b-lactam resistance mechanisms: gram-positive bacteria...

b-Lactam Resistance Mechanisms: Gram-PositiveBacteria and Mycobacterium tuberculosis

Jed F. Fisher and Shahriar Mobashery

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556-5670

Correspondence: [email protected]; [email protected]

The value of the b-lactam antibiotics for the control of bacterial infection has erodedwith time. Three Gram-positive human pathogens that were once routinely susceptibleto b-lactam chemotherapy—Streptococcus pneumoniae, Enterococcus faecium, andStaphylococcus aureus—now are not. Although a fourth bacterium, the acid-fast (but notGram-positive-staining) Mycobacterium tuberculosis, has intrinsic resistance to earlierb-lactams, the emergence of strains of this bacterium resistant to virtually all other antibioticshas compelled the evaluation of newer b-lactam combinations as possible contributorsto the multidrug chemotherapy required to control tubercular infection. The emergingmolecular-level understanding of these resistance mechanisms used by these four bacteriaprovides the conceptual framework for bringing forward new b-lactams, and new b-lactamstrategies, for the future control of their infections.

Bacteria exemplify extraordinary diversity ofsize and shape (Young 2006, 2007). For the

eubacteria, both the shape and integrity ofthe cell are intimately related to the chemicalbonding pattern of the peptidoglycan polymerof their cell walls. Given this direct correlation, itis hardly surprising that many antibiotics targetthe enzymes that assemble the peptidoglycan(Schneider and Sahl 2010; Silhavy et al. 2010;Bugg et al. 2011; Silver 2013). Accordingly, bac-teria have evolved a myriad of mechanisms tosubvert these antibiotics.

Clinical bacterial resistance to antibioticsis often the acquisition of a primary resistancemechanism, abetted in important ways by sec-ondary mechanisms. For the clinically im-portant b-lactam antibiotics (these includethe penicillin, cephalosporin, carbapenem, and

monobactam subfamilies), the primary resis-tance mechanisms used by Gram-positive bac-teria are different from those used by the Gram-negative bacteria. The primary mechanismfor the Gram-negative bacteria is expression ofenzyme(s) that hydrolytically destroy the b-lac-tam, whereas for the Gram-positive bacteria theprimary mechanism is target modification. Thislatter mechanism whereby structural changesto the specific enzyme targets of the b-lactamantibiotics render these enzymes less reactiveto the b-lactam is identical to the mechanismused by the bacterial producers of theb-lactams(Ogawara 2015). The target of the b-lactamsis a family of enzymes still known today by thename given to these enzymes—penicillin-bind-ing proteins (PBPs)—dating from the discoverythat these enzymes are inactivated, by covalent

Editors: Lynn L. Silver and Karen Bush

Additional Perspectives on Antibiotics and Antibiotic Resistance available at www.perspectivesinmedicine.org

Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a025221

Cite this article as Cold Spring Harb Perspect Med 2016;6:a025221

1

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org

on May 20, 2020 - Published by Cold Spring Harbor Laboratory Press http://perspectivesinmedicine.cshlp.org/Downloaded from

mailto:[email protected]




http://www.perspectivesinmedicine.org



http://perspectivesinmedicine.cshlp.org/

modification, by the b-lactams. The PBPs arethe primary catalysts for the synthesis and re-modeling of the peptidoglycan cell wall of bac-teria. All bacteria have a family of PBPs, withsome PBPs essential and others not essential.Recognition of a b-lactam structure by essentialPBPs, leading to a mechanism-based loss of itsenzymatic activity through the b-lactam inacti-vation, invariably culminates in cell lysis (To-masz 1979). Although the evolutionary basisfor the selection of target modification as theprimary resistance mechanism for the Gram-positive bacteria is uncertain, the absence of anexterior membrane (and, thus, the absence of acontrol mechanism for exposure to antibiotics)in the Gram-positive bacteria surely contributesto this mechanism.

Although the peptidoglycan of all eubacte-ria has an identical core structure—a repeatingdisaccharide for the glycan strand with a pep-tide stem on the alternate saccharides of theglycan—each eubacteria tailors its peptidogly-can structure to accommodate the structuralrequirements for its shape, for the mechanismsfor the reproduction inter alia of that shape, andfor antibiotic resistance (Vollmer et al. 2008;Turner et al. 2014). The peptidoglycan ofGram-positive bacteria is a multilayer exoskele-ton above their single membrane, whereas thepeptidoglycan of the Gram-negative bacteria isa thinner (one- or two-layered) structure locat-ed in the periplasmic space between the twomembranes. Two events define the structure ofthe peptidoglycan polymer: glycan strand elon-gation and cross-linking of the glycan strandsby interconnection of the peptide stems onthe alternate glycans. This latter event usesthe terminal – D –Ala– D-Ala structure of thestem as an acyl donor from one strand to anamine acceptor of an adjacent strand. The b-lactam antibiotics have nearly synonymousthree-dimensional disposition to – D–Ala– D-Ala, but whereas the acyl-enzyme derived fromthe – D –Ala– D-Ala structure is a catalytic spe-cies en route to a reaction, the acyl-enzymederived from the b-lactam is not. The transpep-tidase enzymes of peptidoglycan biosynthesisare inactivated by the b-lactam. Figure 1 sum-marizes the molecular events of peptidoglycan

synthesis, with focus on the transpeptidationevent, in the context of the peptidoglycan struc-ture found in Streptococcus pneumoniae. Thecompelling rationale for understanding thesemechanisms is the timeless value of the b-lac-tams as therapeutic agents and the progressiveemergence of resistance in both Gram-negativeand -positive bacteria that were once routinelycontained by b-lactam chemotherapy but arenow clinically resistant.

THE ENZYME TARGETS OF THE b-LACTAMANTIBIOTICS

The Enzymology of Peptidoglycan Assembly

The events of peptidoglycan synthesis involvea multiprotein and multienzyme assembly,whose composition and performance are dis-tinctive to each bacterial species. The catalyticcore of this assembly contains the enzymecatalysts of transglycosylation and transpeptida-tion. As b-lactams inactivate the enzyme cata-lysts of transpeptidation, these enzymes are col-lectively referred to as PBPs (Waxman andStrominger 1983; Sauvage et al. 2008; Spratt2012). The use of “penicillin” in this terminol-ogy is historical: The penicillins were the firstfamily of the b-lactam antibiotics, whereasthe b-lactam antibiotics now used clinically in-clude also the cephalosporins, carbapenems,and monobactams. All bacteria have a familyof PBP enzymes classified by catalytic functionand mass. The low-molecular-mass PBPs (alsocalled class C PBPs) are primarily -D-Ala– D-Ala carboxypeptidases and control the popula-tion of stems competent for cross-linking. Thehigh-molecular-mass PBPs divide into twosubclasses. The first (class A) is bifunctional,having (in separate domains) the two catalyticactivities (transglycosylase and transpeptidase)required for peptidoglycan assembly. Class BPBPs are monofunctional catalysts of D-Ala–D-Ala-dependent transpeptidation. Althoughall PBPs show signature active-site sequencemotifs organized by a characteristic tertiarystructure, in other respects (such as remainingsequence and thus nuance of domain function)they are distinctive, both with respect to each

J.F. Fisher and S. Mobashery

2 Cite this article as Cold Spring Harb Perspect Med 2016;6:a025221

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



other and with respect to the PBPs of other(even closely related) bacterial species. A criticalcorollary is that individual b-lactam antibioticsshow distinctive patterns for PBP inactivation atthe (often) subsaturating conditions encoun-

tered in chemotherapy. This pattern determinesthe selection of theb-lactam to control infectionand the development of b-lactam resistance.We examine the complexity of this nexus—involving b-lactam structure, PBP structure

O

OH

NHAcO

OOOH

NHAc

HOO

ONH

Cross-linkedpeptide stem

NAMNAG

O

OH

NHAcO

OO

OH

NHAcHO

O

O

HN

Me

Me

HN

NH

CO2

O HN

ONH

O

Me

O Me

OMe

O

O

OH

NHAcOOO

OH

NH2

HOO

ONH

O

OH

NHAcOOO

OH

NH2

O

O

HN

Me

Me

HN

NH

CO2

OHN

NH

O Me

O

Me

O

HO

L-Lys–D-Ala–D-Ala

HN

NH

O

Me

NH

OMe

NAMNAG

Peptide stems

L-Ala–L-Alabridge

2

3

4

7

6

2

Glycosyl transferase reaction of the PBPs

Transpeptidase reaction of the PBPs

2

1

6

5

3

Glycan strands

Figure 1. Schematic for the D,D-cross-link of the peptidoglycan of Streptococcus pneumoniae. This schematicindicates the structural connectivity for the normal cross-linking of the peptidoglycan and does not representthree-dimensional structure. The peptidoglycan is assembled by sequential actions of the penicillin-bindingproteins (PBPs). The presumed first action occurs at the glycosyl transferase active site wherein a glycan strand(1) is assembled by polymerization of lipid II as the substrate. The second action is cross-linking of the peptidestems (2) of adjacent glycan strands through transpeptidase catalysis by the PBPs. Completion of both steps givesthe polymeric structure of the peptidoglycan cell wall. Transpeptidase catalysis is the step blocked by theb-lactam antibiotics. This schematic shows two glycan strands and the completed cross-link between them.Key additional features of the peptidoglycan are emphasized. The sequence of the terminal amino acids of thestem is – L-Lys– D-Ala– D-Ala (in which the 3 identifies the penultimate D-Ala). In S. pneumoniae, an L-Ala-L-Aladipeptide bridge (4) is added to the side-chain amine of this lysine. An L-Ser-L-Ala bridge is also encountered inthis bacterium. An L-Ala-L-Ala bridge is also used by Enterococcus faecium, whereas Staphylococcus aureus uses aGly5 pentapeptide bridge. In all of these bacteria, the primary cross-linking event uses the central D-Ala of thestem as an acyl donor to a serine in the transpeptidase active site of the PBP, releasing the terminal D-Ala. Thisacyl-enzyme then transfers the acyl moiety to the terminal amino acid of the bridge, completing the cross-link(5). In this schematic, the upper glycan strand is the acyl-donor strand and the lower strand is the acyl-acceptorstrand. The mechanisms controlling glycan strand length and the termination of glycan elongation (6) arepoorly understood. In S. pneumoniae, a substantial portion of the N-acetyl groups of the glycan strand arehydrolytically removed (7) as a defensive measure against untoward peptidoglycan degradation, such as bylysozyme. An alternative pattern of cross-linking is used by E. faecium as a b-lactam resistance method. PBPs,Penicillin-binding proteins; NAM, N-acetylmuramic acid; NAG, N-acetyl-2-amino-2-deoxyglucosamine.

b-Lactam Resistance Mechanisms

Cite this article as Cold Spring Harb Perspect Med 2016;6:a025221 3

ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



and function, and b-lactam resistance—for thefour Gram-positive pathogens S. pneumoniae,Enterococcus faecium, Staphylococcus aureus,and Mycobacterium tuberculosis.

