Mechanisms of resistance to antibiotics

Mechanisms of resistance to antibiotics

563 Mechanisms of resistance to antibiotics Gerard D Wright Microbial resistance to antibiotics is manifested by changes in antibiotic permeability, ...

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Mechanisms of resistance to antibiotics Gerard D Wright Microbial resistance to antibiotics is manifested by changes in antibiotic permeability, alteration of target molecules, enzymatic degradation of the antibiotics, and efflux of antimicrobials from the cytosol. Bacteria and other microorganisms use all of these mechanisms to evade the toxic effects of antibiotics. Recent research on the molecular aspects of these mechanisms, often informed by atomic resolution structures of proteins, enzymes and nucleic acids involved in these processes, has deepened our understanding of antibiotic action and resistance and, in several cases, spurred the development of strategies to overcome resistance in vitro and in vivo.

assertion. Such events demonstrate that antibiotic management and new discovery must continue in the face of these pressures. A key requirement in the discovery process is a comprehensive understanding of the molecular mechanisms of resistance, and how new drugs may be affected by existing mechanisms and ones that we can anticipate emerging. This review presents some recent findings in the area of the mechanisms of antibiotic resistance of selected antibiotics and includes discussion of some new antimicrobial agents, and strategies that have been developed to overcome resistance.

Addresses Department of Biochemistry, McMaster University, 1200 Main St W, Hamilton, Ontario L8N 3Z5, Canada e-mail: [email protected]

Glycopeptides

Current Opinion in Chemical Biology 2003, 7:563–569 This review comes from a themed issue on Mechanisms Edited by Shahriar Mobashery and John P Richard 1367-5931/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2003.08.004

Abbreviations MIC minimal inhibitory concentration VRE vancomycin-resistant enterococci

Introduction Microbial resistance to antibiotics in the clinic emerged soon after the first use of these agents in the treatment of infectious diseases, and continues to pose a significant challenge for the health care sector. Resistance, which was once primarily associated with health care institutions, has firmly emerged as a problem in the wider community. This is attested by the spread, with associated deaths, of infection by methicillin-resistant Staphylococcus aureus [1,2] and the increased prevalence of drug-resistant Streptococcus pneumoniae in communityacquired pneumonia [3]. While attention commonly focuses on resistance to antibacterial agents, resistance to antifungal [4], antiparasitic [5] and antiviral [6] drugs is also on the rise. Antimicrobial resistance is driven by inescapable evolutionary pressures and is therefore predictable and inevitable. The emergence in the past year of vancomycinresistant S. aureus [7], an event that has been anticipated for the past decade with great dread [8], punctuates this www.current-opinion.com

Glycopeptide antibiotics, such as teicoplanin and vancomycin, bind to the terminal D-Ala-D-Ala dipeptide of bacterial peptidoglycan in the cell wall of Gram-positive bacteria (the lipopolysacchararide outer membrane of Gram-negative bacteria renders this target inaccessible to the drugs, and thus these organisms are intrinsically resistant). Formation of the glycopeptide–peptidoglycan complex results in inhibition of the cell wall transpeptidase that maintains wall integrity and thus arrests cell wall biosynthesis, blocking cell division and growth [9]. The interaction between glycopeptides and the peptidoglycan involves the formation of a non-covalent complex mediated by a hydrogen bond network. This network arises from precise alignment of the antibiotic and the terminal acyl-D-Ala-D-Ala of peptidoglycan and its monomeric precursor, Lipid II (Figure 1a). Resistance to these antibiotics typically is the result of the biosynthesis of peptidoglycan with altered glycopeptide recognition sites (reviewed in [10,11]). Vancomycin-resistant enterococci (VRE), such as Enterococcus faecium and Enterococcus faecalis, that have acquired glycopeptide resistance, display the VanA, VanB or VanD phenotypes, which incorporate the depsipeptide D-Ala-D-lactate into the peptidoglycan. This results in loss of one hydrogen bond in the glycopeptide– Lipid II complex and the unfavorable proximity of repulsive carbonyl and ester oxygens, which combine to generate a 1000-fold decrease of affinity between glycopeptide and depsipeptide [12]. In vitro, this results in increases in the minimal inhibitory concentration (MIC) of antibiotic to over 64 mg/ml, well above clinically achievable concentrations. Other VRE of the VanC, VanE and VanG phenotypes are intrinsically resistant to lower levels of glycopeptides (MIC 2–32 mg/ml), and these incorporate D-Ala-D-Ser into the peptidoglycan. This probably causes steric obstruction between the antibiotic and the peptide and provides a sixfold reduction in binding affinity [13]. Current Opinion in Chemical Biology 2003, 7:563–569

