b-Lactam antibiotic resistance: a current structural perspective

Mark S Wilke1, Andrew L Lovering1 and Natalie CJ Strynadka

Bacterial resistance to b-lactam antibiotics can be achieved by any of three strategies: the production of b-lactam-hydrolyzing b-lactamase enzymes, the utilization of b-lactam-insensitive cell wall transpeptidases, and the active expulsion of b-lactam molecules from Gram-negative cells by way of efux pumps. In recent years, structural biology has contributed signicantly to the understanding of these processes and should prove invaluable in the design of drugs to combat b-lactam resistance in the future.
Addresses Department of Biochemistry and Molecular Biology, and the Center for Blood Research, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 Corresponding author: Strynadka, Natalie CJ (natalie@byron.biochem.ubc.ca) 1 Mark S Wilke and Andrew L Lovering contributed equally to this review.

wall) for the preservation of cell shape and rigidity. This cell wall is comprised of a basic repeating unit of an alternating disaccharide N-acetyl glucosamine and N-acetyl muramic acid. The latter sugar in this disaccharide is modied by a characteristic pentapeptide. This varies amongst the Gram-negative and Gram-positive species, but always terminates in two D-alanine residues. The individual peptidoglycan units are produced inside the cell, but their nal cross-linking is catalyzed outside the cytoplasmic membrane by a group of membraneanchored bacterial enzymes known as the cell-wall transpeptidases. In this cross-linking reaction, a peptide bond is formed between the penultimate D-alanine on one chain and the free amino end of a diamino pimelic acid (Gram-negative) or an L-lysine (Gram-positive) residue on the other chain. The linkage is formed with the penultimate D-alanine, causing the terminal D-alanine to be cleaved in the process. Transpeptidase enzymes utilize an active site serine and perform their catalytic cycle by way of an acylation/ deacylation pathway. b-lactam antibiotics efciently inhibit the bacterial transpeptidases, therefore these enzymes are often termed penicillin binding proteins or PBPs. They are able to do this owing to the stereochemical similarity of the b-lactam moiety with the D-alanineDalanine substrate. In the presence of the antibiotic, the transpeptidases form a lethal covalent penicilloyl enzyme complex that serves to block the normal transpeptidation reaction. This results in weakly cross-linked peptidoglycan, which makes the growing bacteria highly susceptible to cell lysis and death. As with most antimicrobial agents, b-lactams are rendered inactive against bacteria by way of three primary mechanisms of resistance (Figure 1). The most common mechanism is the production of enzymes that degrade or modify the antibiotic before it can reach the appropriate target site. In this case, the b-lactamase family of enzymes degrade b-lactam antibiotics and are found widely disseminated amongst Gram-positive and Gram-negative bacteria. The second mechanism is alteration of the antibiotic target site. In this case, the b-lactam-resistant cell-wall transpeptidases perform this role; this is now a major cause of resistance in several pathogens including the problematic Gram-positive Staphylococcal and Streptococcal species. The nal mechanism is prevention of access of the antibiotic to the target by way of altered permeability or forced efux. For example, this can be performed by the MexA,BOprM antibiotic efux pump, which is a major cause of resistance in Pseudomonas and in other pathogenic Gram-negative species.
Current Opinion in Microbiology 2005, 8:525533

Current Opinion in Microbiology 2005, 8:525533 This review comes from a themed issue on Antimicrobials Edited by Christopher Walsh and Malcolm GP Page Available online 29th August 2005 1369-5274/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.08.016

Introduction
The introduction of b-lactam antibiotics into the health care system in the latter stages of World War II represents one of the most important contributions to medical science in recent history. Today, b-lactams remain the most widely utilized antibiotics owing to their comparatively high effectiveness, low cost, ease of delivery and minimal side effects. b-lactams target transpeptidase enzymes that synthesize the bacterial cell wall. The desirable attributes of this class of antibiotic arise from the facts that these enzymes are localized to the outer leaet of the bacterial cytoplasmic membrane (i.e. are relatively accessible) and that they are specic to bacteria (with no functional or structural counterpart in the human host). In a practical sense, the low cost of production of b-lactam antibiotics allows for a wide availability; thus, it is imperative that we preserve the power of this valuable clinical resource. How do b-lactam antibiotics work? Bacteria of all species rely on a heavily cross-linked peptidoglycan layer (cell
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Figure 1

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In this review we describe seminal new discoveries in which structural biology has provided new insights into these b-lactam antibiotic resistance phenomena as well as new strategies for drug development.

