http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_1 The complete genome sequences of about 300 bacteria (mostly pathogenic) have been determined, and many more such projects are currently in progress. The detection of bacterial genes that are non-homologous to human genes and are essential for the survival of the pathogen represent a promising means of identifying novel drug targets. We present a subtractive genomics approach for the identification of putative drug targets in microbial genomes and demonstrate its execution using Pseudomonas aeruginosa as an example. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This strategy enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. It should be recognized that there are limitations to this computational approach for drug target identification. Distant gene relationships may be missed since the alignment scores are likely to have low statistical significance. In conclusion, the results of such a strategy underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_10 The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_11 Key enzymes that assemble the bacterial cell wall are also the target of the Β-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_12 This chapter is orgTreatment of Gram-negative bacterial infections is complicated by innate and acquired drug resistance resulting in a limited number of effective antibiotics. Several Gram-negative bacteria, for which current therapies are ineffective, have recently been identified as potential bioterror agents. These findings highlight the need for new antibiotics, specifically antibiotics that act on new drug targets to circumvent drug resistance. Potential targets in Gram-negative bacteria include enzymes involved in the biosynthesis of lipopolysaccharides (LPS) that form outer membranes of these organisms. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the committed step in the biosynthesis of the lipid A portion of LPS. Therefore, inhibitors of this enzyme have the potential to serve as antibiotics, and efforts toward the development of LpxC inhibitors are currently underway. Here we describe methods for assaying LpxC inhibitors, including methods for measuring deacetylase activity and binding affinity for LpxC, which will be useful for the development of LpxC inhibitors. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_13 Widespread resistance to antibiotics in current clinical use is increasing at an alarming rate. Novel approaches in antimicrobial therapy will be required in the near future to maintain control of infectious diseases. An enormous array of small cationic peptides exists in nature as part of the innate defense systems of organisms ranging from bacteria to humans. For most naturally occurring linear peptides, such as magainins and cecropins, a common feature is their capacity to form an amphipathic α-helix (with polar and nonpolar groups on opposite faces of the helix), a structural feature believed to be important in their antimicrobial function as membrane-lytic agents. A massive effort over the past two decades has resulted in a better understanding of the molecular mechanism of antimicrobial peptides and the production of more potent analogues. To date, however, few of these peptides have been shown to have clinical efficacy, especially for systemic use, in large part due to insufficient selectivity between target and host cells. Recently, we developed a new strategy in the design of antimicrobial peptides. These linear cationic peptides, which form amphipathic β-sheets rather than α-helices, demonstrated superior selectivity in binding to the lipids contained in bacterial vs. mammalian plasma membranes. Here we describe methods to evaluate the structure and function of cationic antimicrobial peptides. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_14 This chapter describes reliable flow cytometric methods for assessment of two important physiologic characteristics of bacteria, membrane potential and membrane permeability, which can provide indications of the effects of antimicrobial agents on microorganisms. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_15 Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mechanisms utilized by more than 15 species of Gram-negative bacterial cells is the Resistance Nodulation Division (RND) efflux pump, which eliminates several classes of antibiotics such as penicillins and cephalosporin macrolides aminoglycosides, fluoroquinolonesx and tetracyclines. Here we describe a multistep process to identify compounds that inhibit the RND-type efflux pumps. This involves measuring the inhibition of accumulation of ethidium bromide in E. coli or Haemophilus influenzae cells and confirming that the inhibition is specific for the efflux pumps by using genetic constructs and biochemical methods to measure nonspecific inhibition due to e.g. intrinsic antibacterial activity or membrane disruption. In whole bacterial cells synergism antagonism or indifference of the combination of an antibiotic with the putative inhibitor is determined and this is then confirmed by quantitating viable bacterial cells in liquid culture over24h. