiotic resistance.
Annu. Rev. Genet.
PUBLISHED: 09-09-2010
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The need for new antibiotic therapies is acute and growing in large part because of the emergence of drug-resistant pathogens. A vast number of resistance determinants are, however, found in nonpathogenic micro-organisms. The resistance totality in the global microbiota is the antibiotic resistome and includes not only established resistance genes but also genes that have the potential to evolve into resistance elements. We term these proto-resistance genes and hypothesize that they share common ancestry with other functional units known as housekeeping genes. Genomic enzymology is the study of protein structure-function in light of genetic context and evolution of protein superfamilies. This concept is highly applicable to study of antibiotic resistance evolution from proto-resistance elements. In this review, we summarize some of the genomic enzymology evidence for resistance enzymes pointing to common ancestry with genes of other metabolic functions. Genomic enzymology plays a key role in understanding the origins of antibiotic resistance and aids in designing strategies for diagnosis and prevention thereof.
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Antibiotic resistance in the environment: a link to the clinic?
Curr. Opin. Microbiol.
PUBLISHED: 06-21-2010
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The emergence of resistance to all classes of antibiotics in previously susceptible bacterial pathogens is a major challenge to infectious disease medicine. The origin of the genes associated with resistance has long been a mystery. There is a growing body of evidence that is demonstrating that environmental microbes are highly drug resistant. The genes that make up this environmental resistome have the potential to be transferred to pathogens and indeed there is some evidence that at least some clinically relevant resistance genes have originated in environmental microbes. Understanding the extent of the environmental resistome and its mobilization into pathogenic bacteria is essential for the management and discovery of antibiotics.
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The antibiotic resistome.
Expert Opin Drug Discov
PUBLISHED: 06-17-2010
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Antibiotics are essential for the treatment of bacterial infections and are among our most important drugs. Resistance has emerged to all classes of antibiotics in clinical use. Antibiotic resistance has, proven inevitable and very often it emerges rapidly after the introduction of a drug into the clinic. There is, therefore, a great interest in understanding the origins, scope and evolution of antibiotic resistance.
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Acyldepsipeptide antibiotics induce the formation of a structured axial channel in ClpP: A model for the ClpX/ClpA-bound state of ClpP.
Chem. Biol.
PUBLISHED: 05-12-2010
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In ClpXP and ClpAP complexes, ClpA and ClpX use the energy of ATP hydrolysis to unfold proteins and translocate them into the self-compartmentalized ClpP protease. ClpP requires the ATPases to degrade folded or unfolded substrates, but binding of acyldepsipeptide antibiotics (ADEPs) to ClpP bypasses this requirement with unfolded proteins. We present the crystal structure of Escherichia coli ClpP bound to ADEP1 and report the structural changes underlying ClpP activation. ADEP1 binds in the hydrophobic groove that serves as the primary docking site for ClpP ATPases. Binding of ADEP1 locks the N-terminal loops of ClpP in a ?-hairpin conformation, generating a stable pore through which extended polypeptides can be threaded. This structure serves as a model for ClpP in the holoenzyme ClpAP and ClpXP complexes and provides critical information to further develop this class of antibiotics.
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Nucleotide selectivity of antibiotic kinases.
Antimicrob. Agents Chemother.
PUBLISHED: 03-15-2010
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Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3)-IIIa, APH(2)-Ib, and MPH(2)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3)-IIIa exclusively uses ATP, MPH(2)-I exclusively uses GTP, and APH(2)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.
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Crystallization and preliminary X-ray crystallographic analysis of the tetracycline-degrading monooxygenase TetX2 from Bacteroides thetaiotaomicron.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 03-12-2010
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The flavin-dependent monooxygenase TetX2 from Bacteroides thetaiotaomicron confers resistance against tetracyclines in aerobically grown Escherichia coli. TetX2 modifies several tetracycline antibiotics by regioselective hydroxylation of the substrates to 11a-hydroxy-tetracyclines. X-ray diffraction data were collected from a native TetX2 crystal and a TetX2 crystal with incorporated selenomethionine to resolutions of 2.5 and 3.0 A, respectively. The native crystal belonged to the triclinic space group P1, with unit-cell parameters a = 68.55, b = 80.88, c = 87.53 A, alpha = 111.09, beta = 98.98, gamma = 93.38 degrees , whereas the selenomethionine-labelled TetX2 crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 87.34, b = 68.66, c = 152.48 A, beta = 101.08 degrees .