Antibiotic-modifying enzymes: the b-lactamases

The b-lactamases confer signicant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the four-membered b-lactam ring. These enzymes are especially important in Gram-negative bacteria as they constitute the major defense mechanism against b-lactam-based drugs. The spread of b-lactamase genes has been greatly exacerbated by their integration within mobile genetic elements, such as plasmids or transposons, which facilitate the rapid transfer of genetic material between microbes. Even more ominous is the organization of b-lactamase genes within integrons as part of multi-drug resistance cassettes that bestow mechanisms for resistance not only to b-lactams but also to other antibiotic classes such as aminoglycosides, macrolides, sulphonamides and chloramphenicol (for a recent review, see [1]). Once expressed, b-lactamases are secreted into the periplasmic space (in Gram-negative bacteria), bound to the cytoplasmic membrane, or excreted (in Grampositive bacteria).
Structure and mechanism

Classes B1 and B3 are able to bind one or two zinc ions [3], whereas the class B2 enzymes appear to be mononuclear [4]. In the binuclear metallo-b-lactamases, the zinc ions are proximal to each other and are separated by a bridging hydroxide that has been proposed to be the attacking nucleophile in b-lactam hydrolysis. The class B1 and B3 metallo-b-lactamases can also function as mononuclear enzymes, in which a single zinc ion (that occupies the Zn1 site) coordinates the nucleophilic hydroxide; this mechanism has been proposed to predominate in the presence of substrate under physiological conditions [5]. The crystal structure of a class B2 metallo-b-lactamase (CphA from Aeromonas hydrophilia) has only been published recently [4]. Its proposed catalytic mechanism differs from the class B1 and B3 mononuclear mechanisms in that the zinc ion occupies the Zn2 site, a general base activates the nucleophilic water, and the zinc ion forms a bond with the amine nitrogen of the hydrolyzed b-lactam amide (Figure 2).
A new generation of b-lactamases

The >470 b-lactamases known to date [2] are typically organized into four classes (A to D) on the basis of sequence similarity. Crystal structures are currently available for representatives of each class (for a recent review, see [2]). Classes A, C and D share a similar fold and all have a mechanism that involves creation of a serine nucleophile by deprotonation of an active site serine with a general base, nucleophilic attack of the b-lactam ring to form an acyl-enzyme intermediate, and hydrolysis of the intermediate using a general base activated water molecule. The differences between the catalytic mechanisms of the serine b-lactamase classes center around the type of general base residues used in acylation and deacylation. The class B b-lactamases are zinc metalloenzymes and are completely distinct from the serine b-lactamases in terms of sequence, fold and mechanism. There are three subclasses of class B metallo-b-lactamases (B1 to B3).

The b-lactamases are ancient enzymes that were relatively rare until b-lactam antibiotics were introduced into medicine and agriculture half a century ago [6]. The widespread use of carbapenems, the monobactam aztreonam, cephamycins and oxyimino-cephalosporins in the past few decades has led to the evolution of a new generation of b-lactamases, which have an extended substrate spectrum (i.e. extended-spectrum b-lactamases or ESBLs), as well as the development of novel carbapenemases and plasmid-mediated AmpC b-lactamases (for recent reviews, see [2,7,8]). Common ESBLs include varieties from the class A b-lactamases TEM, SHV and CTX-M and the class D b-lactamase OXA. These enzymes are typied by a broad substrate spectrum that includes oxyimino-cephalosporins, aztreonam and, in the case of some OXA and CTX-M enzymes, cefepime. Recently, several CTX-M structures have been made available including inhibitor-bound structures, which provide snapshots of two reaction cycle transition states, and the acyl-enzyme intermediate, which can aid in the design of inhibitors [9] (Figure 3). In addition, several atomic resolution CTX-M structures demonstrate that the enhanced ceftazidimase activity of these enzymes is a result of the increased active site exibility; however, this

(Figure 1 Legend) Structural depiction of proteins involved in b-lactam resistance. The proteins responsible for resistance are sub-divided into the b-lactamase, PBP and efflux pump groups. Also shown are two of the repressors for resistance operons BlaI and MexR (genes are shown as solid arrows). The structures of the glycosyltransferase domain of the class A PBPs and the cytoplasmic protease domain of BlaR that are not yet determined are replaced by blue and green solid shapes, respectively. The diagram also shows the interaction of b-lactams with these subgroups the b-lactam-dependent signaling of BlaR, ring hydrolysis by the b-lactamases, acylation of the PBPs (poor in PBP2x and 2a), and proton antiport of the antibiotics by the efflux pump systems. The recently solved structure of S. pneumoniae PBP1b is used in place of the closely-related resistance determinant PBP1a, and the structures of MexB and OprM are used here as models derived from the co-ordinates of their respective homologues, AcrB and TolC (docked together manually). Details of the precise interaction of MexA with MexBOprM remain unknown, as does the nature of the effector responsible for terminating MexR repression. Abbreviations: gm +/, Gram-positive/negative; IM, inner membrane; OM, outer membrane; PG, peptidoglycan. CTXM9 and CphA are shown as representatives of the >40 available serine and metallo-b-lactamase structures. www.sciencedirect.com Current Opinion in Microbiology 2005, 8:525533

