Target For New Antibacterial Drugs Research Articles

Genetic Engineering in Mycobacteria

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

Genetic Engineering in Mycobacteria

Abstract Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

Bacterial biopolymers: from pathogenesis to advanced materials.

Bacteria are prime cell factories that can efficiently convert carbon and nitrogen sources into a large diversity of intracellular and extracellular biopolymers, such as polysaccharides, polyamides, polyesters, polyphosphates, extracellular DNA and proteinaceous components. Bacterial polymers have important roles in pathogenicity, and their varied chemical and material properties make them suitable for medical and industrial applications. The same biopolymers when produced by pathogenic bacteria function as major virulence factors, whereas when they are produced by non-pathogenic bacteria, they become food ingredients or biomaterials. Interdisciplinary research has shed light on the molecular mechanisms of bacterial polymer synthesis, identified new targets for antibacterial drugs and informed synthetic biology approaches to design and manufacture innovative materials. This Review summarizes the role of bacterial polymers in pathogenesis, their synthesis and their material properties as well as approaches to design cell factories for production of tailor-made bio-based materials suitable for high-value applications.

The Genome-Wide Interaction Network of Nutrient Stress Genes in Escherichia coli.

ABSTRACTConventional efforts to describe essential genes in bacteria have typically emphasized nutrient-rich growth conditions. Of note, however, are the set of genes that become essential when bacteria are grown under nutrient stress. For example, more than 100 genes become indispensable when the model bacterium Escherichia coli is grown on nutrient-limited media, and many of these nutrient stress genes have also been shown to be important for the growth of various bacterial pathogens in vivo. To better understand the genetic network that underpins nutrient stress in E. coli, we performed a genome-scale cross of strains harboring deletions in some 82 nutrient stress genes with the entire E. coli gene deletion collection (Keio) to create 315,400 double deletion mutants. An analysis of the growth of the resulting strains on rich microbiological media revealed an average of 23 synthetic sick or lethal genetic interactions for each nutrient stress gene, suggesting that the network defining nutrient stress is surprisingly complex. A vast majority of these interactions involved genes of unknown function or genes of unrelated pathways. The most profound synthetic lethal interactions were between nutrient acquisition and biosynthesis. Further, the interaction map reveals remarkable metabolic robustness in E. coli through pathway redundancies. In all, the genetic interaction network provides a powerful tool to mine and identify missing links in nutrient synthesis and to further characterize genes of unknown function in E. coli. Moreover, understanding of bacterial growth under nutrient stress could aid in the development of novel antibiotic discovery platforms.

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria.

Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.

Antibacterial Boom And Bust

When David L. Pompliano joined GlaxoSmithKline in 2002 as head of biology for infectious diseases, the pharmaceutical company was ending a largely unsuccessful multiyear screening program to search for novel antibacterial drugs. The program started in the mid-1990s after the publication of the first bacterial genomes. GSK and other pharmaceutical companies thought these genomes held a trove of new targets for antibacterial drugs. The company examined 300 genes, ran 70 high-throughput screening campaigns, and screened between 300,000 and 500,000 compounds per campaign. After seven years, company scientists found just five drug leads. The success rate was about 20–25% lower than rates for other therapeutic areas in the company (Nat. Rev. Drug Discovery 2007, DOI: 10.1038/nrd2201). Looking back on the vast effort and resources spent on the program, the strategy “certainly did not live up to the promise that everyone was proposing,” Pompliano says. The GSK story exemplifies the difficulties of ...

Peptides as Inhibitors of the First Phosphorylation Step of the Streptomyces coelicolor Phosphoenolpyruvate: Sugar Phosphotransferase System

The phosphotransferase system (PTS) controls the use of sugars in bacteria. The PTS is ubiquitous in bacteria, but it does not occur in plants and animals; it modulates catabolite repression, intermediate metabolism, gene expression, and chemotaxis. Its uniqueness and pleiotropic function make the PTS an attractive target for new antibacterial drugs. The PTS is constituted of two general proteins, namely, enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI has two domains: the N-terminal domain (EIN), which binds to HPr, and the C-terminal domain (EIC), which contains the dimerization interface. In this work, we determined the binding affinities of peptides derived from EIN of Streptomyces coelicolor (EIN(sc)) against HPr of the same organism (HPr(sc)), by using nuclear magnetic resonance and isothermal titration calorimetry techniques. Furthermore, we measured the affinity of EIN(sc) for (i) a peptide derived from HPr(sc), containing the active-site histidine, and (ii) other peptides identified previously by phage display and combinatorial chemistry in Escherichia coli [Mukhija, S. L., et al (1998) Eur. J. Biochem. 254, 433-438; Mukhija, S., and Erni, B. (1997) Mol. Microbiol. 25, 1159-1166]. The affinities were in the range of ~10 μM, being slightly higher for the binding of EIN(sc) with peptides derived from HPr(sc), phage display, or combinatorial chemistry (K(D) ~ 5 μM). Because the affinity of intact EIN(sc) for the whole HPr(sc) is 12 μM, we suggest that the assayed peptides might be considered as good hit compounds for inhibiting the interaction between HPr(sc) and EIN(sc).