564 Mechanisms

Figure 1

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Glycopeptide antibiotics. (a) Structure of teicoplanin and complex with acyl-D-Ala-D-Ala. (b) Structure of oritavancin, a semi-synthetic lipophilic glycopeptide antibiotic in phase III clinical trials.

These resistance mechanisms have largely been associated with avirulent intrinsically resistant organisms such as lactobacilli or enterococci, which are intestinal organisms and problematic only in clinical settings. Recently however, the VanA phenotype genes encoding inducible high-level resistance to vancomycin have been found in S. aureus, which is found on the skin, is much more virulent and capable of spread in the community [7]. This is a highly concerning event, and one that may be the harbinger of darker days. Vancomycin resistance in staphylococci to intermediate levels of antibiotic (MICs of 8–16 mg/ml) had already been observed in the mid-to-late 1990s in some isolates. However, this resistance mechanism is quite distinct from the incorporation of depsipeptides in peptidoglycan of VanA and includes the dramatic thickening and release of segments of the bacterial cell wall only in the presence of the antibiotic [14–16]. Generation of this ‘molecular flak’ leads to overproduction of the antibiotic binding site, and a net decrease in the effective concentration of drug, thus enabling the organism to grow in the presence of vancomycin. A new generation of semi-synthetic lipophilic glycopeptide antibiotics, including oritavancin (Figure 1b), currently in phase III clinical trials [17], has been reported. Unlike vancomycin, which is bacteriostatic, these antibiotics are rapidly bactericidal against Gram-positive bacteria including VRE, and apparently have an alternative mode of action to vancomycin’s inhibition of the peptidoglycan transpeptidase. The Kahne group has Current Opinion in Chemical Biology 2003, 7:563–569

reported that in vitro, lipophilic glycopeptide analogues, including those that do not bind to acyl-D-Ala-D-Ala, inhibit the bacterial transglycosylase that is required to extend the peptidoglycan polymer [18,19]. Supporting evidence for such an interaction has been provided by the observation that vancomycin affinity columns and, in particular, the vancosamine sugar, immobilize the Escherichia coli transglycosylase-transpeptidase PBP-1B [20]. A chemical genetics approach has been used to identify the gene yfgL, which encodes a predicted lipoprotein of unknown function in glycopeptide-sensitive E. coli, as important to the bactericidal activity of these homologues [21]. However since there are no obvious orthologous genes in Gram-positive bacteria, including the recently sequenced vancomycin-resistant E. faecalis V583 [22], the relevance of this observation to VRE is not clear.

Aminoglycosides The aminoglycoside antibiotics are cationic inhibitors of bacterial translation that have found clinical use for over half a century. Resistance to these antibiotics is primarily the result of expression of enzymes that covalently modify the antibiotics, either by acetylation, phosphorylation or adenylylation [23]. This modification interferes with binding to the target 16S rRNA in the decoding region of the A-site of the ribosome [24]. 3D structures of representative enzymes from each class have been determined [25–28], and recently an additional aminoglycoside kinase [29] and aminoglycoside acetyltransferase [30] have been reported. The structure of the two known www.current-opinion.com

