- Email: [email protected]
Antimicrobials
Available online at www.sciencedirect.com
Antimicrobials Editorial overview Christopher T Walsh and Gerard D Wright Current Opinion in Microbiology 2009, 12:473–475 Available online 3rd September 2009 1369-5274/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.08.002
Christopher T Walsh Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5701, United States e-mail: [email protected]
Christopher Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and does research on mechanisms of biosynthesis and mode of action of antibiotic natural products. He is the author of Antibiotics: Actions, Origins, Resistance (2001, ASM Press) and many research publications on the biosynthesis of nonribosomal peptide and glycopeptides antibiotics as well as aminocoumarins.
Gerard D Wright M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main St W, Hamilton, ON, Canada L8N 3Z5 e-mail: [email protected]
Gerard Wright is the director of the Michael G DeGroote Institute for Infectious Disease Research at McMaster University. His research is focused on the mechanisms, origins, and inhibition of antibiotic resistance and the biosynthesis of natural products. In particular, he has made contributions to understanding the mechanism of resistance to the aminoglycoside, glycopeptide, tetracycline, and streptogramin antibiotics as well as elaborating the concept and importance of the antibiotic resistome in the genesis and maintenance of resistance in microbial communities.
Infectious disease is a therapeutic arena in which there is a constant need for new drugs to combat waves of resistant microorganisms (bacteria, fungi, parasites, viruses) that are selected for by the widespread application of any microbial agent. For antibacterial agents the problem has been exacerbated over the past decade both by the spread of clinically significant multiply drug-resistant Gram-negative and Gram-positive pathogens and the exit of several major pharmaceutical companies from this therapeutic space. The decreased emphasis on antibacterial drugs by pharma and biotech companies has many reasons but important among them are diminishing returns both from classical screening approaches for natural antibiotics and the lack of robust leads from screens of synthetic compound libraries. Hence new strategies are warranted and are the subject of the reviews in this section. Louis Rice in his article on the clinical consequences of antimicrobial resistance sets the stage by addressing the changes in antimicrobial susceptibility over the past decade both in community settings and for hospitalized patients and the consequent challenges for effective therapeutic strategies. He enumerates problems posed by Gram-positive pathogens such as Streptococcus pneumoniae strains not covered by the heptavalent vaccine and by community-acquired MRSA on the Gram-positive pathogen front. For Gramnegatives he notes that carbapenem-resistant Klebsiella and multidrugresistant Pseudomonas aeruginosa and Acinetobacter baumannii are formidable pathogens for crucially ill patients. The sparse developmental pipeline of new antibacterial agents puts ever-greater premium on understanding pathogen behavior and resistance routes to enable effective clinical management of infected patients and ‘maintain antimicrobial susceptibility patterns at a level we all can live with’. The article by James Collins on the Role of Reactive Oxygen Species in Antibiotic Action and Resistance provides insights that antibiotics induce changes in bacterial metabolism leading to increase in reactive oxygen species (ROS). These drug-induced oxidative stressors turn on heat shock and SOS response pathways and abet resistance development by various routes including selective gene activation, mutagenesis, and acquisition of foreign genes. The systems response is exemplified by E. coli response to the fluoroquinolone norfloxacin. Also b-lactams and aminoglycosides also turn on OH radical mediated killing pathways via alterations in TCA cycle metabolism, NADH depletion, iron misregulation, and FE/S cluster breakdown. To the extent that ROS increases are common downstream sequellae to antibiotic exposure, Collins highlights that therapeutic strategies that target the SOS system could enhance the efficacy of existing classes of antibiotics. Barczak and Hung in their review Productive steps toward an antimicrobial targeting virulence reinforce the view that new paradigms for antibiotic
www.sciencedirect.com
Current Opinion in Microbiology 2009, 12:473–475
474 Antimicrobials