Visible fluorescence detection of type III protein secretion from bacterial pathogens.

Type III protein secretion is essential for many gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.

Defining the Epitope Region of a Peptide from the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System Able to Bind to the Enzyme I

The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr 9–30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPr sc. By using fluorescence and circular dichroism, we first determined qualitatively that HPr sc and HPr 9–30 did bind to EI sc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr 9–30 and HPr sc for EI sc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr 9–30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EI sc and HPr sc is enthalpy driven and in other species is entropy driven; further, the affinity of HPr sc for EI sc was smaller than in other species. However, the affinity of HPr 9–30 for EI sc was only moderately lower than that of EI sc for HPr sc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.

The Helical Structure Propensity in the First Helix of the Histidine Phosphocarrier Protein of Streptomyces coelicolor

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), formed by a cascade of several proteins, mediates the uptake and phosphorylation of carbohydrates, and it is also involved in signal transduction. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or proteins fragments able to interfere the first step of the protein cascade: the phosphorylation of the HPr protein by the enzyme EI. We designed a peptide comprising the active site and the first alpha-helix of HPr of S. coelicolor; we also obtained a fragment of HPr by protein engineering methods, comprising the first forty-eight residues and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure ( approximately 7 %), and an identical free energy of helix formation. In 40 % TFE, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2D-NMR assignments of the shorter peptide, and the presence of similar NOE contacts in both fragments. These findings, with the kinetic results in other members of the HPr family, highlight the importance of short- and long-range interactions during the folding reaction of HPr proteins. Based on the residual helical population, hypothesis about the inhibition capacity of the PTS by both fragments are discussed.

Technique Could Help To Identify Novel Antibacterial Targets

A new “predator-prey” system can help in identifying infection-critical genes of bacterial pathogens such as Klebsiella pneumoniae and also which mutants are less capable of producing pneumonia in mice, according to Mohammed Benghezal and his colleagues from the company Athelas and the University of Geneva, both in Geneva, Switzerland. They presented their findings during the poster session “Pathogenesis I” at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) last December in Washington, D.C. Their poster, “New Targets for Antibacterial Drugs,” received the ICAAC Program Committee Award for research excellence.

Drugs that Inhibit Mycolic Acid Biosynthesis in Mycobacterium tuberculosis - An Update

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis, and infects approximately 32 % of the worlds human population. It is estimated that 8.2 million new TB cases occurred worldwide in the year 2000, with approximately 1.8 million deaths in the same year, and more than 95 % of those were in developing countries. The interruption of centuries of decline in case rates of TB occurred, in most cases, in the late 1980s and involved the USA and some European countries due to increased poverty in urban settings and the immigration from TB high-burden countries. Thus, no sustainable control of TB epidemics can be reached in any country without properly addressing the global epidemic. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multi-drug-resistant (MDR) strains have created much scientific interest in developing new antimycobacterial agents to both treat M. tuberculosis strains resistant to existing drugs, and shorten the duration of short-course treatment to improve patient compliance. Bacterial cell-wall biosynthesis is a proven target for new antibacterial drugs. Mycolic acids, which are key components of the mycobacterial cell wall, are α- alkyl, β-hydroxy fatty acids, with a species-dependent saturated "short" arm of 20-26 carbon atoms and a "long" meromycolic acid arm of 50-60 carbon atoms. The latter arm is functionalized at regular intervals by cyclopropyl, α-methyl ketone, or α-methyl methylethers groups, and, as shown more recently, by unsaturations. The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages: (i) synthesis of C20 to C26 straightchain saturated fatty acids to provide the α-alkyl branch; (ii) synthesis of the meromycolic acid chain to provide the main carbon backbone, (iii) modification of this backbone to introduce other functional groups; (iv) the final Claisen-type condensation step followed by reduction; and (v) various mycolyltransferase processes to cellular lipids. The drugs shown to inhibit mycolic acid biosynthesis are isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. Pyrazinamide was thought to inhibit fatty acid synthase type I, thereby reducing the synthesis of precursor of mycolic acids. However, current experimental evidence indicates that it has no defined target of action. The main focus of our contribution is on new data describing the mode of action of antitubercular drugs that inhibit mycolic acid biosynthesis, and description of inhibitors of fatty acid synthase type II enzymes as potential lead compounds that may allow the development of new antitubercular agents. Keywords: tuberculosis, mycobacteria, cell wall, mycolic acids, inhibitors, drugs

X-ray structure of HI0817 from Haemophilus influenzae: protein of unknown function with a novel fold.