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aminoglycoside kinases [26,29] along with a series of biochemical studies [31–33] support a mechanistic and evolutionary link between these antibiotic-resistance enzymes and Ser/Thr/Tyr protein kinases. Several lines of evidence support a dissociative-like mechanism of phosphate transfer in the aminoglycoside kinase APH(30 )-IIIa [32,33]. Conversely, the reactivity of a series of 40 -difluoro aminoglycosides with APH(30 )-Ia and APH(30 )-IIa favor an associative mechanism (S Mobashery, personal communication). These results suggest unexpected but important mechanistic diversity in these kinases. The structures of AAC(20 )-Ic from Mycobacterium tuberculosis in complex with the aminoglycosides tobramycin, ribostamycin and kanamycin A are the first examples of aminoglycosides binding to an aminoglycoside acetyltransferase, all of which are members of the GCN5 superfamily of acyltransferases [30]. A surprising revelation of these studies is the paucity of direct contacts between the enzyme and the substrates; rather, the majority of contacts occur through intermediary water molecules. This is distinct from the structures of APH(30 ) aminoglycoside kinases and their aminoglycoside substrates that make direct contacts with amino acid residues [29,34]. Both the APH(30 )s and AAC(20 )-Ic share a broad aminoglycoside substrate tolerance, and thus have evolved different molecular strategies to capture a wide range of substrates. Recently, a catalytically inactive acetyltransferase encoded on a Pseudomonas aeruginosa class 1 integron was paradoxically shown to confer aminoglycoside antibiotic resistance [35]. The protein was found to bind aminoglycosides tightly (Kd  1 mM), and the authors conclude that antibiotic sequestration could therefore be the basis for resistance. This is an intriguing new mechanism that requires additional study to rationalize this observation in the context of the concentration of ribosomes, with comparable aminoglycoside Kd, in the bacterial cytosol. The increased knowledge of aminoglycoside modifying enzyme structure and mechanism has been exploited in the synthesis of inhibitory molecules. Recent examples include dimeric neamine derivatives that retain substantial antibiotic activity and inhibit some aminoglycoside kinases [36], and a series of cationic peptides [37]. The latter show broad-spectrum inhibition of enzymes, including those from two classes (kinases and acetyltransferases), and are the first molecules that are capable of inhibition of two distinct aminoglycoside inactivation activities. These peptides have the potential to serve as leads in the development of universal aminoglycoside resistance enzyme inhibitors. The X-ray structure of the aminoglycosides paromomycin, streptomycin and spectinomycin in complex with the 30S subunit of Thermus thermophilus has been reported [38]. This information has informed the synthesis of a www.current-opinion.com

series of O6-aminoalkyl derivatives of neamine that retain antibiotic activity, even against organisms harboring aminoglycoside-resistance enzymes, and bind to the ribosomal A-site RNA [39]. A 3D structure of one of the derivatives in complex with a model A-site RNA has also been recently reported [40]. An NMR study has also identified several small-molecule aminoglycoside surrogates such as a series of 2-aminoquinolines and 2-aminopyridines that bind to a model A-site RNA [41]. These results demonstrate that there remains much to be learned and accomplished within this class of antibiotics, and that new agents of clinical significance are tractable using modern drug design approaches.

Streptogramins and macrolides The FDA approved the antibiotic formulation Synercid in 1999 for the treatment of infections caused by VRE. Synercid is a combination of two semi-synthetic inhibitors of bacterial translation, the type A streptogramin dalfopristin and the type B streptogramin quinupristin. Each component is individually bacteriostatic, but together they have synergistic bactericidal activity against several Gram-positive bacteria and a select group of Gramnegatives. Perhaps as a result of the use of streptogramins in agriculture for several decades, recovery of bacteria resistant to streptogramin antibiotics is not uncommon in commercial meat samples [42–44]. Streptogramin resistance is the result of efflux, target mutation and enzymatic mechanisms. All E. faecalis are intrinsically resistant to Synercid, and the mechanism of this resistance was recently associated with the predicted ABC transporter Lsa [45]. Enzymatic resistance to type A streptogramins is the result of a family of acetyl-CoAdependent O-acetyltransferases and the 3-D structure of one of these, VatD from E. faecium, has been reported in both the ligand-free and acetyl-CoA-bound forms [46]. Streptogramin acetyltransferases are members of the hexapeptide repeat family of acyltransferases that include chloramphenicol acetyltransferases. The structure of VatD revealed the predicted similarity to other hexapeptide repeat acyltransferases, including a trimeric quaternary structure. The active sites lie at the interface of monomers. Enzymatic resistance to the cyclic depsipeptide group B streptogramins occurs through either modification of the antibiotic docking site on the ribosomal 23S RNA by methylation enzymes of the Erm family that also provide cross-resistance to the macrolide and lincosamide antibiotics (MLSB phenotype) [47], or by direct inactivation. The latter mechanism had been previously attributed to hydrolysis of the lactone bridge between a Thr hydroxyl and the C-terminal carboxylate of the antibiotics [48,49]. However, careful analysis of the products of the reaction catalyzed by one of these resistance enzymes, Vgb from S. aureus, has identified a new resistance mechanism Current Opinion in Chemical Biology 2003, 7:563–569