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Figure 2

Figure 3

The fold and active site of CphA bound by the substrate biapenem (PDB code 1X8). (a) Ribbon representation of the CphA fold. (b) Stick representation of several active site residues, colored by atom type (C atoms, yellow; O, red; N, blue; and S, orange). Biapenem is displayed with purple carbon atoms to distinguish it from the protein carbons shown in yellow. A single catalytic zinc and a water molecule are shown as dark grey and cyan spheres, respectively. Hydrogen bonds are represented by green dashes.

The fold and active site of cefoxitin-acylated CTXM9 (PDB code 1YMX). (a) Ribbon representation of the CTXM9 fold. (b) Stick representation of several active site residues, colored by atom type (C atoms, yellow; O, red; N, blue; and S, orange). The covalently-bound cefoxitin is shown with purple carbon atoms to distinguish it from the protein carbons displayed in yellow. An active site water molecule is indicated with a cyan sphere.

increase in exibility is at the cost of protein stability [10]. Carbapenemases are derived from classes A, B and D and they provide resistance to carbapenems as well as to oxyimino-cephalosporins and cephamycins. The class B metallo-b-lactamase CphA (mentioned above) is a carbapenamase. Its crystal structure in complex with the carbapenem substrate biapenem (Figure 2) has been determined; this might prove useful in the design of inhibitors or of non-hydrolyzable antibiotics [4]. Historically, combination therapies in which the action of b-lactams is supplemented with b-lactamase inhibitors
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have successfully restored the antibiotic activity of b-lactam drugs against resistant pathogens. Although outside the scope of this review, several novel compounds have been reported in the past two years that have inhibitory activity against class A, B and C b-lactamases; these molecules include peptides [11], succinic acid derivatives [12], substituted penam sulfones [13], mercaptomethyl-penicillinates [14], bridged bicyclic octanones [15], thiophene-carboxy derivatives [16], and tricylclic 6-methylidene penems [17].
Regulation of resistance

b-lactamase expression is often induced by b-lactams through a novel regulation system that consists of the repressor BlaI and the receptor BlaR (Figure 1). A complete description of the induction process is currently
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unavailable, but recent determination of the structures of BlaI [18,19] and the extracellular PBP-like sensor domain of BlaR [2022] have characterized important features of the b-lactamase regulation machinery. BlaR is a transmembrane protein composed of an extracellular sensor domain that is acylated by b-lactam antibiotics, a transmembrane domain (consisting of four membrane-spanning helices) that transduces the b-lactam-binding signal across the membrane, and an intracellular zymogenic zinc metalloprotease domain that is activated by autoproteolysis upon acylation of the sensor domain. Interestingly, the b-lactam sensor domain of BlaR resembles the b-lactamases in terms of its fold (especially the class D b-lactamases) as well as its use of a serine nucleophile to form an acyl-enzyme intermediate. However, unlike the b-lactamases and akin to the PBPs, the covalent b-lactam adduct is stabilized as it appears to lack a functional general base residue capable of deacylation. No signicant conformational changes accompany acylation of the sensor domain [21] and it has been proposed that the large extracellular BlaR loop designated L2 that is positioned adjacent to the sensor domain in the BlaR receptor might mediate the b-lactambinding signal to the transmembrane domain through an interaction with the BlaR sensor [23]. The cytosolic repressor BlaI forms a dimer and binds an operator in the bla divergon that encodes BlaI, BlaR and the b-lactamase structural gene. Once activated, the BlaR metalloprotease domain elicits cleavage of BlaI within its dimerization domain, which prevents operator binding and permits b-lactamase expression. The BlaI monomer consists of an amino-terminal winged helix DNA-binding domain and a carboxy-terminal dimerization domain, which are separated by a exible hinge region. This exibility allows the BlaI dimer to not only bind the bla operator but bind the operator sequence for the mec divergon as well, allowing BlaR and BlaI to additionally mediate the expression of PBP2a (the protein encoded by mecA) [19]. PBP2a expression in methicillin-resistant Staphylococcus aureus (MRSA) is controlled by a highly similar regulation system that consists of the repressor MecI and receptor MecR. Although many MRSA strains have MecI deletions that inactivate PBP2a repression, these strains appear to be co-regulated by BlaRBlaI [24]. Indeed, the bla or mec regulatory genes appear to be required for the maintenance and expression of mecA, which suggests that PBP2a expression incurs a tness cost on the host [25]. Therefore, the bla and mec regulation machinery might prove useful as targets for the design of inhibitors to counter b-lactamase and PBP2a-mediated resistance, particularly if further structural details are forthcoming. Alternatively, the creation of non-b-lactam lead compounds that circumvent the activation of such regulatory mechanisms are a logical step in inhibitor design, as highlighted recently by Tondi et al. [16] and Zervosen et al. [26].
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Altered antibiotic targets: the cell wall transpeptidases (PBPs)