THE MOLECULAR MECHANSIMS OFb-LACTAM RESISTANCE BY S. pneumoniae

S. pneumoniae is a human commensal of thenasopharyngeal microbiota (Hakenbeck et al.2012; Henriques-Normark and Tuomanen2013; Fischetti and Ryan 2015). Notwithstand-ing the benefit of pneumococcal vaccines(which are themselves forces for clinical strainand resistance-determinant selections) (An-goulvant et al. 2015; van Tonder et al. 2015),invasive infection of S. pneumoniae amonghumans with compromised immune systems re-mains a significant cause of morbidity and mor-tality. In the 40 years since the first appearanceof b-lactam resistance in S. pneumoniae, thisbacterium has responded quickly to clinical in-terventions by recombination with other strep-tococci (Sauerbier et al. 2012; Jensen et al. 2015)to secure resistance not just to the b-lactamsbut to other clinically important antibiotics(Reinert 2009; Croucher et al. 2011). S. pneumo-niae achieves b-lactam resistance through ex-tensive and complementary “mosaic” mutationof three key PBP target enzymes with minimalfitness cost (Albarracin Orio et al. 2011) whileretaining the genetic determinants that makethis bacterium so successful for persistent hu-man colonization (Nobbs et al. 2015). The chal-lenge for successful chemotherapy against thisbacterium is the invention of structure, whetherantibiotic or vaccine, that subverts this balance.We address this challenge as it relates to b-lac-tam control of infection by S. pneumoniae,through consideration of the role of its PBPsin peptidoglycan biosynthesis and evaluationof the effect of the mutations to critical PBPs.

The PBPs of S. pneumoniae

S. pneumoniae is a fitting introduction to thetopic of b-lactam resistance among the Gram-positive pathogens as this bacterium (comparedwith the others to be discussed) arguably pre-sents our best (although far from complete)

understanding of Gram-positive peptidoglycanbiosynthesis (Massidda et al. 2013). Three phas-es of peptidoglycan synthesis complete the ovo-coccal peptidoglycan of this bacterium. Onephase is sidewall growth. The remaining twophases are septal growth and final septation.Septal growth occurs across the middle planeof the ovococcus and culminates with the syn-thesis of a distinctive circular growth—a surfaceannulus—of peptidoglycan growth immediate-ly preceding septation (Wheeler et al. 2011).The regions of old and new peptidoglycan aredistinctly demarcated, and S. pneumoniae growswithout peptidoglycan turnover so as to pre-sumably avoid detection of its peptidoglycanby the innate immune system (Boersma et al.2015). S. pneumoniae apportions six PBPs to thetask of constructing its peptidoglycan. As thedifferent phases of peptidoglycan growth areinterdependent (Philippe et al. 2015), function-al complementarity (and redundancy) amongthese PBPs follows. Three (PBP1a, PBP1b,PBP2a) of the six PBPs are bifunctional classA PBPs, having both transglycosylase and trans-peptidase activities. Two are class B transpepti-dases (PBP2b and PBP2x), whereas the sixthPBP (PBP3) is a class C D,D-carboxypeptidase.The success of b-lactam chemotherapy in con-trolling S. pneumoniae infection establishes thatcertain of these PBPs, and certain events of pep-tidoglycan growth catalyzed by these PBPs, areessential to the bacterium. Unmasking thisidentity is central to the understanding of itsb-lactam resistance.

The complexity of experimental design re-quired for this task cannot be overstated. Weemphasized previously the substantial sequencevariability among the PBPs, apart from theirsignature catalytic motifs, within their con-served tertiary structure. Likewise, the b-lactamantibiotics also encompass structural diversity.Accordingly, the identification of b-lactamstructure useful against a particular bacteriumrests on the empirical discernment of the matchbetween b-lactam structure and the structuralspace of the active site of the “essential” PBPs.It is thus not surprising that there is greatbreadth of b-lactam match, and mismatch, tothe S. pneumoniae PBP ensemble (Kocaoglu



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



et al. 2015). Genetic analyses establish themonofunctional transpeptidases PBP2b (as amember of the elongosome complex used forpeptidoglycan elongation) and PBP2x (as amember of the divisome complex synthesizingthe peptidoglycan of the septum) as the twoessential PBPs of S. pneumoniae (Berg et al.2013; Philippe et al. 2014). Each of the bifunc-tional PBPs (PBP1a, PBP1b, PBP2) can bedeleted individually. Paired PBP1a/PBP2 dele-tions as well as the triple deletion are lethal,whereas other pairwise deletions show mor-phological defects. Deletion of the low-molec-ular-mass PBP3 maintains viability but also atthe cost of morphological defects (Hakenbecket al. 2012). As PBP2b and PBP2x are mono-functional transpeptidases, completion of pep-tidoglycan synthesis requires their pairing witha transglycosylase. As a result of the functionalredundancy among PBP1a, PBP1b, and PBP2,the identities for such pairings (and roles) areuncertain. Indeed, although the elongosomeand divisome complexes colocalize at mid-cellat the start of the growth and division events ofpeptidoglycan synthesis, the PBPs of these com-plexes subsequently follow individual patternsof spatial localization (Land et al. 2013; Tsuiet al. 2014). Resistance to b-lactams by S. pneu-moniae is the consequence of mutations to ei-ther PBP2b or PBP2x. High-level resistance tothe penicillins requires abetting mutations inPBP1a (Zapun et al. 2008).

The b-Lactam-Resistant PBPsof S. pneumoniae

Notwithstanding the preservation of the lysine-serine signature motifs in both the PBP2b andPBP2x D,D-transpeptidases, PBP2x is structur-ally distinct from PBP2b. PBP2b is a “typical”four-domain class A/B PBP: a short amino-terminal cytoplasmic tail; a transmembraneanchor; and protruding into the periplasm, aprotein–protein interaction “pedestal” domainfollowed by the catalytic domain (Contreras-Martel et al. 2009). The cytoplasmic tail andtransmembrane domains are also functional,presumably for protein–protein recognition(Berg et al. 2014). In addition to equivalents

of these four domains, PBP2x has two ad-ditional carboxy-terminal domains—termed“PBP and serine-threonine kinase-associated”(PASTA) domains—that follow the catalytic do-main. The PASTA domains are suggested to re-spond to peptidoglycan structure under phos-phorylation control (Maestro et al. 2011;Morlot et al. 2013). The importance of thePASTA domains for modulation of the catalyticdomain of PBP2x (Dias et al. 2009; Maurer et al.2012) and for its septal localization (Peters et al.2014) has been shown.

The key questions with respect to b-lactamresistance are the mutation(s) to the essentialPBPs, the mechanistic relevance of the muta-tions, and the additional contributors to S.pneumoniae b-lactam resistance. A recent ge-netic analysis addressed the last question. Ge-nome-wide association of single nucleotidepolymorphisms identified, in addition to thegenes for PBP1a, PBP2b, and PBP2x, contri-butions from the mraWand mraY genes withinthe peptidoglycan synthesis pathway, as well asgenes in the cell-division pathway ( ftsL, gpsB),genes encoding chaperones (clpL, clpX), anda gene in the recombination pathway (recU)(Chewapreecha et al. 2014). The gene associa-tions with penicillins were not coincident forthe cephalosporins. The polymorphisms weredistributed both in vaccine-targeted and non-vaccine-targeted lineages, suggested to explainwhy vaccination has failed to reduce b-lactamresistance (Hakenbeck 2014). A key determi-nant with respect to PBP mutation is the b-lactam used for resistance selection. For exam-ple, cefotaxime selectively inactivates the PBP2x(it is unreactive to PBP2b) of susceptibleS. pneumoniae (Kocaoglu et al. 2015). A sin-gle-point mutation in the catalytic domainof PBP2x achieves clinical resistance to cefo-taxime—but not to penicillins—as a result ofa fourfold increase in the minimal-inhibitoryconcentration (MIC) of cefotaxime (Coffeyet al. 1995). The more common experience isexemplified by the comparative structures of thePBP2b isolated from a penicillin-susceptible(minimum inhibitory concentration [MIC] of0.01 mg/mL) and a PBP2b isolated from a high-ly penicillin-resistant (MIC of 6 mg/mL) clini-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



cal strain of S. pneumoniae. This latter enzymehad 58 mutations (Fig. 2A), presenting a mosaicpattern of alteration across both the pedestaland catalytic domains (Contreras-Martel et al.2009). The differentiation between mutationsthat are incidental to resistance and those thatcontribute to resistance cannot be made solelyfrom structural analysis (Hakenbeck et al.2012). Although genetic analyses of the mosaicchanges underscore notable “point” mutations

to PBP2b and PBP2x, as exemplified recently(Ip et al. 2015), the realization of b-lactam re-sistance has a greater dimension than that seenas amino acid change alone.

The intertwining of protein structure, cata-lytic function, and resistance pathways is exem-plified by the “piperacillin paradox” observedfor S. pneumoniae (Philippe et al. 2015). Piper-acillin (a penicillin) is extensively used inthe clinic against S. pneumoniae, and, although

A B

Figure 2. (A) The mosaic pattern of resistance mutations within the periplasm-located domains of S.pneumococcus PBP2b (PDB Code 2WAE; Contreras-Martel et al. 2009). The enzyme crystallizes as a monomer.The 58 residues that undergo mutation during the transformation of this enzyme from ab-lactam-susceptible toa b-lactam-resistant state are shown in space-filling depictions. The catalytic serine used in the acyl-transferreactions of transpeptidation is depicted in green in a space-filling representation. Although mutations in thecatalytic domain predominate, several distal mutations are implicated as critical (as evidenced by the frequencyof their appearance). In general, the differentiation between mutations that are incidental and those mutationsthat contribute directly to resistance by favorably altering the loop “breathing motions” required to enable accessto this serine is exceptionally challenging. (B) The structure of the periplasm-located domains of S. aureusPBP2a with a bound quinazolinone (non-b-lactam) allosteric effector (PDB Code 4CJN; Bouley et al. 2015).The enzyme crystallizes as a dimer. The allosteric effector has intrinsic antibacterial activity and is depicted ingreen in a space-filling depiction (lower right of the structure). A single molecule of the allosteric effector isbound in a groove between the allosteric domain and a so-called pedestal domain. The orientation of this PBP2adimer is approximately 90˚ relative to that of the PBP2b of A. The two catalytic serines are shown in green in aspace-filling depiction. The mauve-colored arrows direct attention to their location. These serines are �60 Adistant from the allosteric site. The structural change that occurs in the allosteric transformation that controlsaccess to these active-site serines is understood (Otero et al. 2013).