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Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1.
J. Antibiot.
PUBLISHED: 03-12-2010
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The bacterial genus Streptomyces is endowed with a remarkable secondary metabolism that generates an enormous number of bioactive small molecules. Many of these genetically encoded small molecules are used as antibiotics, anticancer agents and as other clinically relevant therapeutics. The rise of resistant pathogens has led to calls for renewed efforts to identify antimicrobial activities, including expanded screening of streptomycetes. Indeed, it is known that most strains encode >20 secondary metabolites and that many, perhaps most of these, have not been considered for their possible therapeutic use. One roadblock is that many strains do not express their secondary metabolic gene clusters efficiently under laboratory conditions. As one approach to this problem, we have used alleles of a pleiotropic regulator of secondary metabolism from Streptomyces coelicolor to activate secondary biosynthetic gene clusters in heterologous streptomycetes. In one case, we demonstrate the activation of pulvomycin production in S. flavopersicus, a metabolite not previously attributed to this species. We find that the absA1-engineered strains produced sufficient material for purification and characterization. As a result, we identified new, broad-spectrum antimicrobial activities for pulvomycin, including a potent antimicrobial activity against highly antibiotic-resistant Gram-negative and Gram-positive pathogens.
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A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor.
Nat. Chem. Biol.
PUBLISHED: 03-09-2010
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Inducible resistance to the glycopeptide antibiotic vancomycin requires expression of vanH, vanA and vanX, controlled by a two-component regulatory system consisting of a receptor histidine kinase, VanS, and a response regulator, VanR. The identity of the VanS receptor ligand has been debated. Using a synthesized vancomycin photoaffinity probe, we show that vancomycin directly binds Streptomyces coelicolor VanS (VanSsc) and this binding is correlated with resistance and required for vanH, vanA and vanX gene expression.
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Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli.
Biochemistry
PUBLISHED: 01-07-2010
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Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.
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Making sense of antisense in antibiotic drug discovery.
Cell Host Microbe
PUBLISHED: 09-15-2009
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In an era of increasing antibiotic resistance, growing clinical need for new drugs, and few lead molecules, new research is helping to identify new targets and the mechanism of action of lead molecules.
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Structure and mechanism of the lincosamide antibiotic adenylyltransferase LinB.
Structure
PUBLISHED: 08-07-2009
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Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.
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The tetracycline resistome.
Cell. Mol. Life Sci.
PUBLISHED: 07-06-2009
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Resistance to tetracycline emerged soon after its discovery six decades ago. Extensive clinical and non-clinical uses of this class of antibiotic over the years have combined to select for a large number of resistant determinants, collectively termed the tetracycline resistome. In order to impart resistance, microbes use different molecular mechanisms including target protection, active efflux, and enzymatic degradation. A deeper understanding of the structure, mechanism, and regulation of the genes and proteins associated with tetracycline resistance will contribute to the development of tetracycline derivatives that overcome resistance. Newer generations of tetracyclines derived from engineering of biosynthetic genetic programs, semi-synthesis, and in particular recent developments in their chemical synthesis, together with a growing understanding of resistance, will serve to retain this class of antibiotic to combat pathogens.
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Isolation of flavonoids from the heartwood and resin of Prunus avium and some preliminary biological investigations.
Phytochemistry
PUBLISHED: 05-21-2009
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An investigation of the constituents in heartwood and resin of Prunus avium is reported. A mini-library of structurally diverse flavanones and flavones was screened for human cytochrome P450 1A1, 3A4 and 19 (aromatase) inhibition, and for antifungal activity against a panel of pathogenic fungi. The defensive role of these natural plant flavonoids as antifungal phytoalexins and phytoanticipins is discussed.
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Noncanonical vancomycin resistance cluster from Desulfitobacterium hafniense Y51.
Antimicrob. Agents Chemother.