PBPs are divided into two subgroups: low molecular mass (LMM) and high molecular mass (HMM) enzymes. The HMM enzymes are further subdivided into the bifunctional class A enzymes (which possess a b-lactam-insensitive transglycosylase domain and the traditional b-lactam-sensitive transpeptidase domain) and the monofunctional transpeptidase class B enzymes. The soluble LMM PBPs have no identied role in b-lactam resistance and are primarily involved in carboxypeptidase reactions and peptidoglycan trimming. Despite their low sequence identity with the more essential HMM counterparts, the LMM PBPs have been a common vehicle for inhibitor studies owing to their soluble nature. Inhibitors that have been most recently used include peptidoglycanmimetic b-lactams and b-sultams [27,28]. By contrast, inhibitor studies of the membrane-anchored HMM PBPs have been hindered by their under-representation in the Protein Data Bank (PDB) structural database, primarily owing to the difculty of working with membrane proteins and the scarce lipid II substrate. However, structures of the major determinants in class B PBP-associated b-lactam resistance have recently become available to increase understanding of these phenomena, for example, PBP2x from the penicillin-resistant Streptococcus pneumoniae (PRSP) [2931], PBP2a from MRSA [32] and PBP5fm from the naturally resistant Enterococcus faecium [33]. The transpeptidase domain of the class A S. pneumoniae PBP1b has also been solved [34] and might provide a basis on which to model mutations in the resistant PBP1a enzyme. There are several PBP-mediated mechanisms of b-lactam resistance, including: acquisition of a new less-sensitive enzyme, mutation of an endogenous PBP to lessen the reaction with b-lactams (while maintaining some transpeptidase activity), or upregulation of PBP expression. The latter option appears to be the least effective, although the converse (lowering expression of some resistant PBPs) might prove to be useful in the control of b-lactam-resistant bacteria [35].
PBP2x structures

The mutation of PBP2x in PRSP has been studied extensively [2931] (Figure 4). Resistance occurs in a mosaic pattern, with many mutations occurring in different clinical isolates. This can cause problems in isolation of the main determinants of resistance and also presents the requirement for screening crystallization conditions anew. The structures of PBP2x from several resistant strains have been determined [2931]. Strain sp328 harbors the most clinically important mutation, T338A, which was found to result in the loss of an important active site water molecule, thus weakening the hydrogen bonding network that stabilizes the acylCurrent Opinion in Microbiology 2005, 8:525533

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Figure 4

enzyme complex [31]. The S389L and N514H mutations that are also present in this strain were found to sterically hinder favorable interactions with the b-lactam, reducing the acylation rate. An additional mutation, M339F, confers higher-level resistance to strains that possess the T338A mutation. The structure of this variant was found to re-orientate the S337 nucleophile and lower the reaction rate by 410-fold [29]. PBP2x from the PRSP strain sp5259 has also been structurally characterized [30] and appears to offer an alternative mechanism of resistance that shares features with other class B enzymes. The mutation of Q552 to glutamate introduces a negative charge near the edge of the active site; this might act like a similarly positioned residue in PBP5fm, disfavoring interaction with negatively charged b-lactams. Other mutations in this strain act in a similar way to residues from PBP2a, altering the conformation of b-strand 3 so that an energetically costly rearrangement is required for acylation. However, the observation that resistant class B PBPs share these features might not assist in the development of new broadspectrum antibiotics. Molecular dynamics simulations on a group of enzymes that share the transpeptidase fold raised the possibility that there are different mechanisms of acylation dependent on the particular combination of enzyme and antibiotic [36]; this leaves bacteria many possibilities to develop resistance against new b-lactams.
Other PBP structures