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



it has greater potency against PBP2x (halfminimal inhibitory concentration (IC50) of0.02 mg/mL in susceptible S. pneumoniae), itselects preferentially for resistance mutationsin PBP2b (IC50 of 0.18 mg/mL) (Kocaoglu etal. 2015). The proposed explanation for thisparadox is that partial loss of PBP2b functionin elongation is sufficient to arrest growth,whereas the more complete loss of PBP2x func-tion in septation does not. Acquisition of a low-affinity PBP2b, thus, suffices to maintain cellmultiplication (Philippe et al. 2015).

The molecular basis for the low affinity inthe PBP targets has been extensively explored bycrystallography. Correlation of the observedmutations to the locations within these struc-tures shows preference for mutation at or nearthe active site. The cumulative effect of thesemutations is interpreted as a conformation ad-justment within the cleft that opens so as tofavor substrate over the b-lactam inactivator.This concept is discussed with respect to theparticular structures of S. pneumoniae PBP1a(Contreras-Martel et al. 2006; Job et al. 2008),PBP1b (Macheboeuf et al. 2005), PBP2b (Con-treras-Martel et al. 2009), and PBP2x (Gordonet al. 2000; Dessen et al. 2001; Chesnel et al.2003; Carapito et al. 2006; Maurer et al. 2008;Yamada et al. 2008). In selected cases, the mo-lecular basis of the mutation is understood.Cephalosporins have poor PBP2b affinity butgood PBP2x affinity and, thus, select PBP2xmutations. In response to cefotaxime challenge,under both laboratory and clinical conditions,mutation within the active site of PBP2x of thenoncatalytic threonine-550 residue to alanineconfers cefotaxime resistance (T550A, 20-folddecrease in acylation). The basis for cefotaximeresistance in these PBP2x mutants is interpretedas a result of the loss of a key hydrogen bondthat is used for recognition of the cephalospo-rins (Gordon et al. 2000). The T550A PBP2x is,however, more susceptible to inactivation bypenicillins (Mouz et al. 1999). Computationalevaluations are now used in which such simplecorrelations are not possible, as with clinicalmosaic mutations (Ge et al. 2012; Ramalingamet al. 2013). An aspect missing from these anal-yses is the effect of mutations on the transpep-

tidase reaction itself (as in vitro PBP assay of thisreaction is not possible) and on the integrationof the PBP into the elongosome and divisomecomplexes (Zerfass et al. 2009).

Antibiotic resistance often corresponds tomultiple adaptation mechanisms and, here aswell, S. pneumoniae shows its capability forb-lactam resistance. Its murMN operon encodestransferases that add an L-Ala-L-Ala (or L-Ser-L-Ala) cross-bridge extension to the L-Lys residueof the peptidoglycan stem (Filipe et al. 2002).These extensions contribute to b-lactam resis-tance (Hakenbeck et al. 2012), possibly byimproving the efficacy of PBP2b-dependenttranspeptidation (Berg et al. 2013). MurM/MurN-dependent cross-bridge extension isalso important for proper control of pneumo-lysin release from the peptidoglycan to supportvirulence (Greene et al. 2015). An additionalenzyme of peptidoglycan biosynthesis, MurE(the catalyst of L-Lys addition to the stem inthe course of lipid II biosynthesis), also enhanc-es—for unknown reasons—the b-lactam resis-tance of S. pneumoniae, as it also does forS. aureus (Todorova et al. 2015). S. pneumoniaefurther exemplifies the increasing recognitionthat antibiotic discovery in the future will re-quire evaluations beyond that of the interactionof the antibiotic with its target. Although werecognize that all targets (and especially thePBPs) function within a confluence of regulatedmetabolic pathways, we equally well recognizethat we understand neither the key componentsof these pathways nor how these componentsinterrelate. For example, the roles for Ser/Thr kinase control of not just PBP2x, but ofthe peptidoglycan biosynthetic pathway (Diaset al. 2009) and the pathways for cell growthand division (Falk and Weisblum 2013; Fleurieet al. 2014), remain essentially unknown. Anexample of the value provided by a whole bacte-rium perspective on antibiotic selection is thestudy of the growth response of antibiotic-sus-ceptible S. pneumoniae following exposure toantibiotics that were either bacteriostatic, bac-tericidal, or bactericidal as a result of lysis (i.e.,b-lactams) (Sorg and Veening 2015). Exposureof S. pneumoniae to these antibiotics at the“F10” concentration of the antibiotic (in which



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



F10 is the antibiotic concentration that achievesa 10-fold suppression of growth over a 10-h pe-riod and is a concentration typically somewhatgreater than the MIC) confirmed the advantageof a bactericidal over a bacteriostatic mecha-nism. However, remarkable differences wereseen among different antibiotics. Among thethree b-lactams compared (ampicillin, MIC of0.018 mg/mL; penicillin G, MIC of 0.015 mg/mL; and cephalexin, MIC of 0.22 mg/mL), am-picillin was the most efficacious. The basis forits superiority was interpreted in terms of itspossession of a narrow concentration range forefficacy (corresponding to a narrow mutant se-lection window) coinciding with suppressionof heterogeneous phenotype selection. In addi-tion, the b-lactam data further suggested a re-lationship between heteroresistance and cellmorphology (Sorg and Veening 2015). Theseobservations, although fully consistent with theuniqueness of b-lactam structure coincidingwith a unique profile for inhibition among thePBP family (Kocaoglu et al. 2015), affirmthe growing recognition of the limitation ofthe MIC value (alone) as the criterion to defineantibiotic efficacy.

THE MOLECULAR MECHANISMS OFb-LACTAM RESISTANCE BY E. faecium

Before the introduction of the antibiotics, theenterococci were innocuous commensal bacte-ria of the gut and were infrequently associatedwith infection (Arias and Murray 2012; Hen-drickx et al. 2013; Werner et al. 2013; Kristichet al. 2014). Coincident with the introductionof the antibiotics, rapid genetic diversificationculminated in the emergence 30 years ago ofthe enterococci as insidious, multidrug-resis-tant nosocomial pathogens. Whereas the keypathogen at the time of this emergence was En-terococcus faecalis, infections by E. faecium andE. faecalis now are equally prevalent. A key ob-servation made during this transition was thatthe enterococci had intrinsic resistance to thecephalosporin class of b-lactam antibiotics.The transition of these bacteria from high- tolow-penicillin susceptibility—to the point to-day in which nosocomial infection by the en-

terococci in the United States is presumed to beboth b-lactam- and vancomycin-resistant—hasbeen addressed from the vantages of genomics,structural biology, and enzymology (Palmeret al. 2010; Hendrickx et al. 2013; Lebretonet al. 2013; Werner et al. 2013). The origin ofthe b-lactam resistance of E. faecium (the morepathogenic of the two, in large part as a result ofits greater b-lactam resistance) exemplifies themultifactorial resistance mechanisms now usedby resistant bacteria.

Despite the identity of S. pneumoniae, theenterococci, and S. aureus as Gram-positivecocci, the structural similarities of their pepti-doglycans, and the in vitro ability to function-ally interchange the peptidoglycan biosyntheticpathways of these cocci (Arbeloa et al. 2004a),each chooses a different PBP mechanism to at-tain b-lactam resistance. The ability of the en-terococci to assimilate pathways in support ofantibiotic resistance and virulence was exempli-fied sharply by the appearance of vancomycinresistance as a result of the acquisition of theself-resistance mechanism used by vancomy-cin-producing bacteria. Vancomycin resistanceresults from the remodeling of peptidoglycansynthesis so as to replace the vancomycin-bind-ing D-Ala-D-Ala stem terminus normally usedfor transpeptidation (Cattoir and Leclercq2013). b-Lactam resistance by E. faecium in-volves a low-affinity PBP for catalysis of trans-peptidation. Mutation of the essential (andintrinsically cephalosporin-unreactive) PBP5of E. faecium gives the requisite low-affinity en-zyme PBP5fm (Zorzi et al. 1996; Arbeloa et al.2004b; Lebreton et al. 2013; Pietta et al. 2014).PBP5fm pairs with one of several transglycosy-lases to complete peptidoglycan synthesis (Riceet al. 2009). The basis for the low affinity, visu-alized from the vantage of the location of themutations in the PBP5fm structure, is suggestedas restricted motion in the loop that controls byan opening motion access to the active site(Sauvage et al. 2002). This mechanism parallelsthe observation for the key PBPs of S. pneumo-niae and also parallels the resistance mechanismof PBP2a of S. aureus.

The contribution of PBP5fm to theb-lactam resistance of E. faecium is central



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



but not exclusive. All bacteria revamp theirmetabolism in response to cell-wall stress. Forthe enterococci, these additional changes in-clude mitigation of reactive species to attenuatethe bactericidal effect of antibiotics in general(Ladjouzi et al. 2013; Djoric and Kristich2015), consistent with the emerging hy-potheses for the bactericidal effect of antibiot-ics (Lobritz et al. 2015). A genome-wide eval-uation of ampicillin resistance in E. faeciumconfirmed the central role of PBP5fm andidentified a supporting contribution arisingfrom an alternative mechanism for peptidogly-can cross-linking (Zhang et al. 2012). Here, thecooperative activity of a PBP carboxypeptidase(to remove the terminal D-Ala from the peptidestem) and an L,D-peptidoglycan transpeptidase,enabling use of the L-Lys-D-Ala stem segmentas the acyl-donor for peptidoglycan cross-link-ing and, thus, the bypass of the b-lactam-sensitive use of D-Ala-D-Ala as the acyl donor

(Fig. 3). An identical enzyme function contrib-utes to b-lactam resistance in M. tuberculosis.An L,D-transpeptidase is encoded in yet otherGram-positive bacterium (such as Clostridiumdifficile) but is not present in S. pneumoniaeand E. faecalis. The operation of this pathwayis under both two-component and Ser/Thrkinase control (Sacco et al. 2014). The struc-ture, mechanism, and versatility (with respectto peptidoglycan structure) of this L,D-trans-peptidase are characterized in Mainardi et al.(2005), Biarrotte-Sorin et al. (2006), Cremniteret al. (2006), and Magnet et al. (2007). Not-withstanding substantive catalytic differencescompared with the PBPs (a catalytic cysteine,rather than a serine), this L,D-transpeptidaseis inactivated by carbapenems via acylation ofthe catalytic cysteine. Penicillins do not in-activate and cephalosporins weakly inactivate(Dubee et al. 2012; Lecoq et al. 2013; Tribouletet al. 2013, 2015).