discovery and development are needed. Small molecule inhibitors of virulence have been shown to alter the course of disease in animal models of bacterial infection. From this perspective there are novel targets in bacterial signaling pathways and bacterial defense against host immunity. These authors discuss the strengths and weaknesses of targeting virulence. They suggest focus on functions required for in vivo survival and for the ones that cause tissue damage and disease, for example virulence factors, such as Mtb isocitrate lyase required in macrophages and V. cholerae vibriobactin for mouse model diarrheal disease. Virulence factors include Salmonella SopB,E and also Mtb dihydrolipoyl acyl transferase for the detoxification of host peroxynitrite. They review recent examples on blocking cell to cell bacterial signaling, blockade of Type III secretion systems in Salmonella infections, blockade of adhesion in Gram-negatives by pilicides and glycodendrimers and the prospect of sortase inhibitors in Gram-positives. A challenge for the field will be the development of broad-spectrum antivirulence compounds active against a range of Gram-positive and, especially, Gram-negative pathogens. Falconer and Brown in their article New Screens and targets in antibacterial drug discovery take on the challenge of pressing need for new antibiotic discovery. Since classical screening approaches have yielded diminished returns there must be renewed emphasis on novel screening strategies. These authors review new phenotypic-based screens to identify active molecules that may have novel mechanisms and/or novel targets. Examples noted include inhibitors for P. aeruginosa virulence protein ExoS, identified in a yeast screen and also the use of the nematode C. elegans exposed to Enterococcus faecalis as a live-animal infection model for small molecule screens. Also reviewed is work on cell wall synthesis inhibition, including LpxC inhibitors, structure-based studies with moenomycin analogs on transglycosylase and on enzymes for teichoic acid biogenesis in Gram-positive bacteria. The authors also discuss current prospects for bacteriophage as therapeutic agents. Zhang, Fisher, and Mobashery focus in on The Bifunctional Enzymes of Antibiotic Resistance and raise the question of whether such double-headed resistance catalysts are a harbinger of the future. They note that a classic case is the transpeptidase–transglycosylase bifunctional enzyme in peptidoglycan polymerization and cell wall extension. While the best known class of bifunctional enzymes in antibiotic resistance are the aminoglycoside resistance enzymes, they call out the recently discovered bifunctional b-lactamases Tp47 from Treponema palladium (causative agent of syphilis) which is a PBP and a b-lactamase and blaLRA-13 which is a class C-lactamase fused to a class D-lactamase. The class C activity confers resistance to penicillins, and the class D to cephalexin. The bifunctional aminoglycoside resistance enzymes generate a Current Opinion in Microbiology 2009, 12:473–475
broadened resistance profile, for example coupling acetyltransferase domains with kinase domains to inactivate aminoglycosides by regiospecific acetylations and/or phosphorylations. Different combinations have evolved to couple acetylation with phosphorylation, acetylation with nucleotidylation, and even pairs of regioselective acetyltransferases. The authors also remark on a new enzyme that acetylates both aminoglycosides and the fluorquinolone ciprofloxacin. The merger of two genes that confer complementary modification chemistry on antibiotic scaffolds can take out the whole family of antibiotic variants and provide resistance against many generations of antibiotic scaffolds. The three major mechanisms for antibiotic resistance by bacterial pathogens are typically alteration of the antibiotic during or after uptake (e.g. b-lactamases); alteration of the target as in vancomycin-resistant enterococci, and the export of antibiotics by efflux pumps. Blair and Piddock provide an authoritative summary on this third mechanism in their article on Structure, Function, and Inhibition of RND efflux pumps in Gram Negative Bacteria: an update. The acronym RND = resistance nodulation division and this class encompasses ubiquitously expressed efflux pump proteins. The RND pumps have tripartite machinery in Gram-negatives, with inner membrane/periplasm/outer membrane protein components to span both membrane barriers. The RND efflux pumps transport a wide variety of small molecules and have key impact on multidrug resistance as providers of innate resistance. Pump expression levels correlate with drug/ antibiotic resistance. ArcB of E. coli and MexB of P. aeruginosa have been solved by X-ray crystallography and the structures illuminate trimers as the functional unit and support a rotational model for pump function. Energy is provided by proton motive force via a proton translocation domain at an allosteric site. The hydrophobic substrate-binding pocket has been characterized by mutagenesis. A periplasmic docking domain interacts with periplasmic