It is also prudent to note that PBP1a and PBP2b are involved in PRSP resistance. Sanbongi et al. [37] sequenced six S. pneumoniae PBPs, all of which are present in 40 clinical isolates, and thereby conrmed the correlation of mutations in these proteins to b-lactam minimum inhibitory concentrations (MICs). Mutations in PBP2b resulted in higher MICs for penicillins and carbapenems, whereas MIC increases for cephalosporins were associated with PBP2x. Alteration of the class A PBP1a conferred additional resistance to strains already bearing mutations in PBPs 2b and 2x. PBP2a of MRSA is encoded by the mecA gene, which is believed to have arisen as a result of horizontal transfer from an undisclosed species. When challenged with b-lactams, MRSA will utilize the transglycosylase activity of PBP2 (the only class A enzyme of S. aureus) and the transpeptidase functionality of PBP2a to synthesize the cell wall. It has recently been shown that PBP2 is able to mutate to a resistant form in the laboratory [38], but thankfully it is not responsible for the emergence of a second alternate form of MRSA in the environment. The structure of PBP2a [32] suggested that the poor acylation by b-lactams was caused by a b-strand 3 alteration, and a route to more effective antibiotics could be to increase the length of the b-lactam compound to improve non-covalent interactions. Several such new compounds have been
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The fold and active site of S. pneumoniae PBP2x. (a) Surface representation overlaid with protein fold. The transpeptidase domain is colored red and the other domains blue. (b) Detail of active site topology. This shows the S337 nucleophile and residues responsible for the development of resistance to b-lactams. Residues are represented in stick format and are colored according to atom type (C atoms, yellow; O, red; N, blue; and S, orange). The protein co-ordinates used are from PDB 1RP5, with a molecule of cefuroxime (C atoms, yellow in [a], magenta in [b]) placed in an equivalent position to that observed in the complex from PDB 1QMF. Note that because 1RP5 is used, the native amino acid Q552 is mutated to a glutamate residue. Current Opinion in Microbiology 2005, 8:525533

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described at length by Bush et al. [39], including BAL9141, which is active against both PRSP and MRSA. The use of specic chemical groups to mimic the differences in peptidoglycan sub-structure between various bacterial pathogens, or indeed to facilitate the conformational changes required for acylation, might also prove fruitful, as suggested recently by Fuda et al. [40]. In contrast to S. aureus and S. pneumoniae, the bacterium E. faecium is naturally resistant to b-lactam antibiotics. The PBP responsible for this resistance, PBP5fm, has been structurally characterized [33], although no co-ordinates for this enzyme are available in the PDB at present. The reason for the endogenous low-level afnity for b-lactams in E. faecium isnt immediately apparent, but could be owing to the reduced active site accessibility and also to charge repulsion with the b-lactam carboxylate group. E. faecium can demonstrate higher-level resistance by mutation of PBP5fm [41], including an insertion after S466 that is present in a loop that shifts upon acylation.
Bifunctional PBPs

tics that target the essential transglycosylase domain. Such inhibitors would undoubtedly work well in combination therapy with b-lactams, as shown by the essential nature of PBP2 and PBP2a co-operation in MRSA strains.

Altered antibiotic permeability and efux: the Gram-negative efux pumps

With the exception of some strains of the Streptococci, Enterococci and Staphylococci superbugs, Gram-negative bacteria are generally more resistant to a large variety of antibiotics and chemotherapeutic agents than are Grampositive bacteria. It is now recognized that a major contribution to antibiotic resistance in Gram-negative species is the presence of broad-specicity drug-efux pumps. One of the best-characterized of these is the drug efux system MexABOprM (recently reviewed in [43]) of the opportunistic pathogen, Pseudomonas aeruginosa. This tripartite pump (composed of the inner membrane RND transporter pump MexB, the outer membrane porin OprM, and the soluble periplasmic MexA) acts on a wide range of antibiotics, including tetracycline, chloramphenicol, quinilones, novobiocin, macrolides and trimethoprim, as well as b-lactams and b-lactamase inhibitors such as clavulanic acid. The past four years have seen a tremendous increase in our understanding of the structural features of the individual components of the tripartite efux pumps (for a recent review see [44]); the orthologous outermembrane porin and innermembrane pump components TolC and AcrB from E. coli have been determined to 2.1 and 3.5 A resolution, respectively, and the periplasmic component MexA from Pseudomonas aer uginosa to 3.5 A resolution. A structure of the inner membrane pump AcrB in the presence of several hydrophobic small molecule compounds has also been determined [45], which implies a diverse binding mode for individual ligands, at least in this component of the efux pump. Although these structures have provided a tremendous new level of understanding of the distinct architecture of the three proteins that make up these pumps, there are still many unanswered questions with regard to the way in which these components interact to form a single path for extruded antibiotic ligands. These systems represent logical targets for novel antibiotic design, and development of lead compounds in this area is evolving rapidly (as reviewed recently by Kaatz [46]).