PUBLISHED: 05-04-2009
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The glycopeptide vancomycin is a drug of last resort for infection with gram-positive organisms, and three genes are vital to resistance: vanH, vanA, and vanX. These genes are found in a vanHAX cluster, which is conserved across pathogenic bacteria, glycopeptide antibiotic producers, and other environmental bacteria. The genome sequence of the anaerobic, gram-positive, dehalogenating bacterium Desulfitobacterium hafniense Y51 revealed a predicted vanA homolog; however, it exists in a vanAWK-murFX cluster, unlike those of other vancomycin-resistant organisms. Using purified recombinant VanA from D. hafniense Y51, we determined its substrate specificity and found it to have a 42-fold preference for D-lactate over D-alanine, confirming its activity as a D-Ala-D-Lac ligase and its annotation as VanA. Furthermore, we showed that D. hafniense Y51 is highly resistant to vancomycin, with a MIC for growth of 64 microg/ml. Finally, vanA(Dh) is expressed during growth in vancomycin, as demonstrated by reverse transcription-PCR. This finding represents a new glycopeptide antibiotic resistance gene cluster and expands the genetic diversity of resistance to this important class of antibiotic.
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Diversity-oriented synthesis and preliminary biological screening of highly substituted five-membered lactones and lactams originating from an allyboration of aldehydes and imines.
J Comb Chem
PUBLISHED: 02-28-2009
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alpha-exo-Methylene-gamma-lactones and alpha-exo-methylene-gamma-lactams are key structural units in a wide variety of natural products. These substances exhibit a high degree of bioactivity against numerous biological targets that play important roles in several diseases. A library of functionalized gamma-lactones and gamma-lactams containing both unsaturated and saturated side chains at the alpha position of the ring was synthesized. The generation of this library first involves sequential allylation of aldehydes or imines with 2-alkoxycarbonyl allylboronates, followed by ring closure to give alpha-exo-methylene-gamma-lactones or alpha-exo-methylene-gamma-lactams, which are subjected to various transition metal catalyzed coupling reactions to introduce additional diversity. A subset of the library was screened for inhibition of homoserine transacetylase (HTA) from Haemophilus influenzae and showed promising initial activity profiles.
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Structure and function of the glycopeptide N-methyltransferase MtfA, a tool for the biosynthesis of modified glycopeptide antibiotics.
Chem. Biol.
PUBLISHED: 02-06-2009
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There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.
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Opportunities for Synthetic Biology in Antibiotics: Expanding Glycopeptide Chemical Diversity.
ACS Synth Biol
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Synthetic biology offers a new path for the exploitation and improvement of natural products to address the growing crisis in antibiotic resistance. All antibiotics in clinical use are facing eventual obsolesce as a result of the evolution and dissemination of resistance mechanisms, yet there are few new drug leads forthcoming from the pharmaceutical sector. Natural products of microbial origin have proven over the past 70 years to be the wellspring of antimicrobial drugs. Harnessing synthetic biology thinking and strategies can provide new molecules and expand chemical diversity of known antibiotic scaffolds to provide much needed new drug leads. The glycopeptide antibiotics offer paradigmatic scaffolds suitable for such an approach. We review these strategies here using the glycopeptides as an example and demonstrate how synthetic biology can expand antibiotic chemical diversity to help address the growing resistance crisis.
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Glycopeptide sulfation evades resistance.
J. Bacteriol.
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The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compounds ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.
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Bacterial inactivation of the anticancer drug doxorubicin.
Chem. Biol.
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Microbes are exposed to compounds produced by members of their ecological niche, including molecules with antibiotic or antineoplastic activities. As a result, even bacteria that do not produce such compounds can harbor the genetic machinery to inactivate or degrade these molecules. Here, we investigated environmental actinomycetes for their ability to inactivate doxorubicin, an aminoglycosylated anthracycline anticancer drug. One strain, Streptomyces WAC04685, inactivates doxorubicin via a deglycosylation mechanism. Activity-based purification of the enzymes responsible for drug inactivation identified the NADH dehydrogenase component of respiratory electron transport complex I, which was confirmed by gene inactivation studies. A mechanism where reduction of the quinone ring of the anthracycline by NADH dehydrogenase leads to deglycosylation is proposed. This work adds anticancer drug inactivation to the enzymatic inactivation portfolio of actinomycetes and offers possibilities for novel applications in drug detoxification.