adaptor proteins (ArcA, MexA), whose structures have also just been completed. Structures of several outer membrane channels in the RND complex are known, including TolC trimers, OprM, and Vce to allow visualization of the tripartite, double membrane-spanning pump architecture. The current state of knowledge suggests a peristaltic, iris opening model to move cytoplasmic hydrophobic substrates down the tunnel to the extracellular space. Some RND pumps function as virulence factors by the export of host metabolites such as bile acids, fatty acids, and steroids. Host antimicrobial peptides are substrates for some RND-mediated efflux pumps (MtrCDE of Neisseria gonorrhea). The remarkable advancements in understanding the structures of RND efflux pumps set www.sciencedirect.com
Antimicrobials Walsh and Wright 475
the stage for new rounds of inhibitor design and specificity analysis. The last article by Michael Fischbach, Antibiotics from Microbes: Converging to Kill, is a thoughtful perspective on how biosynthetic pathways for small molecule antibiotics produced by microbes have emerged and converged. Evolutionary convergence on a target is exemplified by antibiotics that bind to distinct sites on large and small subunits of bacterial ribosomes, to distinct enzymes and substrates involved in the extracellular stages on peptidoglycan maturation and crosslinking, including sequestration of the carrier molecule lipid II. Fischbach also emphasizes convergence to an effective molecular scaffold for antibiotics, notably to fashion b-lactams by distinct collections of enzymes that make the 4/5 fused ring system by different strategies. Fischbach then explores the evolutionary steps of merging biosynthetic gene clusters into superclusters. These can generate a pair of antibiotics acting synergistically and under coordinate regulation. Streptomyces clavuligerus makes both the cephalosporin cephamycin and the lactamase inactivator clavulanic acid with the genes arranged in a contiguous 90-kb DNA region, enabling coordinate regu-
www.sciencedirect.com
lation. An alternate merger is for the fusion of separate biosynthetic gene clusters into one pathway that borrows elements from previously separate pathways; the DNA gyrase-targeting simocyclinone is a hybrid of aminocoumarin and anthracycline glycoside scaffolds. Fischbach notes that most antibiotic gene clusters are cryptic (repressed) under standard culture conditions and suggests the cosmopolitan distribution of antibiotic biosynthetic gene clusters via horizontal gene transfer bears further examination and may lead to more readily malleable hosts for in vivo engineering of newly altered antibiotic scaffolds. These reviews provide an excellent snapshot of the state of the field of antimicrobial agents. Technological advances in protein structure determination, rapid genome sequencing, and in high-throughput and systems biology are illuminating the modes and mechanisms of antibiotic action and resistance as never before. In many ways we are in a new Golden Era of antibiotic research. Paradoxically however, the clinical challenges of antibiotic resistance and the emergence of new pathogens remain acute and are growing. How this new knowledge and technology might be harnessed for the development of new drugs that can be effectively used to treat disease is the challenge now facing us all.
Current Opinion in Microbiology 2009, 12:473–475
Antimicrobials Editorial overview Christopher T Walsh and Gerard D Wright Current Opinion in Microbiology 2009, 12:473–475 Available online 3rd September 2009 1369-5274/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.08.002
Christopher T Walsh Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5701, United States e-mail: [email protected]
Christopher Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and does research on mechanisms of biosynthesis and mode of action of antibiotic natural products. He is the author of Antibiotics: Actions, Origins, Resistance (2001, ASM Press) and many research publications on the biosynthesis of nonribosomal peptide and glycopeptides antibiotics as well as aminocoumarins.
Gerard D Wright M.G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main St W, Hamilton, ON, Canada L8N 3Z5 e-mail: [email protected]
Gerard Wright is the director of the Michael G DeGroote Institute for Infectious Disease Research at McMaster University. His research is focused on the mechanisms, origins, and inhibition of antibiotic resistance and the biosynthesis of natural products. In particular, he has made contributions to understanding the mechanism of resistance to the aminoglycoside, glycopeptide, tetracycline, and streptogramin antibiotics as well as elaborating the concept and importance of the antibiotic resistome in the genesis and maintenance of resistance in microbial communities.