The rst crystal structure of a transpeptidase domain from a class A enzyme PBP1b from S. pneumoniae has recently been reported [34]. The role of PBP1b in b-lactam resistance is minor at best, but its structure can be used as a model for mutational effects in the resistant PBP1a enzyme (45% sequence identity between the transpeptidase region of PBP1a and PBP1b). It is also noted that crystallization conditions for PBP1a have been reported [42]. Like PBPs 2x, 2a and 5fm, the enzyme possesses a classical transpeptidase domain, which is anked by regions of unknown function that might play a role in association with other cell wall modifying proteins. The postulation of multi-enzyme complexes for HMM PBPs is particularly interesting when applied to this structure as the active site appears closed and activity might be regulated by interaction with other proteins. One consequence of open and closed active sites in these enzymes is that resistant PBPs are expected to favor open conformations, allowing transpeptidation of the bulkier substrates that result from inhibition of the LMM PBP carboxypeptidases by b-lactams. The resistance-conferring mutation of T371S/A in PBP1a is analogous to that of T338A described for PBP2x. Other mutations of b-lactam-insensitive PBP1a can be mapped to a conserved proline near the classical SXN motif as well as to a range of mutations along b-strands 4 and 5 [34]. Given the structural data thus far and the requirement of conformational changes in catalysis of the HMM but not of the LMM PBPs, there is some concern about using the latter as model systems for inhibitor design. It is therefore important to obtain structures of other HMM PBPs to better understand the commonality of this active site plasticity. In addition, structures of the full-length class A enzymes might enable the generation of new antibiowww.sciencedirect.com

Conclusions
Recent years have witnessed a substantial increase in our understanding of the mechanisms responsible for b-lactam resistance. The structures of the molecular determinants of resistance particularly in complex with antibiotics or inhibitors are poised not only to explain resistance, but also to inspire novel methods of combating it. The b-lactam class of antibiotics has proven itself to be invaluable in the treatment of bacterial infections, and structural biology will undoubtedly play a
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central role in ensuring that b-lactams remain therapeutically effective.

13. Phillips OA, Reddy AV, Setti EL, Spevak P, Czajkowski DP, Atwal H, Salama S, Micetich RG, Maiti SN: Synthesis and biological evaluation of penam sulfones as inhibitors of beta-lactamases. Bioorg Med Chem 2005, 13:2847-2858. 14. Buynak JD, Chen H, Vogeti L, Gadhachanda VR, Buchanan CA, Palzkill T, Shaw RW, Spencer J, Walsh TR: Penicillin-derived inhibitors that simultaneously target both metallo- and serinebeta-lactamases. Bioorg Med Chem Lett 2004, 14:1299-1304. 15. Bonnefoy A, Dupuis-Hamelin C, Steier V, Delachaume C, Seys C, Stachyra T, Fairley M, Guitton M, Lampilas M: In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor. J Antimicrob Chemother 2004, 54:410-417. 16. Tondi D, Morandi F, Bonnet R, Costi MP, Shoichet BK: Structurebased optimization of a non-beta-lactam lead results in inhibitors that do not up-regulate beta-lactamase expression in cell culture. J Am Chem Soc 2005, 127:4632-4639. 17. Venkatesan AM, Gu Y, Dos Santos O, Abe T, Agarwal A, Yang Y, Petersen PJ, Weiss WJ, Mansour TS, Nukaga M et al.: Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum betalactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates. J Med Chem 2004, 47:6556-6568. 18. Melckebeke HV, Vreuls C, Gans P, Filee P, Llabres G, Joris B, Simorre JP: Solution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance. J Mol Biol 2003, 333:711-720. 19. Safo MK, Zhao Q, Ko TP, Musayev FN, Robinson H, Scarsdale N,  Wang AH, Archer GL: Crystal structures of the BlaI repressor from Staphylococcus aureus and its complex with DNA: insights into transcriptional regulation of the bla and mec operons. J Bacteriol 2005, 187:1833-1844. This report describes the rst crystal structures of BlaI in a free form and in complex with the mec operator. Although the BlaI structures closely resemble the published structures of MecI, the authors describe an upand-down model of repressor-binding to the mec operon (versus the side-by-side model for the bla operon) and propose that proteolytic cleavage of BlaI or MecI preferentially occurs in the DNA-bound form. 20. Kerff F, Charlier P, Colombo ML, Sauvage E, Brans A, Frere JM, Joris B, Fonze E: Crystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis. Biochemistry 2003, 42:12835-12843. 21. Wilke MS, Hills TL, Zhang HZ, Chambers HF, Strynadka NC: Crystal structures of the Apo and penicillin-acylated forms of the BlaR1 beta-lactam sensor of Staphylococcus aureus. J Biol Chem 2004, 279:47278-47287. 22. Birck C, Cha JY, Cross J, Schulze-Briese C, Meroueh SO, Schlegel HB, Mobashery S, Samama JP: X-ray crystal structure of the acylated beta-lactam sensor domain of BlaR1 from Staphylococcus aureus and the mechanism of receptor activation for signal transduction. J Am Chem Soc 2004, 126:13945-13947. 23. Hanique S, Colombo ML, Goormaghtigh E, Soumillion P, Frere JM, Joris B: Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction. J Biol Chem 2004, 279:14264-14272. 24. Rosato AE, Kreiswirth BN, Craig WA, Eisner W, Climo MW, Archer GL: mecA-blaZ corepressors in clinical Staphylococcus aureus isolates. Antimicrob Agents Chemother 2003, 47:1460-1463. 25. Katayama Y, Zhang HZ, Hong D, Chambers HF: Jumping the barrier to beta-lactam resistance in Staphylococcus aureus. J Bacteriol 2003, 185:5465-5472. 26. Zervosen A, Lu WP, Chen Z, White RE, Demuth TP Jr, Frere JM: Interactions between penicillin-binding proteins (PBPs) and two novel classes of PBP inhibitors, arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones. Antimicrob Agents Chemother 2004, 48:961-969. 27. Llinas A, Ahmed N, Cordaro M, Laws AP, Frere JM, Delmarcelle M, Silvaggi NR, Kelly JA, Page MI: Inactivation of bacterial DDpeptidase by beta-sultams. Biochemistry 2005, 44:7738-7746. www.sciencedirect.com