The enormous amount of sequence data isbeing derived from 160 completed genomes (http://www.cbs.dtu.dk/services/GenomeAtlas/index.php) andfrom over 170 genome sequencing projects in progress(http://www.ncbi.nlm.nih.gov/genomes/MICROBES/InProgress.html). For many protein sequences, which areannotated as “hypothetical proteins,” neither a functionnor a three-dimensional structure is available. The deter-mination of the structures and functions of these proteinsoffer the basis for important discoveries such as newantibacterial drug targets and a better understanding ofcellularprocesses.OurexperiencewithStructuralGenom-ics of 43 such proteins reveals an enrichment of novel foldscompared with the general set of proteins entering theProtein Data Bank (PDB).As of this writing, the

Bacterial mimics of eukaryotic GTPase-activating proteins (GAPs)

Bacterial GTPase-activating proteins (GAPs) subvert their host's eukaryotic Rho GTPases to their own advantage. Studies of bacterial GAPs extend our understanding of the action of eukaryotic GAPs, provide new tools for studies of cytoskeletal dynamics and offer new targets for anti-bacterial drugs.

Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.

Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis.

Tuberculosis resurged in the late 1980s and now kills more than 2 million people a year. The reemergence of tuberculosis as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, and the proliferation of multi-drug-resistant (MDR) strains have created much scientific interest in developing new antimycobacterial agents to both treat Mycobacterium tuberculosis strains resistant to existing drugs, and shorten the duration of short-course treatment to improve patient compliance. Bacterial cell-wall biosynthesis is a proven target for new antibacterial drugs. Mycolic acids, which are key components of the mycobacterial cell wall, are alpha-alkyl, beta-hydroxy fatty acids, with a species-dependent saturated "short" arm of 20-26 carbon atoms and a "long" meromycolic acid arm of 50-60 carbon atoms. The latter arm is functionalized at regular intervals by cyclopropyl, alpha-methyl ketone, or alpha-methyl methylethers groups. The mycolic acid biosynthetic pathway has been proposed to involve five distinct stages: (i) synthesis of C20 to C26 straight-chain saturated fatty acids to provide the alpha-alkyl branch; (ii) synthesis of the meromycolic acid chain to provide the main carbon backbone, (iii) modification of this backbone to introduce other functional groups; (iv) the final Claisen-type condensation step followed by reduction; and (v) various mycolyltransferase processes to cellular lipids. The drugs shown to inhibit mycolic acid biosynthesis are isoniazid, ethionamide, isoxyl, thiolactomycin, and triclosan. In addition, pyrazinamide was shown to inhibit fatty acid synthase type I which, in turn, provides precursors for fatty acid elongation to long-chain mycolic acids by fatty acid synthase II. Here we review the biosynthesis of mycolic acids and the mechanism of action of antimicrobial agents that act upon this pathway. In addition, we describe molecular modeling studies on InhA, the bona-fide target for isoniazid, which should improve our understanding of the amino acid residues involved in the enzyme's mechanism of action and, accordingly, provide a rational approach to the design of new drugs.

Mutational Analysis of UMP Kinase from Escherichia coli

UMP kinase from Escherichia coli is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of E. coli UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).

Phage display selection of peptides against enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system (PTS).

The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. Uniqueness and pleiotropic function make the PTS a target for new antibacterial drugs. Enzyme I is the first component of the divergent protein phosphorylation cascade of the PTS. It transfers phosphoryl groups from phosphoenolpyruvate to the general phosphoryl carrier protein HPr. Six 15-mer, nine 10-mer and nine 6-mer peptides that inhibit enzyme I were selected from phage display libraries. Of these, 16 were synthesized and characterized. The majority of the peptides contain a histidine with an adjacent arginine. Two peptides were found to contain cysteines but no histidine. All peptides are rich in basic residues and lack acidic amino acids. The peptides inhibit the phosphotransferase system in vitro with IC50 of between 10 microM and 2 mM. Some, but not all, of the peptides inhibit cell growth in the agar diffusion test by an as yet undefined mechanism. All peptides are phosphorylated by enzyme I, and some are regenerated by slow autocatalytic hydrolysis of the phospho-peptide bond.