Infectious disease is a therapeutic arena in which there is a constant need for new drugs to combat waves of resistant microorganisms (bacteria, fungi, parasites, viruses) that are selected for by the widespread application of any microbial agent. For antibacterial agents the problem has been exacerbated over the past decade both by the spread of clinically significant multiply drug-resistant Gram-negative and Gram-positive pathogens and the exit of several major pharmaceutical companies from this therapeutic space. The decreased emphasis on antibacterial drugs by pharma and biotech companies has many reasons but important among them are diminishing returns both from classical screening approaches for natural antibiotics and the lack of robust leads from screens of synthetic compound libraries. Hence new strategies are warranted and are the subject of the reviews in this section. Louis Rice in his article on the clinical consequences of antimicrobial resistance sets the stage by addressing the changes in antimicrobial susceptibility over the past decade both in community settings and for hospitalized patients and the consequent challenges for effective therapeutic strategies. He enumerates problems posed by Gram-positive pathogens such as Streptococcus pneumoniae strains not covered by the heptavalent vaccine and by community-acquired MRSA on the Gram-positive pathogen front. For Gramnegatives he notes that carbapenem-resistant Klebsiella and multidrugresistant Pseudomonas aeruginosa and Acinetobacter baumannii are formidable pathogens for crucially ill patients. The sparse developmental pipeline of new antibacterial agents puts ever-greater premium on understanding pathogen behavior and resistance routes to enable effective clinical management of infected patients and ‘maintain antimicrobial susceptibility patterns at a level we all can live with’. The article by James Collins on the Role of Reactive Oxygen Species in Antibiotic Action and Resistance provides insights that antibiotics induce changes in bacterial metabolism leading to increase in reactive oxygen species (ROS). These drug-induced oxidative stressors turn on heat shock and SOS response pathways and abet resistance development by various routes including selective gene activation, mutagenesis, and acquisition of foreign genes. The systems response is exemplified by E. coli response to the fluoroquinolone norfloxacin. Also b-lactams and aminoglycosides also turn on OH radical mediated killing pathways via alterations in TCA cycle metabolism, NADH depletion, iron misregulation, and FE/S cluster breakdown. To the extent that ROS increases are common downstream sequellae to antibiotic exposure, Collins highlights that therapeutic strategies that target the SOS system could enhance the efficacy of existing classes of antibiotics. Barczak and Hung in their review Productive steps toward an antimicrobial targeting virulence reinforce the view that new paradigms for antibiotic
www.sciencedirect.com
Current Opinion in Microbiology 2009, 12:473–475
474 Antimicrobials
discovery and development are needed. Small molecule inhibitors of virulence have been shown to alter the course of disease in animal models of bacterial infection. From this perspective there are novel targets in bacterial signaling pathways and bacterial defense against host immunity. These authors discuss the strengths and weaknesses of targeting virulence. They suggest focus on functions required for in vivo survival and for the ones that cause tissue damage and disease, for example virulence factors, such as Mtb isocitrate lyase required in macrophages and V. cholerae vibriobactin for mouse model diarrheal disease. Virulence factors include Salmonella SopB,E and also Mtb dihydrolipoyl acyl transferase for the detoxification of host peroxynitrite. They review recent examples on blocking cell to cell bacterial signaling, blockade of Type III secretion systems in Salmonella infections, blockade of adhesion in Gram-negatives by pilicides and glycodendrimers and the prospect of sortase inhibitors in Gram-positives. A challenge for the field will be the development of broad-spectrum antivirulence compounds active against a range of Gram-positive and, especially, Gram-negative pathogens. Falconer and Brown in their article New Screens and targets in antibacterial drug discovery take on the challenge of pressing need for new antibiotic discovery. Since classical screening approaches have yielded diminished returns there must be renewed emphasis on novel screening strategies. These authors review new phenotypic-based screens to identify active molecules that may have novel mechanisms and/or novel targets. Examples noted include inhibitors for P. aeruginosa virulence protein ExoS, identified in a yeast screen and also the use of the nematode C. elegans exposed to Enterococcus faecalis as a live-animal infection model for small molecule screens. Also reviewed is work on cell wall synthesis inhibition, including LpxC inhibitors, structure-based studies with moenomycin analogs on transglycosylase and on enzymes for teichoic acid biogenesis in Gram-positive bacteria. The authors also discuss current prospects for bacteriophage as therapeutic agents. Zhang, Fisher, and Mobashery focus in on The Bifunctional Enzymes of Antibiotic Resistance and raise the question of whether such double-headed resistance catalysts are a harbinger of the future. They note that a classic case is the transpeptidase–transglycosylase bifunctional enzyme in peptidoglycan polymerization and cell wall extension. While the best known class of bifunctional enzymes in antibiotic resistance are the aminoglycoside resistance enzymes, they call out the recently discovered bifunctional b-lactamases Tp47 from Treponema palladium (causative agent of syphilis) which is a PBP and a b-lactamase and blaLRA-13 which is a class C-lactamase fused to a class D-lactamase. The class C activity confers resistance to penicillins, and the class D to cephalexin. The bifunctional aminoglycoside resistance enzymes generate a Current Opinion in Microbiology 2009, 12:473–475