Acknowledgements
We are grateful for support from the Howard Hughes Medical Institute (to NS), the Canadian Institute of Health Research (to NS and MW), and the Michael Smith Foundation for Health Research (to MW and AL).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Weldhagen GF: Integrons and beta-lactamases a novel perspective on resistance. Int J Antimicrob Agents 2004, 23:556-562.

2. 

Fisher JF, Meroueh SO, Mobashery S: Bacterial resistance to beta-lactam antibiotics: compelling opportunism, compelling opportunity. Chem Rev 2005, 105:395-424. An excellent and comprehensive review about the mechanisms of blactam resistance. 3. Heinz U, Adolph HW: Metallo-beta-lactamases: two binding sites for one catalytic metal ion? Cell Mol Life Sci 2004, 61:2827-2839.

4. 

Garau G, Bebrone C, Anne C, Galleni M, Frere JM, Dideberg O: A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem. J Mol Biol 2005, 345:785-795. This article reports the crystal structures of wild-type CphA, its N220G mutant, and a complex of N220G CphA with the carbapenem substrate biapenem. As well as providing the rst structures of a class B2 metallob-lactamase, the authors propose a novel monozinc carbapenemase mechanism based on the details of the CphA active site in complex with substrate. 5. Wommer S, Rival S, Heinz U, Galleni M, Frere JM, Franceschini N, Amicosante G, Rasmussen B, Bauer R, Adolph HW: Substrateactivated zinc binding of metallo-beta-lactamases: physiological importance of mononuclear enzymes. J Biol Chem 2002, 277:24142-24147. Palumbi SR: Humans as the worlds greatest evolutionary force. Science 2001, 293:1786-1790. Poole K: Resistance to beta-lactam antibiotics. Cell Mol Life Sci 2004, 61:2200-2223. Jacoby GA, Munoz-Price LS: The new beta-lactamases. N Engl J Med 2005, 352:380-391.

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Chen Y, Shoichet B, Bonnet R: Structure, function, and inhibition along the reaction coordinate of CTX-M betalactamases. J Am Chem Soc 2005, 127:5423-5434. The crystal structures of CTX-M-9 and/or CTX-M-14 are reported in complex with glycylboronic acid transition state analogues and with the inhibitor cefoxitin. These structures represent snapshots of CTX-M progressing through b-lactam hydrolysis, (i.e. in an acylation transition state, as an acyl-enzyme intermediate, and in a deacylation transition state). To explain the inhibitory activity of cefoxitin, steric hindrance owing to the 7a-group of the cefoxitin was implicated in blocking formation of the deacylation transition state. 10. Chen Y, Delmas J, Sirot J, Shoichet B, Bonnet R: Atomic resolution structures of CTX-M beta-lactamases: extended spectrum activities from increased mobility and decreased stability. J Mol Biol 2005, 348:349-362. 11. Sanschagrin F, Levesque RC: A specic peptide inhibitor of the class B metallo-beta-lactamase L-1 from Stenotrophomonas maltophilia identied using phage display. J Antimicrob Chemother 2005, 55:252-255. 12. Moloughney JG, Thomas JD, Toney JH: Novel IMP-1 metallobeta-lactamase inhibitors can reverse meropenem resistance in Escherichia coli expressing IMP-1. FEMS Microbiol Lett 2005, 243:65-71.