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The origins of antibiotic resistance.
Handb Exp Pharmacol
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Antibiotics remain one of our most important pharmacological tools for the control of infectious disease. However, unlike most other drugs, the use of antibiotics selects for resistant organisms and erodes their clinical utility. Resistance can emerge within populations of bacteria by mutation and be retained by subsequent selection or by the acquisition of resistance elements laterally from other organisms. The source of these resistance genes is only now being understood. The evidence supports a large bacterial resistome-the collection of all resistance genes and their precursors in both pathogenic and nonpathogenic bacteria. These genes have arisen by various means including self-protection in the case of antibiotic producers, transport of small molecules for various reasons including nutrition and detoxification of noxious chemicals, and to accomplish other goals, such as metabolism, and demonstrate serendipitous selectivity for antibiotics. Regardless of their origins, resistance genes can rapidly move through bacterial populations and emerge in pathogenic bacteria. Understanding the processes that contribute to the evolution and selection of resistance is essential to mange current stocks of antibiotics and develop new ones.
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Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to ?-lactams.
ACS Chem. Biol.
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Rising drug resistance is limiting treatment options for infections by methicillin-resistant Staphylococcus aureus (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie ?-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of ?-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking tarO. The combination was also effective in a Galleria mellonella model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the N-acetylglucosamine-1-phosphate transferase encoded in gene tarO and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of S. aureus. This approach represents a new paradigm to tackle MRSA infection.
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Characterization of a rifampin-inactivating glycosyltransferase from a screen of environmental actinomycetes.
Antimicrob. Agents Chemother.
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Identifying and understanding the collection of all antibiotic resistance determinants presented in the global microbiota, the antibiotic resistome, provides insight into the evolution of antibiotic resistance and critical information for the development of future antimicrobials. The rifamycins are broad-spectrum antibiotics that target bacterial transcription by inhibition of RNA polymerase. Although mutational alteration of the drug target is the predominant mechanism of resistance to this family of antibiotics in the clinic, a number of diverse inactivation mechanisms have also been reported. In this report, we investigate a subset of environmental rifampin-resistant actinomycete isolates and identify a diverse collection of rifampin inactivation mechanisms. We describe a single isolate, WAC1438, capable of inactivating rifampin by glycosylation. A draft genome sequence of WAC1438 (most closely related to Streptomyces speibonae, according to a 16S rRNA gene comparison) was assembled, and the associated rifampin glycosyltransferase open reading frame, rgt1438, was identified. The role of rgt1438 in rifampin resistance was confirmed by its disruption in the bacterial chromosome, resulting in a loss of antibiotic inactivation and a 4-fold decrease in MIC. Interestingly, examination of the RNA polymerase ?-subunit sequence of WAC1438 suggests that it harbors a resistant target and thus possesses dual mechanisms of rifamycin resistance. Using an in vitro assay with purified enzyme, Rgt1438 could inactivate a variety of rifamycin antibiotics with comparable steady-state kinetics constants. Our results identify rgt1438 as a rifampin resistance determinant from WAC1438 capable of inactivating an assortment of rifamycins, adding a new element to the rifampin resistome.
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Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold.
Proc. Natl. Acad. Sci. U.S.A.
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Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of anti-infective agents in the treatment of serious gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3-phosphoadenosine 5-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3-phosphoadenosine 5-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.
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A forward chemical screen identifies antibiotic adjuvants in Escherichia coli.
ACS Chem. Biol.