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Structure and inactivation of streptogramin antibiotics. The two semi-synthetic components quinupristin and dalfopristin comprise the drug Synercid. These can be broken down by the enzymes Vgb and Vat, respectively.

whereby the peptide is linearized by an elimination reaction rather than by hydrolysis (Figure 2) [50]. This has important implications in any attempts to design antibiotics that are not susceptible to this mechanism. As noted above, modification of the 23S rRNA by the Erm methylases, specifically mono- and di-methylation of A2058 of domain V (E. coli numbering), also results in resistance to the macrolide antibiotics such as erythromycin. This modification prevents effective binding of macrolides in the peptide exit tunnel of the ribosome. Current Opinion in Chemical Biology 2003, 7:563–569

A new semi-synthetic macrolide, telithromycin, was approved by the FDA for clinical use in 2003. This new class of antibiotic replaces the sugar-substituted hydroxyl at position 3 of most macrolides with a keto group, generating a ‘ketolide’ (Figure 3). Telithromycin is active against bacteria encoding erm genes, in part as a result of increased affinity of the ribosome over macrolides at a site distinct from the domain V Erm methylation site (domain II) [51] and retention of affinity in monomethylated domain V [52]. Furthermore, ketolides such as telithromycin fail to induce the expression of inducible www.current-opinion.com

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Structure of telithromycin, an example of a ketolide. The erythromycin structure is shown for comparison.

erm genes, such as those predominately found in the important respiratory pathogen Streptococcus pneumoniae [53,54]. These antibiotics therefore overcome resistance in many ways, which should result in a decreased rate of developing resistance.

Future directions The rise of antibiotic resistance in the clinic continues unabated and, as can be seen in this brief review, microorganisms can deploy a myriad of mechanisms in the face of use of these toxic agents. Alteration of drug target, changes in target accessibility, degradation by enzymes and efflux (reviewed elsewhere in this volume), can all be utilized by microorganisms to evade antibiotics. There is much promise, however, in new strategies described in the literature, new agents in development, and new antibiotics recently introduced to the marketplace that resistance can be met and overcome. A key element in this strategy is knowledge of mechanism of action and resistance, and this remains the foundation of new cycles of antibiotic discovery.

Acknowledgements The author is supported by a Canada Research Chair in Antibiotic Biochemistry.