broadened resistance profile, for example coupling acetyltransferase domains with kinase domains to inactivate aminoglycosides by regiospecific acetylations and/or phosphorylations. Different combinations have evolved to couple acetylation with phosphorylation, acetylation with nucleotidylation, and even pairs of regioselective acetyltransferases. The authors also remark on a new enzyme that acetylates both aminoglycosides and the fluorquinolone ciprofloxacin. The merger of two genes that confer complementary modification chemistry on antibiotic scaffolds can take out the whole family of antibiotic variants and provide resistance against many generations of antibiotic scaffolds. The three major mechanisms for antibiotic resistance by bacterial pathogens are typically alteration of the antibiotic during or after uptake (e.g. b-lactamases); alteration of the target as in vancomycin-resistant enterococci, and the export of antibiotics by efflux pumps. Blair and Piddock provide an authoritative summary on this third mechanism in their article on Structure, Function, and Inhibition of RND efflux pumps in Gram Negative Bacteria: an update. The acronym RND = resistance nodulation division and this class encompasses ubiquitously expressed efflux pump proteins. The RND pumps have tripartite machinery in Gram-negatives, with inner membrane/periplasm/outer membrane protein components to span both membrane barriers. The RND efflux pumps transport a wide variety of small molecules and have key impact on multidrug resistance as providers of innate resistance. Pump expression levels correlate with drug/ antibiotic resistance. ArcB of E. coli and MexB of P. aeruginosa have been solved by X-ray crystallography and the structures illuminate trimers as the functional unit and support a rotational model for pump function. Energy is provided by proton motive force via a proton translocation domain at an allosteric site. The hydrophobic substrate-binding pocket has been characterized by mutagenesis. A periplasmic docking domain interacts with periplasmic adaptor proteins (ArcA, MexA), whose structures have also just been completed. Structures of several outer membrane channels in the RND complex are known, including TolC trimers, OprM, and Vce to allow visualization of the tripartite, double membrane-spanning pump architecture. The current state of knowledge suggests a peristaltic, iris opening model to move cytoplasmic hydrophobic substrates down the tunnel to the extracellular space. Some RND pumps function as virulence factors by the export of host metabolites such as bile acids, fatty acids, and steroids. Host antimicrobial peptides are substrates for some RND-mediated efflux pumps (MtrCDE of Neisseria gonorrhea). The remarkable advancements in understanding the structures of RND efflux pumps set www.sciencedirect.com
Antimicrobials Walsh and Wright 475
the stage for new rounds of inhibitor design and specificity analysis. The last article by Michael Fischbach, Antibiotics from Microbes: Converging to Kill, is a thoughtful perspective on how biosynthetic pathways for small molecule antibiotics produced by microbes have emerged and converged. Evolutionary convergence on a target is exemplified by antibiotics that bind to distinct sites on large and small subunits of bacterial ribosomes, to distinct enzymes and substrates involved in the extracellular stages on peptidoglycan maturation and crosslinking, including sequestration of the carrier molecule lipid II. Fischbach also emphasizes convergence to an effective molecular scaffold for antibiotics, notably to fashion b-lactams by distinct collections of enzymes that make the 4/5 fused ring system by different strategies. Fischbach then explores the evolutionary steps of merging biosynthetic gene clusters into superclusters. These can generate a pair of antibiotics acting synergistically and under coordinate regulation. Streptomyces clavuligerus makes both the cephalosporin cephamycin and the lactamase inactivator clavulanic acid with the genes arranged in a contiguous 90-kb DNA region, enabling coordinate regu-
www.sciencedirect.com
lation. An alternate merger is for the fusion of separate biosynthetic gene clusters into one pathway that borrows elements from previously separate pathways; the DNA gyrase-targeting simocyclinone is a hybrid of aminocoumarin and anthracycline glycoside scaffolds. Fischbach notes that most antibiotic gene clusters are cryptic (repressed) under standard culture conditions and suggests the cosmopolitan distribution of antibiotic biosynthetic gene clusters via horizontal gene transfer bears further examination and may lead to more readily malleable hosts for in vivo engineering of newly altered antibiotic scaffolds. These reviews provide an excellent snapshot of the state of the field of antimicrobial agents. Technological advances in protein structure determination, rapid genome sequencing, and in high-throughput and systems biology are illuminating the modes and mechanisms of antibiotic action and resistance as never before. In many ways we are in a new Golden Era of antibiotic research. Paradoxically however, the clinical challenges of antibiotic resistance and the emergence of new pathogens remain acute and are growing. How this new knowledge and technology might be harnessed for the development of new drugs that can be effectively used to treat disease is the challenge now facing us all.
Current Opinion in Microbiology 2009, 12:473–475