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Multi-drug-resistant infections caused by Gram-negative pathogens are rapidly increasing, highlighting the need for new chemotherapies. Unlike Gram-positive bacteria, where many different chemical classes of antibiotics show efficacy, Gram-negatives are intrinsically insensitive to many antimicrobials including the macrolides, rifamycins, and aminocoumarins, despite intracellular targets that are susceptible to these drugs. The basis for this insensitivity is the presence of the impermeant outer membrane of Gram-negative bacteria in addition to the expression of pumps and porins that reduce intracellular concentrations of many molecules. Compounds that sensitize Gram-negative cells to "Gram-positive antibiotics", antibiotic adjuvants, offer an orthogonal approach to addressing the crisis of multi-drug-resistant Gram-negative pathogens. We performed a forward chemical genetic screen of 30,000 small molecules designed to identify such antibiotic adjuvants of the aminocoumarin antibiotic novobiocin in Escherichia coli. Four compounds from this screen were shown to be synergistic with novobiocin including inhibitors of the bacterial cytoskeleton protein MreB, cell wall biosynthesis enzymes, and DNA synthesis. All of these molecules were associated with altered cell shape and small molecule permeability, suggesting a unifying mechanism for these antibiotic adjuvants. The potential exists to expand this approach as a means to develop novel combination therapies for the treatment of infections caused by Gram-negative pathogens.
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Antibiotic resistance is prevalent in an isolated cave microbiome.
PLoS ONE
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Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.
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Diversity of integron- and culture-associated antibiotic resistance genes in freshwater floc.
Appl. Environ. Microbiol.
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Clinically important antibiotic resistance genes were detected in culturable bacteria and class 1 integron gene cassettes recovered from suspended floc, a significant aquatic repository for microorganisms and trace elements, across freshwater systems variably impacted by anthropogenic activities. Antibiotic resistance gene cassettes in floc total community DNA differed appreciably in number and type from genes detected in bacteria cultured from floc. The number of floc antibiotic resistance gene cassette types detected across sites was positively correlated with total (the sum of Ag, As, Cu, and Pb) trace element concentrations in aqueous solution and in a component of floc readily accessible to bacteria. In particular, concentrations of Cu and Pb in the floc component were positively correlated with floc resistance gene cassette diversity. Collectively, these results identify suspended floc as an important reservoir, distinct from bulk water and bed sediment, for antibiotic resistance in aquatic environments ranging from heavily impacted urban sites to remote areas of nature reserves and indicate that trace elements, particularly Cu and Pb, are geochemical markers of resistance diversity in this environmental reservoir. The increase in contamination of global water supplies suggests that aquatic environments will become an even more important reservoir of clinically important antibiotic resistance in the future.
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Mechanism and diversity of the erythromycin esterase family of enzymes.
Biochemistry
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Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.
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Determining the mode of action of bioactive compounds.
Bioorg. Med. Chem.
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Matching bioactive molecules with molecular targets is key to understanding their modes of action (MOA). Moving beyond the mere discovery of drugs, investigators are now just beginning to integrate both biochemical and chemical-genetic approaches for MOA studies. Beginning with simple screens for changes in cell phenotype upon drug treatment, drug bioactivity has been traditionally explored with affinity chromatography, radiolabeling, and cell-based affinity tagging procedures. However, such approaches can present an oversimplified view of MOA, especially in light of the recent realization of the extent of polypharmacology and the unexpected complexity of drug-target interactions. With the advent of more sophisticated tools for genetic manipulation, a flood of powerful techniques has been used to create characteristic drug MOA fingerprints. In particular, whole genome expression profiling and deletion and overexpression libraries have greatly enhanced our understanding of bioactive compounds in vivo. Here we highlight challenges and advances in studying bioactive compound-target interactions.
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Antibiotic resistance is ancient: implications for drug discovery.
Trends Microbiol.
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An unfailing observation over the past 70 years is that resistance to all antibiotics emerges eventually after use in the clinic. Where does this resistance come from? Recent work has shown that antibiotic resistance genes are common in metagenomes of ancient sediments. This prevalence of resistance, well before the use of antibiotics, denotes the importance of taking microbial chemical ecology and deep metagenomic profiling into account in the development and use of antibiotics.
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Antibiotics: a new hope.
Chem. Biol.
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Antibiotic resistance is one of the most significant challenges to the health care sector in the 21st century. A myriad of resistance mechanisms have emerged over the past decades and are widely disseminated worldwide through bacterial populations. At the same time there have been ever fewer new antibiotics brought to market, and the pharmaceutical industry increasingly sees antibiotics as a poor investment. Paradoxically, we are in a Golden Age of understanding how antibiotics work and where resistance comes from. This knowledge is fueling a renaissance of interest and innovation in antibiotic discovery, synthesis, and mechanism that is poised to inform drug discovery to address pressing clinical needs.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.