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30. Vetting MW, Hegde SS, Javid-Majd F, Blanchard JS, Roderick SL:  Aminoglycoside 2(-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates. Nat Struct Biol 2002, 9:653-658. The authors present the first structures of aminoglycosides bound in the active site of an aminoglycoside acetyltransferase. 31. Daigle DM, McKay GA, Thompson PR, Wright GD: Aminoglycoside antibiotic phosphotransferases are also serine protein kinases. Chem Biol 1999, 6:11-18. 32. Boehr DD, Thompson PR, Wright GD: Molecular mechanism of aminoglycoside antibiotic kinase APH(3()-IIIa: roles of conserved active site residues. J Biol Chem 2001, 276:23929-23936. 33. Thompson PR, Boehr DD, Berghuis AM, Wright GD: Mechanism of aminoglycoside antibiotic kinase APH(3()-IIIa: role of the nucleotide positioning loop. Biochemistry 2002, 41:7001-7007. 34. Fong DH, Berghuis AM: Substrate promiscuity of an  aminoglycoside antibiotic resistance enzyme via target mimicry. EMBO J 2002, 21:2323-2331. The authors present the first structures of aminoglycosides bound in the active site of an aminoglycoside kinase. They conclude that the antibiotics bind to the resistance enzymes in similar conformations as the ribosomal target. 35. Magnet S, Smith TA, Zheng R, Nordmann P, Blanchard JS: Aminoglycoside resistance resulting from tight drug binding to an altered aminoglycoside acetyltransferase. Antimicrob Agents Chemother 2003, 47:1577-1583. 36. Sucheck SJ, Wong AL, Koeller KM, Boehr DD, Draker K-a, Sears P, Wright GD, Wong C-H: Design of bifunctional antibiotics that target bacterial rRNA and inhibit resistance-causing enzymes. J Am Chem Soc 2000, 122:5230-5231. 37. Boehr DD, Draker K, Koteva K, Bains M, Hancock RE, Wright GD:  Broad-spectrum peptide inhibitors of aminoglycoside antibiotic resistance enzymes. Chem Biol 2003, 10:189-196. The authors describe the first inhibitory molecules that block both aminoglycoside kinase and acetyltransferase activity. 38. Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V: Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 2000, 407:340-348. 39. Haddad J, Kotra LP, Llano-Sotelo B, Kim C, Azucena EF Jr, Liu M,  Vakulenko SB, Chow CS, Mobashery S: Design of novel antibiotics that bind to the ribosomal acyltransfer site. J Am Chem Soc 2002, 124:3229-3237. First reported ‘rational’ design of aminoglycoside antibiotics. 40. Russell RJ, Murray JB, Lentzen G, Haddad J, Mobashery S: The complex of a designer antibiotic with a model aminoacyl site of the 30S ribosomal subunit revealed by X-ray crystallography. J Am Chem Soc 2003, 125:3410-3411. 41. Yu L, Oost TK, Schkeryantz JM, Yang J, Janowick D, Fesik SW: Discovery of aminoglycoside mimetics by NMR-based screening of Escherichia coli A-site RNA. J Am Chem Soc 2003, 125:4444-4450.

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46. Sugantino M, Roderick SL: Crystal structure of Vat(D): an  acetyltransferase that inactivates streptogramin group A antibiotics. Biochemistry 2002, 41:2209-2216. This is the first structure of this class of antibiotic resistance enzyme that inactivates group A streptogramins, which have only been approved for human use in North America since 1999. 47. Weisblum B: Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother 1995, 39:577-585. 48. Allignet J, Loncle V, Mazodier P, el Sohl N: Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics. Plasmid 1988, 20:271-275. 49. Le Goffic F, Capmau ML, Abbe J, Cerceau C, Dublanchet A, Duval J: Plasmid mediated pristinamycin resistance: PH 1A, a pristinamycin 1A hydrolase. Ann Microbiol (Paris) 1977, 128B:471-474. 50. Mukhtar TA, Koteva KP, Hughes DW, Wright GD: Vgb from  Staphylococcus aureus inactivates streptogramin B antibiotics

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by an elimination mechanism not hydrolysis. Biochemistry 2001, 40:8877-8886. The authors describe a surprising elimination mechanism for inactivation of the cyclic group b streptogramin depsipeptides, correcting the previous literature that indicated that these enzymes were hydrolases. 51. Douthwaite S, Hansen LH, Mauvais P: Macrolide-ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA. Mol Microbiol 2000, 36:183-193. 52. Liu M, Douthwaite S: Activity of the ketolide telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob Agents Chemother 2002, 46:1629-1633. 53. Bonnefoy A, Girard AM, Agouridas C, Chantot JF: Ketolides lack inducibility properties of MLS(B) resistance phenotype. J Antimicrob Chemother 1997, 40:85-90. 54. Clarebout G, Leclercq R: Fluorescence assay for studying the ability of macrolides to induce production of ribosomal methylase. Antimicrob Agents Chemother 2002, 46:2269-2272.

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