O

O

NHAcO

OO

OH

NHAc

HOO

ONH

O

OH

NHAcO

OO

OH

NHAcHO

O

O

HN

Me

Me

HN

NH

C(O)NH2

O HN

OO

Me

O

Me

O

O

O

NHAcOOO

OH

NHAcHO

O

ONH

O

OH

NHAcOOO

OH

NHAc

O

O

HN

Me

Me

HN

NH

C(O)NH2

O

NH

NH

OMe

O

HO

L-Lys–D-Ala

HN OO

NH2

OMe

MeO

L,D-Dipeptidelink

The reaction of the L,D-transpeptidases of E. faecium

Figure 3. Schematic for the L,D-cross-link of the peptidoglycan of E. faecium. The peptidoglycan found insusceptible strains of E. faecium has D,D-cross-linking, wherein a – D-Ala– D-Ala-derived acyl moiety is trans-ferred to the a-amine of the iso-D-Asx (depicted as iso-Asn) residue of the stem bridge. Following in vitroselection for ampicillin resistance, E. faecium that is fully resistant to bothb-lactams and vancomycin is obtainedby peptidoglycan synthesis using a non-PBP-dependent L,D-cross-link. Here, trimming of terminal D-Ala of thecross-link the stem permits the use of the – L-Lys– D-Ala moiety of the stem for acyl transfer to thea-amine of theiso-D-Asx to achieve the cross-link. The six-position of the MurNAc saccharide is acetylated, as occurs frequentlyin the enterococci as a lysozyme-resistance adaptation (Pfeffer et al. 2006).



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



The minimal fitness cost of these resistancepathways (Foucault et al. 2010; Starikova et al.2013; Gilmore et al. 2015) and the ability of theenterococci to further assimilate resistance tonew therapeutic agents (such as daptomycin)account for the modern challenge of enter-ococci chemotherapy (Kristich et al. 2014).Later cephalosporins against E. faecalis andb-lactam-containing antibiotic combinationsagainst E. faecium exemplify the direction forfuture b-lactam therapy of enterococcal infec-tion (Henry et al. 2013; Hindler et al. 2015;Smith et al. 2015a,b; Werth et al. 2015).

THE MOLECULAR MECHANISMS OFb-LACTAM RESISTANCE BY S. aureus

S. aureus is a Gram-positive coccus and humancommensal (Missiakas and Schneewind 2015).Invasive infection by b-lactam-resistant S. au-reus following surgery and increasingly withinthe community to the soft tissue remains asdifficult a challenge for chemotherapeutic con-trol today as in prior decades (de Lencastre et al.2007; Stryjewski and Corey 2014; Knox et al.2015). New b-lactams, again acting to compro-mise the integrity of the peptidoglycan of thecell wall, remain a critical means of surmount-ing the resistance mechanisms used by S. aureus(Holmes and Howden 2014; Peyrani and Ra-mirez 2015). These mechanisms correspondto a complexity of regulatory mechanisms andcenter on an acquired PBP known today asPBP2a. This acquired PBP2a completes the syn-thesis of the peptidoglycan of S. aureus followingincapacitation of its other PBPs by a b-lactam.The cell wall of S. aureus is thicker than manyother Gram-positive bacteria, and, indeed, in-creased cell-wall thickness is a resistance mech-anism used by S. aureus against other cell-walltargeting antibiotics, notably vancomycin (Ca-zares-Dominguez et al. 2015). Its peptidoglycanis exceptionally cross-linked (as much as 80% ofthe available stem peptide). Growth of the pep-tidoglycan of S. aureus involves synthesis of across-wall septum to create a pair of nascent,hemisphere-shaped bacteria. Rapid remodelingof the peptidoglycan provides the sphericalshape of the mature bacterium. Microscopic

analysis of this transformation suggests a con-centric growth pattern, followed by loss of theconcentric features coinciding with the pepti-doglycan remodeling (Turner et al. 2010, 2014;Bailey et al. 2014). At the molecular level, a dis-tinctive feature of the peptidoglycan of b-lac-tam-resistant S. aureus is the presence of a pen-taglycine cross-bridge extension to lysine of thepeptidoglycan stem. Addition of this pentagly-cine extension involves catalysis by the enzymesof the Fem (“factors enhancing methicillin” re-sistance) pathway (Dare and Ibba 2012). A com-parison of the mature cell wall of S. aureus andthe cell wall made by S. aureus having disrup-tions in the Fem pathway shows distinct differ-ences in the polymeric structure (Kim et al.2015; Singh et al. 2015). Nonetheless, the dif-ference at the molecular level for the correlationof the pentaglycine extension to methicillin re-sistance is not known.

There is no uncertainty for the core mech-anism of b-lactam resistance by S. aureus. Thisbacterium acquired two resistance mechanisms,with each acquisition occurring early in the50-year history of this resistance (Chambers2005; Moellering 2012; Peacock and Pater-son 2015; Tong et al. 2015). Following theclinical introduction of the earliest penicillins,penicillin-resistant S. aureus appeared as a resultof expression of a penicillin-specific b-lacta-mase. This b-lactamase was, and remains today,a “penicillinase” of modest catalytic abilityby comparison to the pan-b-lactam capabilityof the b-lactamases now endemic amongthe Gram-negative bacteria. This penicillinasenonetheless provided resistance to S. aureusagainst these early penicillin structures. In re-sponse, medicinal chemists discovered thatpenicillins substituted with sterically large arylgroups, exemplified by methicillin, were poorsubstrates of the penicillinase. The therapeuticvalue of methicillin was not long-lived. S. aure-us, in short order, acquired methicillin resis-tance by acquisition from environmental cocciof a new PBP (Zhou et al. 2008; Antignac andTomasz 2009; Tsubakishita et al. 2010). This ac-quired transpeptidase (PBP2a) integrates intothe enzyme assembly for peptidoglycan syn-thesis as a b-lactam-insensitive catalyst. PBP2a



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



distinguishes between the peptidoglycan as asubstrate and against the b-lactam as an inacti-vator. As this ability extends to all structuralclasses of b-lactams (not just methicillin), thissecond resistance mechanism has perseveredas a powerful resistance mechanism againstall but the newest guises of b-lactam structure.The empirically derived structures of the anti-MRSA (methicillin-resistant S. aureus) cepha-losporins, exemplified by ceftobiprole andceftaroline (Fernandez et al. 2014; Stryjewskiand Corey 2014), are successful antibioticsagainst S. aureus (and other Gram-positive bac-teria) as a direct result of their ability to evadethis structural discrimination by PBP2a. How-ever, although the framework for our discussionis PBP2a, we also provide a perspective on therole of the auxiliary mechanisms.

The central question is the uniqueness ofPBP2a. Methicillin-sensitive S. aureus encodeseight enzymes for peptidoglycan synthesis.MRSA has nine (now including PBP2a). Thecore eight enzymes are PBP1 (a monofunctionaltranspeptidase, active in cell division and sepa-ration), PBP2 (a bifunctional transglycosylaseand transpeptidase), PBP3 (a transpeptidase),PBP4 (a low-molecular-mass transpeptidase),two monofunctional transglycosylases, andtwo “auxiliary” transpeptidases (FmtA andFmtB). Genetic deletion of these activities,most recently using a MRSA strain, showsthat only two—PBP1 and PBP2—are requiredfor the normal growth and normal shape of S.aureus, albeit with loss ofb-lactam resistance, in-creased lysozyme susceptibility, and decreasedvirulence (Reed et al. 2015). Assembly of theMRSA peptidoglycan in the presence of theb-lactam challenge requires cooperative PBP2-dependent transglycosylation and PBP2a-de-pendent transpeptidation (Pinho et al. 2001).The presumption that the peptidoglycan bene-fits from cross-linking by a second transpepti-dase is supported by evidence implicating bothPBP4 and FmtA. PBP4 is involved in peripheralwall peptidoglycan remodeling (Leski and To-masz 2005; Loskill et al. 2014; Qiao et al. 2014;Gautam et al. 2015). PBP4 is essential for b-lac-tam resistance in the community strains ofMRSA (Memmi et al. 2008). Ab-lactam (cefox-

itin) with high PBP4 affinity is synergistic withoxacillin (Memmi et al. 2008). Although thecatalytic role of FmtA remains to be fully clari-fied, its presumed function is transpeptidationunder conditions of cell-wall stress (Qamar andGolemi-Kotra 2012).

Peptidoglycan biosynthesis is highly regu-lated at numerous levels, including by kinasephosphorylation, stress-response pathways,and the transmembrane delivery of lipid II asthe substrate for its PBPs (Lages et al. 2013).Notwithstanding the catalytic competence ofPBP2a in MRSA, PBP2a expression is inducedfollowing the irreversible acylation of the ex-posed cell-surface domain of the transmem-brane sensor protein MecR by a b-lactam anti-biotic. PBP2a is made only when circumstancesdemand its presence. The MecR protein is struc-turally and functionally homologous to a BlaRsensor protein, which itself controls expressionof the S. aureus penicillinase. Cross talk betweenthe two pathways adds additional complexity tothe poorly understood and multi-event cascadesultimately inducing PBP2a (and/or penicillin-ase) expression (Oliveira and de Lencastre 2011;Amoroso et al. 2012; Llarrull and Mobashery2012; Peacock and Paterson 2015; Staude et al.2015). A molecular-level understanding of thesepathways is anticipated to identify new targetsfor antibiotic synergy with b-lactams. An im-portant advance with respect to PBP2a is thediscovery that its active site is under allostericcontrol with respect to two loop motions thatopen the active site in response to its peptido-glycan substrate (Otero et al. 2013; Fishovitzet al. 2014). b-Lactams that appropriately mim-ic peptidoglycan structure, such as ceftaroline,have good MRSA activity by their ability to ef-fect this allosteric opening. The appreciable dis-tance between the newly discovered allostericsite and transpeptidase active site is evidentfrom the crystal structure of the non-b-lactambound to PBP2a (Fig. 2B). As a consequence oftheir allosteric-induced opening of the activesite, the concentrations achieved by these b-lac-tams in vivo coincide with the concentrationrequired for PBP2a inactivation (Fishovitzet al. 2014, 2015). Subversion of the allostericmechanism also has been achieved in vitro us-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



ing a threefold b-lactam combination (Gonza-les et al. 2015). Non-b-lactam allosteric effec-tors capable of b-lactam synergy have also beenidentified (Bouley et al. 2015).