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b-Lactam antibiotic resistance Wilke, Lovering and Strynadka 533

28. Silvaggi NR, Josephine HR, Kuzin AP, Nagarajan R, Pratt RF, Kelly JA: Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a perfect penicillin. J Mol Biol 2005, 345:521-533. 29. Chesnel L, Pernot L, Lemaire D, Champelovier D, Croize J, Dideberg O, Vernet T, Zapun A: The structural modications induced by the M339F substitution in PBP2x from Streptococcus pneumoniae further decreases the susceptibility to beta-lactams of resistant strains. J Biol Chem 2003, 278:44448-44456. 30. Pernot L, Chesnel L, Le Gouellec A, Croize J, Vernet T, Dideberg O,  Dessen A: A PBP2x from a clinical isolate of Streptococcus pneumoniae exhibits an alternative mechanism for reduction of susceptibility to beta-lactam antibiotics. J Biol Chem 2004, 279:16463-16470. Following on from the work done on PRSP strain sp328 [31], the structure of PBP2x from strain sp5259 was obtained. In this case, the mutation of residues serves more to mimic resistance effects observed in other lactam-tolerant enzymes, including the re-arrangement of strain b3 (akin to PBP2a from S. aureus) and the introduction of negative charge near the active site (akin to PBP5fm from E. faecium). In tandem with the mutants from sp328 that act to modify the environment around the active site nucleophile, these results highlight the different techniques used to confer resistance in PBP2x. 31. Dessen A, Mouz N, Gordon E, Hopkins J, Dideberg O: Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations. J Biol Chem 2001, 276:45106-45112. 32. Lim D, Strynadka NC: Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat Struct Biol 2002, 9:870-876. 33. Sauvage E, Kerff F, Fonze E, Herman R, Schoot B, Marquette JP, Taburet Y, Prevost D, Dumas J, Leonard G et al.: The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin. Cell Mol Life Sci 2002, 59:1223-1232. 34. Macheboeuf P, Di Guilmi AM, Job V, Vernet T, Dideberg O,  Dessen A: Active site restructuring regulates ligand recognition in class A penicillin-binding proteins. Proc Natl Acad Sci USA 2005, 102:577-582. The rst structural information obtained for a class A PBP, both in the unliganded form and when complexed with b-lactams. The structure is representative of the classical PBP fold, but also provides information on the interaction with the transglycosylase domain and the regulation of enzymatic activity by way of active site opening. This work also discusses the structural effects of mutations in the related enzyme PBP1a, which is implicated in b-lactam-resistant forms of S. pneumoniae.

35. Tajima Y: Polyoxotungstates reduce the beta-lactam resistance of methicillin-resistant Staphylococcus aureus. Mini Rev Med Chem 2005, 5:255-268. 36. Oliva M, Dideberg O, Field MJ: Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study. Proteins 2003, 53:88-100. 37. Sanbongi Y, Ida T, Ishikawa M, Osaki Y, Kataoka H, Suzuki T, Kondo K, Ohsawa F, Yonezawa M: Complete sequences of six penicillin-binding protein genes from 40 Streptococcus pneumoniae clinical isolates collected in Japan. Antimicrob Agents Chemother 2004, 48:2244-2250. 38. Leski TA, Tomasz A: Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A. J Bacteriol 2005, 187:1815-1824. 39. Bush K, Macielag M, Weidner-Wells M: Taking inventory: antibacterial agents currently at or beyond phase 1. Curr Opin Microbiol 2004, 7:466-476. 40. Fuda C, Hesek D, Lee M, Morio K, Nowak T, Mobashery S: Activation for catalysis of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by bacterial cell wall. J Am Chem Soc 2005, 127:2056-2057. 41. Rice LB, Bellais S, Carias LL, Hutton-Thomas R, Bonomo RA, Caspers P, Page MG, Gutmann L: Impact of specic pbp5 mutations on expression of beta-lactam resistance in Enterococcus faecium. Antimicrob Agents Chemother 2004, 48:3028-3032. 42. Job V, Di Guilmi AM, Martin L, Vernet T, Dideberg O, Dessen A: Structural studies of the transpeptidase domain of PBP1a from Streptococcus pneumoniae. Acta Crystallogr D Biol Crystallogr 2003, 59:1067-1069. 43. Poole K: Efux-mediated antimicrobial resistance. J Antimicrob Chemother 2005, 56:20-51. 44. Eswaran J, Koronakis E, Higgins MK, Hughes C, Koronakis V: Threes company: component structures bring a closer view of tripartite drug efux pumps. Curr Opin Struct Biol 2004, 14:741-747. 45. Yu EW, McDermott G, Zgurskaya HI, Nikaido H, Koshland DE Jr: Structural basis of multiple drug-binding capacity of the AcrB multidrug efux pump. Science 2003, 300:976-980. 46. Kaatz GW: Bacterial efux pump inhibition. Curr Opin Investig Drugs 2005, 6:191-198.

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Current Opinion in Microbiology 2005, 8:525533