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_16 Fatty acid biosynthesis is one of the relatively newer targets in antibacterial drug discovery. The presence of distinct fatty acid synthases (FAS) in mammals and bacteria and the fact that most bacterial FAS enzymes are essential for viability make this a very attractive antimicrobial drug target. The enzyme β-ketoacyl ACP synthase (KASIII or FabH) is the key enzyme that initiates fatty acid biosynthesis in a type II dissociated FAS. This enzyme catalyzes the condensation of acyl CoA and malonyl ACP (acyl carrier protein) to form a β-ketoacyl ACP product, which is further processed to form mature fatty acids that are involved in various essential cellular processes and structures like phospholipid biosynthesis, cell wall formation, etc. Herein we describe a new assay for the Mycobacterium tuberculosis FabH (mtFabH) enzyme involved in a key initiation step in the synthesis of mycolic acids, which are an integral component of the cell wall. The assay eliminates the need for the cumbersome washing steps or specialty scintillation proximity assay beads and the preparation of acyl carrier proteins required in other assay formats. This discontinuous assay involves the reduction of radiolabled long-chain β-ketoacyl CoA product to its dihydroxy derivative, which partitions into a nonpolar phase for quantitation, while the reduced radiolabeled substrate derivative remains in the aqueous phase. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_17 Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups–-sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination were found to act elsewhere instead; a possible reason for this is that full-length sensor kinases are integral membrane proteins whose activity might reflect interaction with the cell membrane or with membrane components. We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_18 Resistance to antibiotics that target the bacterial ribosome is often conferred by methylation at specific nucleotides in the rRNA. The nucleotides that become methylated are invariably key sites of antibiotic interaction. The addition of methyl groups to each of these nucleotides is catalyzed by a specific methyltransferase enzyme. The Erm methyltransferases are a clinically prevalent group of enzymes that confer resistance to the therapeutically important macrolide, lincosamide, and streptogramin B (MLSB) antibiotics. The target for Erm methyltransferases is at nucleotide A2058 in 23S rRNA, and methylation occurs before the rRNA has been assembled into 50S ribosomal particles. Erm methyltransferases occur in a phylogenetically wide range of bacteria and differ in whether they add one or two methyl groups to the A2058 target. The dimethylated rRNA confers a more extensive MLSB resistance phenotype. We describe here a method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to determine the location and number of methyl groups added at any site in the rRNA. The method is particularly suited to studying in vitro methylation of RNA transcripts by resistance methyltransferases such as Erm. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_19 The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn+2-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of β-lactam antibiotics in clinical medicine. This chapter concerns procedures for the evaluation of the catalytic activity of β-lactamases as a means to screen compounds for their inhibitory potency. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_2 DNA gyrase and DNA topoisomerase (topo) IV are the bacterial targets of coumarin and quinolone antimicrobial agents. Widespread resistance to clinically important antibiotics such as beta-lactams and macrolides has stimulated the development of novel gyrase and topo IV inhibitors especially against Streptococcus pneumoniae and other Gram-positive pathogens. Here, we describe how gyrase and topo IV activities are measured and how inhibitors of these enzymes may be assayed, focusing as a paradigm on DNA supercoiling by S. pneumoniae gyrase, DNA decatenation by S. pneumoniae topo IV, and DNA cleavage by both enzymes. These approaches provide mechanistic insight on inhibitor action and allow identification of dual gyrase/topo IV targeting agents that can minimize the emergence of bacterial resistance. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_20 Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_3 The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_4 RNA polymerase is essential to the viability of bacteria in all phases of growth and development and is a proven chemotherapeutic target as the cellular target of the rifamycin class of antibiotics. However, despite the characterization of multiple different classes of natural products that selectively target bacterial RNA polymerase, and the identification of a limited number of synthetic compound inhibitors, only agents of the rifamycin class have been developed and approved for human clinical use as antibiotics. Herein we describe a scintillation proximity assay (SPA) for identifying and characterizing inhibitors of bacterial RNA polymerases and that is applicable to de novo drug discovery programs through application of automated high-throughput screening methods. In addition, we describe gel electrophoresis-based methods that are applicable to the detailed characterization of inhibitors of transcriptional initiation or elongation by bacterial RNA polymerases. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_5 Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We focused on phenylalanyl (Phe)-tRNA synthetase (Phe-RS), but the described methods are not restricted to Phe-RS and might be adapted to other aa-RS. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_6 The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_7 In Escherichia coli, the molecular chaperone HSP70 (DnaK) is necessary for 30S and 50S ribosomal subunit assembly at temperatures above 37°C. Inhibitors of DnaK should therefore hinder ribosome biogenesis, in addition to all of the other DnaK-dependent cellular functions. An easily testable phenotype of DnaK is described here based on α-complementation of β-galactosidase. This protein fragment complementation requires a functional DnaK in vivo, offering a suitable method for screening for DnaK inhibitors. Subsequently, it will be of great importance to check whether inhibitors of bacterial DnaK selected in this way have an effect (inhibitory or stimulatory) on the activities of eukaryotic HSP70 and HSC70 chaperones, because of the universal conservation in all biota of these chaperones in both their structural and functional properties. This question is important due to their implication in many pathways in immunology, cancer biology, and neurodegenerative disorders. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_8 While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, “universal” model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this “universal” mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-246-5_9 The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the peptidyl transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The peptidyl transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the radiolabled product, now joined to the biotin group, using the scintillation proximity assay technology. Binding of the tritiated radioligand to streptavidin-coated beads causes the excitation of the bead-embedded scintillant, thus resulting in the detection of radioactivity. Since no product purification step is required, SPARK is amenable to simple automation, which makes it useful in high-throughput screens of natural or synthetic compound libraries in the search for novel antibiotics. 2007-12-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-570-1_1 Forward genetics has led to many “breakthrough” discoveries, and with the mouse genome almost fully sequenced, the creation of phenotypes through random germline mutagenesis has become an efficient means by which to find the function of yet undescribed genes. In this chapter, we will provide a practical guideline for performing germline mutagenesis in mice. In particular, we will focus on the application of this technology to identify genes that are essential to innate immune defense. 2007-11-19T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-570-1_10 Dendritic cells (DC) are widely regarded as the most potent cellular inducers of the adaptive immune response; so, immunotherapy through DC manipulation is a promising option in the future fight against many human ailments. We have developed a method of isolating DC from the mouse that involves efficient extraction from tissues, followed by the selection of the lightest density cells, then depletion of non-DC through a cocktail of monoclonal antibodies and anti-immunoglobulin magnetic beads. Finally, purification and segregation into DC subtypes is achieved by immunofluorescent labeling and sorting. This has demonstrated a network of DC populations differing in surface phenotype and function. We can now produce larger numbers of many of these DC subpopulations from their precursors using bone marrow cultures supplemented with fms-like tyrosine kinase 3 ligand (Flt3L). The culture-generated DC can be aligned with the populations directly isolated from tissues. Combining the in vivo and in vitro systems will make study of murine DC and their alignment to their human counterparts an easier break process. 2007-11-19T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-570-1_11 Typical assays for natural killer (NK) cell function assess the responses of entire NK cell populations. It is now possible to determine the responses of individual NK cells. Herein, two representative assays are described along with examples of how they have helped clarify current understanding of NK cell biology. 2007-11-19T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-570-1_12 Natural killer (NK) cells were originally defined as mediators of spontaneous cytotoxicity against virus-infected and tumor cells. In human peripheral blood, the majority of NK cells can mediate cell lysis mainly through perforin and granzymes. It has been recently shown, however, that the majority of NK cells in human secondary lymphoid organs are primarily immunoregulatory by secreting cytokines immediately after activation and do not express perforin and granzymes. Because lymph nodes (LN) harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized NK cell compartment in LN and related tissues probably outnumbers perforin+cytolytic NK cells in our body. Although human NK cell biology has so far mainly studied peripheral blood NK cells, we have lately focused on human NK cells harbored in lymphoid tissues and identified procedures for their optimal isolation as well as their phenotypic and functional characterization. 2007-11-19T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-570-1_13 Eosinophils are multifunctional leukocytes classically described as being involved in helminth parasitic infections and allergic diseases. Previously restricted to an exclusive role in the release of cytotoxic mediators, they are now also considered to be immunoregulatory cells and potential effectors in innate immune responses. Eosinophils are mainly found in tissues, so specific procedures are needed for their isolation from venous blood and for functional assays. Murine models are very useful for the dissection of eosinophil physiology in vivo. But murine eosinophils significantly differ from human ones. A complete understanding of eosinophil biology therefore requires comparative study of eosinophils from different mammalian species. We summarize here the main experimental protocols used to study human, mouse, and rat eosinophil biology. We focus on technical improvements of existing methods that optimize purification and in vitro functional studies of eosinophils. 2007-11-19T05:00:00Z