Genomic technologies identify additionalloci that synergize with b-lactams (Roemeret al. 2012), including within the peptidoglycanbiosynthesis pathway (Mann et al. 2013), theFtsZ-dependent organization of the peptido-glycan biosynthetic machinery (Tan et al.2012), and the coordination of the synthesis ofthe wall teichoic acids with that of the peptido-glycan (Atilano et al. 2010; Pasquina et al. 2013;Wang et al. 2013; Sewell and Brown 2014; Win-stel et al. 2014). The targets identified within thewall teichoic acid pathway may have special sig-nificance as the synergistic pairing of interven-tion against both biosynthetic pathways mayextend to other Gram-positive bacteria (Hen-drickx et al. 2013). The translation of successfulin vitro combination therapy into successfulclinical therapy is never straightforward (Bush2015). Nonetheless, intervention at coupledbinding sites (such as by simultaneous occu-pancy of the allosteric site and active site ofPBP2a by two b-lactams) and at intersectingpathways (such as synergy between simultane-ous disruption of peptidoglycan and teichoicacid biosynthesis by two separate inhibitors) isemerging as a credible (if not yet viable, apartfrom b-lactam–b-lactamase pairs) strategy tocontrol resistant bacterial pathogens.

THE MOLECULAR MECHANISMS OFb-LACTAM RESISTANCE BY M. tuberculosis

The non-Gram-positive staining mycobacteriapossess a cell envelope structure that is funda-mentally different from and structurally morecomplex compared with the cell envelope ofeither the Gram-positive or -negative bacterium(Jackson et al. 2013; Alderwick et al. 2015; Na-taraj et al. 2015). A consequence of the nuancedlayers of this cell envelope is impermeability toantibiotic structure. Indeed, the challenge ofchemotherapeutic control of mycobacterial in-fection is legendary (Chakraborty and Rhee2015). Important additional factors contrib-uting toward the b-lactam insensitivity of

M. tuberculosis (in addition to impermeability)are the expression of efflux transporters, theversatility of this bacterium with respect to thesynthesis of alternate peptidoglycan structures,and expression of a robust—sufficiently so, as torepresent a possible means of detection ofM. tuberculosis infection (Cheng et al. 2014)—b-lactamase. For these reasons, the b-lactamshave never been among the many antibioticsused to control M. tuberculosis infection. None-theless, the combination of the emergence ofhighly resistant M. tuberculosis strains (Seunget al. 2015) with recent studies showing somepromise for b-lactams against M. tuberculosis(Hugonnet et al. 2009; Hazra et al. 2014; Wivagget al. 2014) has justified reconsideration of theb-lactams.

Although M. tuberculosis has the expectedPBP family for peptidoglycan biosynthesis(Prigozhin et al. 2014), as a matter of routine,it uses both PBP-catalyzed (and, thus, b-lac-tam-sensitive) D,D-transpeptidation and theb-lactam-insensitive L,D-transpeptidation forthis task (Goffin and Ghuysen 2002). The in-trinsic b-lactam unreactivity by some of thesePBPs (Bansal et al. 2015), a greater dependenceon L,D-transpeptidation in the presence of b-lactams (Gupta et al. 2010; Kumar et al. 2012;Schoonmaker et al. 2014), and reactivity as BlaCsubstrates explain the historic therapeutic fail-ure of the penicillin b-lactams. There is, how-ever, promise for carbapenem combinations.As discussed previously for E. faecium, the car-bapenems not only inactivate these L,D-trans-peptidases (Lavollay et al. 2008; Triboulet et al.2011; Cordillot et al. 2013; Schoonmaker et al.2014; Brammer Basta et al. 2015; Wivagg et al.2016) but have excellent activity against severalof the essential PBPs of M. tuberculosis (Cham-bers et al. 2005; Kumar et al. 2012) and are poorsubstrates for its BlaC b-lactamase (Tremblayet al. 2010; Hazra et al. 2014; Horita et al.2014). The sensitivity of BlaC-type enzymesto inactivation by clavulanate (Tremblay et al.2008) and the diazabicyclooctanone class (Xuet al. 2012; Dubee et al. 2015) indicates promiseforb-lactam pairing. What remains to be seen iswhether clinical use would give a BlaC mutationthat diminishes the efficacy of clavulanate (Ve-



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



ziris et al. 2011; Feiler et al. 2013; Kurz et al.2013; Egesborg et al. 2015; Soroka et al. 2015),and whether a diazabicyclooctanone derivativewill be identified having adequate clinical reac-tivity. Among the exploratory pairings reportedare meropenem-clavulanate, faropenem-cla-vulanate, amoxacillin-clavulanate, ceftaroline-clavulanate, ceftaroline-avibactam, and mero-penem–sulbactam (Hugonnet et al. 2009; Gon-zalo and Drobniewski 2013; Solapure et al.2013; Dhar et al. 2015; Dubee et al. 2015; Zhanget al. 2015). Although some data for these pair-ings are promising, it is certain that such pairswill require incorporation into a multidrug reg-imen. The composition of such regimen maycorrespond to proven, emerging, or new drugsas may be identified against new targets (someof unknown identity) as recognized by genomicsynthetic lethality screening (Lun et al. 2014).

CONCLUSION

The power of the b-lactams as antibiotics hasbeen diminished, but surely not lost, by thebreadth of resistance mechanisms now encoun-tered in both the Gram-positive and -negativebacteria. The most recent generations of cepha-losporin and carbapenem structures (in somecases, now paired with b-lactamase inhibitors),in particular, show promise against many of themost resistance-capable bacteria now encoun-tered. This outcome argues that the structuralspace of the b-lactams needed to subvert tar-get-based (and other) resistance mechanisms,as has been discussed here for the Gram-positivebacteria, is not exhausted. We now understandthat the target modification of the Gram-posi-tive PBPs does not stand alone but is supportedby underlying pathways that may secure synergywith b-lactams if cocompromised. Even a bac-terium historically regarded as impervious totheb-lactams, M. tuberculosis, may be made vul-nerable. Yet, even with the molecular dissectionof the resistant targets and resistance pathways,the future exploration of the structural spacearound the b-lactams will still demand invest-ment in empirical structure-activity develop-ment at a time when commercial interest in em-pirical antibiotic discovery has waned. This

obligation is no less true for the complementarytargets of these bacteria. We arguably are enter-ing an era in which the most intractable bacteriawill be treated with multiantibiotic regimens, asalways has been the case for M. tuberculosis. It issmall comfort to offer assurance that when theimpasse with respect to investing in antibioticdiscovery and development is surmounted, thetargets and strategies to secure the future place ofyet-undiscovered b-lactam antibiotics will be inplace. The substance of this review attests to thisassurance, although we are compelled to omitthe identity of such structures.

ACKNOWLEDGMENTS

The authors are supported by grants from theNational Institutes of Health (AI104987 andGM61629).

REFERENCES

Albarracin Orio AG, Pinas GE, Cortes PR, Cian MB, Eche-nique J. 2011. Compensatory evolution of pbp mutationsrestores the fitness cost imposed byb-lactam resistance inS. pneumoniae. PLoS Pathog 7: e1002000.

Alderwick LJ, Harrison J, Lloyd GS, Birch HL. 2015. Themycobacterial cell wall—Peptidoglycan and arabinoga-lactan. Cold Spring Harb Perspec Med 5: a021113.

Amoroso A, Boudet J, Berzigotti S, Duval S, Teller N, Men-gin-Lecreulx D, Luxen A, Simorre JP, Joris B. 2012. Apeptidoglycan fragment triggers b-lactam resistance inB. licheniformis. PLoS Pathog 8: e1002571.

Angoulvant F, Cohen R, Doit C, Elbez A, Werner A, Bechet S,Bonacorsi S, Varon E, Levy C. 2015. Trends in antibioticresistance of S. pneumoniae and H. influenzae isolatedfrom nasopharyngeal flora in children with acute otitismedia in France before and after 13 valent pneumococcalconjugate vaccine introduction. BMC Infect Dis 15: 236.

Antignac A, Tomasz A. 2009. Reconstruction of the pheno-types of methicillin-resistant S. aureus by replacement ofthe staphylococcal cassette chromosome mec with a plas-mid-borne copy of S. sciuri pbpD gene. Antimicrob AgentsChemother 53: 435–441.

Arbeloa A, Hugonnet JE, Sentilhes AC, Josseaume N, Du-bost L, Monsempes C, Blanot D, Brouard JP, Arthur M.2004a. Synthesis of mosaic peptidoglycan cross-bridgesby hybrid peptidoglycan assembly pathways in Gram-positive bacteria. J Biol Chem 279: 41546–41556.

Arbeloa A, Segal H, Hugonnet JE, Josseaume N, Dubost L,Brouard JP, Gutmann L, Mengin-Lecreulx D, Arthur M.2004b. Role of class A PBPs in PBP5-mediated b-lactamresistance in E. faecalis. J Bacteriol 186: 1221–1228.

Arias CA, Murray BE. 2012. The rise of the Enterococcus:Beyond vancomycin resistance. Nat Rev Microbiol 10:266–278.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Atilano ML, Pereira PM, Yates J, Reed P, Veiga H, Pinho MG,Filipe SR. 2010. Teichoic acids are temporal and spatialregulators of peptidoglycan cross-linking in S. aureus.Proc Natl Acad Sci 107: 18991–18996.

Bailey RG, Turner RD, Mullin N, Clarke N, Foster SJ, HobbsJK. 2014. The interplay between cell wall mechanicalproperties and the cell cycle in S. aureus. Biophys J 107:2538–2545.

Bansal A, Kar D, Murugan RA, Mallick S, Dutta M, PandeySD, Chowdhury C, Ghosh AS. 2015. A putative low-mo-lecular mass (LMM) PBPof M. smegmatis exhibits prom-inent physiological characters of DD-carboxypeptidaseand b-lactamase. Microbiology 161: 1081–1091.

Berg KH, Stamsas GA, Straume D, Havarstein LS. 2013.Effects of low PBP2b levels on cell morphology and pep-tidoglycan composition in S. pneumoniae R6. J Bacteriol195: 4342–4354.

Berg KH, Straume D, Havarstein LS. 2014. The function ofthe transmembrane and cytoplasmic domains of pneu-mococcal PBP2x and PBP2b extends beyond that of sim-ple anchoring devices. Microbiology 160: 1585–1598.

Biarrotte-Sorin S, Hugonnet JE, Delfosse V, Mainardi JL,Gutmann L, Arthur M, Mayer C. 2006. Crystal structureof a novel b-lactam-insensitive peptidoglycan transpep-tidase. J Mol Biol 359: 533–538.

Boersma MJ, Kuru E, Rittichier JT, VanNieuwenhze MS,Brun YV, Winkler ME. 2015. Minimal peptidoglycan(PG) turnover in wild-type and pg hydrolase and celldivision mutants of S. pneumoniae D39 growing plank-tonically and in host-relevant biofilms. J Bacteriol 197:3472–3485.

Bouley R, Kumarasiri M, Peng Z, Otero LH, Song W, SuckowMA, Schroeder VA, Wolter WR, Lastochkin E, AntunesNT, et al. 2015. Discovery of antibiotic (E)-3-(3-carbox-yphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one. J AmChem Soc 137: 1738–1741.

Brammer Basta LA, Ghosh A, Pan Y, Jakoncic J, Lloyd EP,Townsend CA, Lamichhane G, Bianchet M. 2015. A lossof a functionally and structurally distinct LD-transpepti-dase, LdtMt5, compromises cell wall integrity in M. tu-berculosis. J Biol Chem 290: 25670–25685.

Bugg TD, Braddick D, Dowson CG, Roper DI. 2011. Bacte-rial cell wall assembly: Still an attractive antibacterialtarget. Trends Biotechnol 29: 167–173.

Bush K. 2015. Antibiotics: Synergistic MRSA combinations.Nat Chem Biol 11: 832–833.

Carapito R, Chesnel L, Vernet T, Zapun A. 2006. Pneumo-coccal b-lactam resistance due to a conformationalchange in PBP2x. J Biol Chem 281: 1771–1777.

Cattoir V, Leclercq R. 2013. Twenty-five years of shared lifewith vancomycin-resistant enterococci: Is it time to di-vorce? J Antimicrob Chemother 68: 731–742.

Cazares-Dominguez V, Cruz-Cordova A, Ochoa SA, Esca-lona G, Arellano-Galindo J, Rodriguez-Leviz A, Hernan-dez-Castro R, Lopez-Villegas EO, Xicohtencatl-Cortes J.2015. Vancomycin-tolerant, methicillin-resistant S. aure-us reveals the effects of vancomycin on cell wall thicken-ing. PLoS ONE 10: e0118791.

Chakraborty S, Rhee KY. 2015. Tuberculosis drug develop-ment: History and evolution of the mechanism-basedparadigm. Cold Spring Harb Perspect Med 5: a021147.

Chambers HF. 2005. Community-associated MRSA—Resis-tance and virulence converge. N Engl J Med 352: 1485–1487.

Chambers HF, Turner J, Schecter G, Kawamura M, Hope-well PC. 2005. Imipenem for treatment of tuberculosisin mice and humans. Antimicrob Agents Chemother 49:2816–2821.

Cheng Y, Xie H, Sule P, Hassounah H, Graviss EA, Kong Y,Cirillo JD, Rao J. 2014. Fluorogenic probes with substi-tutions at the 2 and 7 positions of cephalosporin arehighly BlaC-specific for rapid M. tuberculosis detection.Angew Chem, Int Ed 53: 9360–9364.

Chesnel L, Pernot L, Lemaire D, Champelovier D, Croize J,Dideberg O, Vernet T, Zapun A. 2003. The structuralmodifications induced by the M339F substitution inPBP2x from St. pneumoniae further decreases the sus-ceptibility to b-lactams of resistant strains. J Biol Chem278: 44448–44456.

Chewapreecha C, Marttinen P, Croucher NJ, Salter SJ, HarrisSR, Mather AE, Hanage WP, Goldblatt D, Nosten FH,Turner C, et al. 2014. Comprehensive identification ofsingle nucleotide polymorphisms associated with b-lac-tam resistance within pneumococcal mosaic genes. PLoSGenet 10: e1004547.

Coffey TJ, Daniels M, McDougal LK, Dowson CG, TenoverFC, Spratt BG. 1995. Genetic analysis of clinical isolates ofS. pneumoniae with high-level resistance to expanded-spectrum cephalosporins. Antimicrob Agents Chemother39: 1306–1313.

Contreras-Martel C, Job V, Di Guilmi AM, Vernet T, Dide-berg O, Dessen A. 2006. Crystal structure of PBP1a revealsa mutational hotspot implicated inb-lactam resistance inS. pneumoniae. J Mol Biol 355: 684–696.

Contreras-Martel C, Dahout-Gonzalez C, Martins Ados S,Kotnik M, Dessen A. 2009. PBPactive site flexibility as thekey mechanism for b-lactam resistance in pneumococci.J Mol Biol 387: 899–909.

Cordillot M, Dubee V, Triboulet S, Dubost L, Marie A,Hugonnet JE, Arthur M, Mainardi JL. 2013. In vitrocross-linking of M. tuberculosis peptidoglycan by L,D-transpeptidases and inactivation of these enzymes by car-bapenems. Antimicrob Agents Chemother 57: 5940–5945.

Cremniter J, Mainardi JL, Josseaume N, Quincampoix JC,Dubost L, Hugonnet JE, Marie A, Gutmann L, Rice LB,Arthur M. 2006. Novel mechanism of resistance to gly-copeptide antibiotics in E. faecium. J Biol Chem 281:32254–32262.

Croucher NJ, Harris SR, Fraser C, Quail MA, Burton J, vander Linden M, McGee L, von Gottberg A, Song JH, KoKS, et al. 2011. Rapid pneumococcal evolution in re-sponse to clinical interventions. Science 331: 430–434.

Dare K, Ibba M. 2012. Roles of tRNA in cell wall biosyn-thesis. Wiley Interdiscip Rev RNA 3: 247–264.

de Lencastre H, Oliveira D, Tomasz A. 2007. Antibiotic re-sistant S. aureus: A paradigm of adaptive power. CurrOpin Microbiol 10: 428–435.

Dessen A, Mouz N, Gordon E, Hopkins J, Dideberg O. 2001.Crystal structure of PBP2x from a highly penicillin-resis-tant S. pneumoniae clinical isolate: A mosaic frameworkcontaining 83 mutations. J Biol Chem 276: 45106–45112.

Dhar N, Dubee V, Ballell L, Cuinet G, Hugonnet JE, Signo-rino-Gelo F, Barros D, Arthur M, McKinney JD. 2015.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Rapid cytolysis of M. tuberculosis by faropenem, an orallybioavailable b-lactam antibiotic. Antimicrob Agents Che-mother 59: 1308–1319.

Dias R, Felix D, Canica M, Trombe MC. 2009. The highlyconserved serine threonine kinase StkP of S. pneumoniaecontributes to penicillin susceptibility independentlyfrom genes encoding PBPs. BMC Microbiol 9: 121.

Djoric D, Kristich CJ. 2015. Oxidative stress enhances ceph-alosporin resistance of E. faecalis through activation ofa two-component signaling system. Antimicrob AgentsChemother 59: 159–169.

Dubee V, Arthur M, Fief H, Triboulet S, Mainardi JL, Gut-mann L, Sollogoub M, Rice LB, Etheve-Quelquejeu M,Hugonnet JE. 2012. Kinetic analysis of E. faecium L,D-transpeptidase inactivation by carbapenems. AntimicrobAgents Chemother 56: 3409–3412.

Dubee V, Bernut A, Cortes M, Lesne T, Dorchene D, LefebvreAL, Hugonnet JE, Gutmann L, Mainardi JL, HerrmannJL, et al. 2015. b-Lactamase inhibition by avibactam inM. abscessus. J Antimicrob Chemother 70: 1051–1058.

Egesborg P, Carlettini H, Volpato JP, Doucet N. 2015.Combinatorial active-site variants confer sustainedclavulanate resistance in BlaC b-lactamase from M. tu-berculosis. Protein Sci 24: 534–544.

Falk SP, Weisblum B. 2013. Phosphorylation of the S. pneu-moniae cell wall biosynthesis enzyme MurC by a eukary-otic-like ser/thr kinase. FEMS Microbiol Lett 340: 19–23.

Feiler C, Fisher AC, Boock JT, Marrichi MJ, Wright L,Schmidpeter PA, Blankenfeldt W, Pavelka M, Delisa MP.2013. Directed evolution of M. tuberculosis b-lactamasereveals gatekeeper residue that regulates antibiotic resis-tance and catalytic efficiency. PLoS ONE 8: e73123.

Fernandez R, Paz LI, Rosato RR, Rosato AE. 2014. Ceftaro-line is active against heteroresistant methicillin-resistantStaphylococcus aureus clinical strains despite associatedmutational mechanisms and intermediate levels of resis-tance. Antimicrob Agents Chemother 58: 5736–5746.

Filipe SR, Severina E, Tomasz A. 2002. The murMN operon:A functional link between antibiotic resistance and anti-biotic tolerance in S. pneumoniae. Proc Natl Acad Sci 99:1550–1555.

Fischetti VA, Ryan P. 2015. Streptococcus. In Practical hand-book of microbiology, 3rd ed. (ed. Goldman E, Green LH),pp. 411–427. CRC, Boca Raton, FL.

Fishovitz J, Rojas-Altuve A, Otero LH, Dawley M, Carrasco-Lopez C, Chang M, Hermoso JA, Mobashery S. 2014.Disruption of allosteric response as an unprecedentedmechanism of resistance to antibiotics. J Am Chem Soc136: 9814–9817.

Fishovitz J, Taghizadeh N, Fisher JF, Chang M, Mobashery S.2015. The Tipper-Strominger hypothesis and triggeringof allostery in PBP2a of methicillin-resistant S. aureus(MRSA). J Am Chem Soc 137: 6500–6505.

Fleurie A, Manuse S, Zhao C, Campo N, Cluzel C, LavergneJP, Freton C, Combet C, Guiral S, Soufi B, et al. 2014.Interplay of the serine/threonine-kinase StkP and theparalogs DivIVA and GpsB in pneumococcal cell elonga-tion and division. PLoS Genet 10: e1004275.

Foucault ML, Depardieu F, Courvalin P, Grillot-CourvalinC. 2010. Inducible expression eliminates the fitness costof vancomycin resistance in Enterococci. Proc Natl AcadSci 107: 16964–16969.

Gautam S, Kim T, Spiegel DA. 2015. Chemical probes revealan extraseptal mode of cross-linking in S. aureus. J AmChem Soc 137: 7441–7447.

Ge Y, Wu J, Xia Y, Yang M, Xiao J, Yu J. 2012. Moleculardynamics simulation of the complex PBP-2x with drugcefuroxime to explore the drug resistance mechanism ofS. suis R61. PLoS ONE 7: e35941.

Gilmore MS, Rauch M, Ramsey MM, Himes PR, Varahan S,Manson JM, Lebreton F, Hancock LE. 2015. Pheromonekilling of multidrug-resistant E. faecalis V583 by nativecommensal strains. Proc Natl Acad Sci 112: 7273–7278.

Goffin C, Ghuysen JM. 2002. Biochemistry and comparativegenomics of SxxK superfamily acyltransferases offer aclue to the mycobacterial paradox: Presence of penicil-lin-susceptible target proteins versus lack of efficiency ofpenicillin as therapeutic agent. Microbiol Mol Biol Rev 66:702–738.

Gonzales PR, Pesesky MW, Bouley R, Ballard A, Biddy BA,Suckow MA, Wolter WR, Schroeder VA, Burnham CD,Mobashery S, et al. 2015. Synergistic, collaterally sensitiveb-lactam combinations suppress resistance in MRSA.Nat Chem Biol 11: 855–861.

Gonzalo X, Drobniewski F. 2013. Is there a place for b-lac-tams in the treatment of multidrug-resistant/extensivelydrug-resistant tuberculosis? Synergy between merope-nem and amoxicillin/clavulanate. J Antimicrob Chemo-ther 68: 366–369.

Gordon E, Mouz N, Duee E, Dideberg O. 2000. The crystalstructure of the penicillin-binding protein 2x fromS. pneumoniae and its acyl-enzyme form: Implicationin drug resistance. J Mol Biol 299: 477–485.

Greene NG, Narciso AR, Filipe SR, Camilli A. 2015.Peptidoglycan branched stem peptides contribute toS. pneumoniae virulence by inhibiting pneumolysin re-lease. PLoS Pathog 11: e1004996.

Gupta R, Lavollay M, Mainardi JL, Arthur M, Bishai WR,Lamichhane G. 2010. The M. tuberculosis protein LdtMt2is a nonclassical transpeptidase required for virulenceand resistance to amoxicillin. Nat Med 16: 466–469.

Hakenbeck R. 2014. Discovery of b-lactam-resistant vari-ants in diverse pneumococcal populations. GenomeMed 6: 72.

Hakenbeck R, Bruckner R, Denapaite D, Maurer P. 2012.Molecular mechanisms of b-lactam resistance in S.pneumoniae. Future Microbiol 7: 395–410.

Hazra S, Xu H, Blanchard JS. 2014. Tebipenem, a new car-bapenem antibiotic, is a slow substrate that inhibits theb-lactamase from M. tuberculosis. Biochemistry 53:3671–3678.

Hendrickx APA, van Schaik W, Willems RJL. 2013. The cellwall architecture of E. faecium: From resistance to path-ogenesis. Future Microbiol 8: 993–1010.

Henriques-Normark B, Tuomanen EI. 2013. The pneumo-coccus: Epidemiology, microbiology, and pathogenesis.Cold Spring Harb Perspect Med 3: a010215.

Henry X, Verlaine O, Amoroso A, Coyette J, Frere JM, JorisB. 2013. Activity of ceftaroline against E. faecium PBP5.Antimicrob Agents Chemother 57: 6358–6360.

Hindler JA, Wong-Beringer A, Charlton CL, Miller SA, Ke-lesidis T, Carvalho M, Sakoulas G, Nonejuie P, Pogliano J,Nizet V, et al. 2015. In vitro activity of daptomycin in



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



combination with b-lactams, gentamicin, rifampin, andtigecycline against daptomycin-nonsusceptible entero-cocci. Antimicrob Agents Chemother 59: 4279–4288.

Holmes NE, Howden BP. 2014. What’s new in the treatmentof serious MRSA infection? Curr Opin Infect Dis 27:471–478.

Horita Y, Maeda S, Kazumi Y, Doi N. 2014. In vitro suscept-ibility of M. tuberculosis isolates to an oral carbapenemalone or in combination with b-lactamase inhibitors.Antimicrob Agents Chemother 58: 7010–7014.

Hugonnet JE, Tremblay LW, Boshoff HI, Barry CE, Blan-chard JS. 2009. Meropenem-clavulanate is effectiveagainst extensively drug-resistant M. tuberculosis. Science323: 1215–1218.

Ip M, Ang I, Liyanapathirana V, Ma H, Lai R. 2015. Geneticanalyses of PBP determinants in multidrug-resistantS. pneumoniae serogroup 19 CC320/271 clone withhigh-level resistance to third-generation cephalosporins.Antimicrob Agents Chemother 59: 4040–4045.

Jackson M, McNeil MR, Brennan PJ. 2013. Progress in tar-geting cell envelope biogenesis in Mycobacterium tuber-culosis. Future Microbiol 8: 855–875.

Jensen A, Valdorsson O, Frimodt-Møller N, Hollingshead S,Kilian M. 2015. Commensal streptococci serve as a res-ervoir for b-lactam resistance genes in S. pneumoniae.Antimicrob Agents Chemother 59: 3529–3540.

Job V, Carapito R, Vernet T, Dessen A, Zapun A. 2008. Com-mon alterations in PBP1a from resistant S. pneumoniaedecrease its reactivity toward b-lactams: Structural in-sights. J Biol Chem 283: 4886–4894.

Kim SJ, Chang J, Singh M. 2015. Peptidoglycan architectureof Gram-positive bacteria by solid-state NMR. BiochimBiophys Acta 1848: 350–362.

Knox J, Uhlemann AC, Lowy FD. 2015. S. aureus infections:Transmission within households and the community.Trends Microbiol 23: 437–444.

Kocaoglu O, Tsui HCT, Winkler ME, Carlson EE. 2015.Profiling of b-lactam selectivity for penicillin-bindingproteins in S. pneumoniae D39. Antimicrob Agents Che-mother 59: 3548–3555.

Kristich CJ, Rice LB, Arias CA., 2014. Enterococcal infec-tion—Treatment and antibiotic resistance. In Enterococci:From commensals to leading causes of drug resistant infec-tion (ed. Gilmore MS, Clewell DB, Ike Y, Shankar N), pp.1–46. Massachusetts Eye and Ear Infirmary, Boston.

Kumar P, Arora K, Lloyd JR, Lee IY, Nair V, Fischer E, Bo-shoff HI, Barry CE. 2012. Meropenem inhibits D,D-carboxypeptidase activity in M. tuberculosis. Mol Micro-biol 86: 367–381.

Kurz SG, Wolff KA, Hazra S, Bethel CR, Hujer AM, SmithKM, Xu Y, Tremblay LW, Blanchard JS, Nguyen L, et al.2013. Can inhibitor resistant substitutions in theM. tuberculosis b-lactamase BlaC lead to clavulanate re-sistance? A biochemical rationale for the use of b-lactamb-lactamase inhibitor combinations. Antimicrob AgentsChemother 57: 6085–6096.

Ladjouzi R, Bizzini A, Lebreton F, Sauvageot N, Rince A,Benachour A, Hartke A. 2013. Analysis of the tolerance ofpathogenic Enterococci and S. aureus to cell wall activeantibiotics. J Antimicrob Chemother 68: 2083–2091.

Lages MC, Beilharz K, Morales Angeles D, Veening J-W,Scheffers DJ. 2013. The localization of key Bacillus subtilispenicillin binding proteins during cell growth is deter-mined by substrate availability. Environ Microbiol 15:3272–3281.

Land AD, Tsui HC, Kocaoglu O, Vella SA, Shaw SL, Keen SK,Sham LT, Carlson EE, Winkler ME. 2013. Requirement ofessential Pbp2x and GpsB for septal ring closure inS. pneumoniae D39. Mol Microbiol 90: 939–955.

Lavollay M, Arthur M, Fourgeaud M, Dubost L, Marie A,Veziris N, Blanot D, Gutmann L, Mainardi JL. 2008. Thepeptidoglycan of stationary-phase M. tuberculosis pre-dominantly contains cross-links generated by L,D-trans-peptidation. J Bacteriol 190: 4360–4366.

Lebreton F, van Schaik W, McGuire AM, Godfrey P, GriggsA, Mazumdar V, Corander J, Cheng L, Saif S, Young S,et al. 2013. Emergence of epidemic multidrug-resistantE. faecium from animal and commensal strains. MBio 4:e00534-13.

Lecoq L, Dubee V, Triboulet S, Bougault C, Hugonnet JE,Arthur M, Simorre JP. 2013. Structure of E. faecium L,D-transpeptidase acylated by ertapenem provides insightinto the inactivation mechanism. ACS Chem Biol 8:1140–1146.

Leski TA, Tomasz A. 2005. Role of PBP2 in the antibioticsusceptibility and cell wall cross-linking of S. aureus: Ev-idence for the cooperative functioning of PBP2, PBP4,and PBP2A. J Bacteriol 187: 1815–1824.

Llarrull LI, Mobashery S. 2012. Dissection of events in theresistance to b-lactam antibiotics mediated by the pro-tein BlaR1 from S. aureus. Biochemistry 51: 4642–4649.

Lobritz MA, Belenky P, Porter CBM, Gutierrez A, Yang JH,Schwarz EG, Dwyer DJ, Khalil AS, Collins JJ. 2015. An-tibiotic efficacy is linked to bacterial cellular respiration.Proc Natl Acad Sci 112: 8173–8180.

Loskill P, Pereira PM, Jung P, Bischoff M, Herrmann M,Pinho MG, Jacobs K. 2014. Reduction of the peptidogly-can crosslinking causes a decrease in stiffness of theS. aureus cell envelope. Biophys J 107: 1082–1089.

Lun S, Miranda D, Kubler A, Guo H, Maiga MC, Winglee K,Pelly S, Bishai WR. 2014. Synthetic lethality revealsmechanisms of M. tuberculosis resistance to b-lactams.mBio 5: e01767.14.

Macheboeuf P, Di Guilmi AM, Job V, Vernet T, Dideberg O,Dessen A. 2005. Active site restructuring regulates ligandrecognition in class A PBPs. Proc Natl Acad Sci 102: 577–582.

Maestro B, Novakova L, Hesek D, Lee M, Leyva E, Mobash-ery S, Sanz JM, Branny P. 2011. Recognition of pepti-doglycan and b-lactam antibiotics by the extracellulardomain of the Ser/Thr protein kinase StkP from S.pneumoniae. FEBS Lett 585: 357–363.

Magnet S, Arbeloa A, Mainardi JL, Hugonnet JE, FourgeaudM, Dubost L, Marie A, Delfosse V, Mayer C, Rice LB, et al.2007. Specificity of L,D-transpeptidases from Gram-pos-itive bacteria producing different peptidoglycan chemo-types. J Biol Chem 282: 13151–13159.

Mainardi JL, Fourgeaud M, Hugonnet JE, Dubost L,Brouard JP, Ouazzani J, Rice LB, Gutmann L, ArthurM. 2005. A novel peptidoglycan cross-linking enzymefor a b-lactam-resistant transpeptidation pathway. J BiolChem 280: 38146–38152.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Mann PA, Muller A, Xiao L, Pereira PM, Yang C, Lee SH,Wang H, Trzeciak J, Schneeweis J, Dos Santos MM, et al.2013. Murgocil is a highly bioactive staphylococcal-spe-cific inhibitor of the peptidoglycan glycosyltransferaseenzyme MurG. ACS Chem Biol 8: 2442–2451.

Massidda O, Novakova L, Vollmer W. 2013. From models topathogens: How much have we learned about S. pneumo-niae cell division? Environ Microbiol 15: 3133–3157.

Maurer P, Koch B, Zerfass I, Krauss J, van der Linden M,Frere JM, Contreras-Martel C, Hakenbeck R. 2008.PBP2x of S. pneumoniae: Three new mutational pathwaysfor remodelling an essential enzyme into a resistance de-terminant. J Mol Biol 376: 1403–1416.

Maurer P, Todorova K, Sauerbier J, Hakenbeck R. 2012.Mutations in S. PBP2x: Importance of the C-terminalPBP and serine/threonine kinase-associated domainsfor b-lactam binding. Microb Drug Resist 18: 314–321.

Memmi G, Filipe SR, Pinho MG, Fu Z, Cheung A. 2008.S. aureus PBP4 is essential for b-lactam resistance incommunity-acquired methicillin-resistant strains. Anti-microb Agents Chemother 52: 3955–3966.

Missiakas D, Schneewind O. 2015. S. aureus and relatedstaphylococci. In Practical handbook of microbiology, 3rded. (ed. Goldman E, Green LH), pp. 383–409. CRC, BocaRaton, FL.

Moellering RC Jr. 2012. MRSA: The first half century. JAntimicrob Chemother 67: 4–11.

Morlot C, Bayle L, Jacq M, Fleurie A, Tourcier G, Galisson F,Vernet T, Grangeasse C, Di Guilmi AM. 2013. Interactionof PBP2x and Ser/Thr protein kinase StkP, two key play-ers in S. pneumoniae R6 morphogenesis. Mol Microbiol90: 88–102.

Mouz N, Di Guilmi AM, Gordon E, Hakenbeck R, DidebergO, Vernet T. 1999. Mutations in the active site of penicil-lin-binding protein PBP2x from S. pneumoniae. Role inthe specificity for b-lactam antibiotics. J Biol Chem 274:19175–19180.

Nataraj V, Varela C, Javid A, Singh A, Besra GS, Bhatt A.2015. Mycolic acids: Deciphering and targeting the Achil-les’ heel of the tubercle bacillus. Mol Microbiol 98: 7–16.

Nobbs AH, Jenkinson HF, Everett DB. 2015. Generic deter-minants of Streptococcus colonization and infection. In-fect Genet Evol 33: 361–370.

Ogawara H. 2015. PBPs in Actinobacteria. J Antibiot 68:223–245.

Oliveira DC, de Lencastre H. 2011. Methicillin-resistance inS. aureus is not affected by the overexpression in trans ofthe mecA gene repressor: A surprising observation. PLoSONE 6: e23287.

Otero LH, Rojas-Altuve A, Llarrull LI, Carrasco-Lopez C,Kumarasiri M, Lastochkin E, Fishovitz J, Dawley M, He-sek D, Lee M, et al. 2013. How allosteric control ofS. aureus PBP2a enables methicillin resistance and phys-iological function. Proc Natl Acad Sci 110: 16808–16813.

Palmer KL, Kos VN, Gilmore MS. 2010. Horizontal genetransfer and the genomics of enterococcal antibiotic re-sistance. Curr Opin Microbiol 13: 632–639.

Pasquina LW, Santa Maria JP, Walker S. 2013. Teichoic acidbiosynthesis as an antibiotic target. Curr Opin Microbiol16: 531–537.

Peacock SJ, Paterson GK. 2015. Mechanisms of methicillinresistance in S. aureus. Annu Rev Biochem 84: 577–601.

Peters K, Schweizer I, Beilharz K, Stahlmann C, Veening JW,Hakenbeck R, Denapaite D. 2014. S pneumoniae PBP2xmid-cell localization requires the C-terminal PASTA do-mains and is essential for cell shape maintenance. MolMicrobiol 92: 733–755.

Peyrani P, Ramirez J. 2015. What is the best therapeuticapproach to methicillin-resistant Staphylococcus aureuspneumonia. Curr Opin Infect Dis 28: 164–170.

Pfeffer JM, Strating H, Weadge JT, Clarke AJ. 2006. Pepti-doglycan O-acetylation and autolysin profile of E. faecalisin the viable but nonculturable state. J Bacteriol 188:902–908.

Philippe J, Vernet T, Zapun A. 2014. The elongation of ovo-cocci. Microb Drug Resist 20: 215–221.

Philippe J, Gallet B, Morlot C, Denapaite D, Hakenbeck R,Chen Y, Vernet T, Zapun A. 2015. Mechanism ofb-lactamaction in S. pneumoniae: The piperacillin paradox. Anti-microb Agents Chemother 59: 609–621.

Pietta E, Montealegre MC, Roh JH, Cocconcelli PS, MurrayBE. 2014. E faecium PBP5-S/R, the missing link betweenPBP5-S and PBP5-R. Antimicrob Agents Chemother 58:6978–6981.

Pinho MG, Filipe SR, de Lencastre H, Tomasz A. 2001.Complementation of the essential peptidoglycan trans-peptidase function of PBP2 by the drug resistance proteinPBP2A in S. aureus. J Bacteriol 183: 6525–6531.

Prigozhin DM, Krieger IV, Huizar JP, Mavrici D, Waldo GS,Hung L, Sacchettini JC, Terwilliger TC, Alber T. 2014.Subfamily-specific adaptations in the structures of twoPBPs from M. tuberculosis. PLoS ONE 9: e116249.

Qamar A, Golemi-Kotra D. 2012. Dual roles of FmtA inS. aureus cell wall biosynthesis and autolysis. AntimicrobAgents Chemother 56: 3797–3805.

Qiao Y, Lebar MD, Schirner K, Schaefer K, Tsukamoto H,Kahne D, Walker S. 2014. Detection of lipid-linked pep-tidoglycan precursors by exploiting an unexpected trans-peptidase reaction. J Am Chem Soc 136: 14678–14681.

Ramalingam J, Vennila J, Subbiah P. 2013. Computationalstudies on the resistance of PBP2B of wild-type and mu-tant strains of S. pneumoniae against b-lactam antibiot-ics. Chem Biol Drug Des 82: 275–289.

Reed P, Atilano ML, Alves R, Hoiczyk E, Sher X, ReichmannNT, Pereira PM, Roemer T, Filipe SR, Pereira-Leal JB,et al. 2015. S. aureus survives with a minimal peptidogly-can synthesis machine but sacrifices virulence and anti-biotic resistance. PLoS Pathog 11: e1004891.

Reinert RR. 2009. The antimicrobial resistance profile ofS. pneumoniae. Clin Microbiol Infect 15: 7–11.

Rice LB, Carias LL, Rudin S, Hutton R, Marshall S, HassanM, Josseaume N, Dubost L, Marie A, Arthur M. 2009.Role of class A PBPs in the expression of b-lactam resis-tance in E. faecium. J Bacteriol 191: 3649–3656.

Roemer T, Davies J, Giaever G, Nislow C. 2012. Bugs, drugsand chemical genomics. Nat Chem Biol 8: 46–56.

Sacco E, Cortes M, Josseaume N, Rice LB, Mainardi JL,Arthur M. 2014. Serine/threonine protein phosphatase-mediated control of the peptidoglycan cross-linking L,D-transpeptidase pathway in E. faecium. mBio 5: e01446.14.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Sauerbier J, Maurer P, Rieger M, Hakenbeck R. 2012. S pneu-moniae R6 interspecies transformation: Genetic analysisof penicillin resistance determinants and genome-widerecombination events. Mol Microbiol 86: 692–706.

Sauvage E, Kerff F, Fonze E, Herman R, Schoot B, MarquetteJP, Taburet Y, Prevost D, Dumas J, Leonard G, et al. 2002.The 2.4-A crystal structure of the PBP-binding proteinPBP5fm from E. faecium in complex with benzylpenicil-lin. Cell Mol Life Sci 59: 1223–1232.

Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. 2008. ThePBPs: Structure and role in peptidoglycan biosynthesis.FEMS Microbiol Rev 32: 234–258.

Schneider T, Sahl HG. 2010. An oldie but a goodie—Cellwall biosynthesis as antibiotic target pathway. Int J MedMicrobiol 300: 161–169.

Schoonmaker MK, Bishai WR, Lamichhane G. 2014. Non-classical transpeptidases of M. tuberculosis alter cell size,morphology, the cytosolic matrix, protein localization,virulence, and resistance to b-lactams. J Bacteriol 196:1394–1402.

Seung KJ, Keshavjee S, Rich ML. 2015. Multidrug-resistanttuberculosis and extensively drug-resistant tuberculosis.Cold Spring Harb Perspect Med 5: a017863.

Sewell EWC, Brown ED. 2014. Taking aim at wall teichoicacid synthesis: New biology and new leads for antibiotics.J Antibiot (Tokyo) 67: 43–51.

Silhavy TJ, Kahne D, Walker S. 2010. The bacterial cell en-velope. Cold Spring Harb Perspect Biol 2: a000414.

Silver LL. 2013. Viable screening targets related to the bac-terial cell wall. Ann NY Acad Sci 1277: 29–53.

Singh M, Kim SJ, Sharif S, Preobrazhenskaya M, Schaefer J.2015. REDOR constraints on the peptidoglycan latticearchitecture of S. aureus and its FemA mutant. BiochimBiophys Acta 1848: 363–368.

Smith JR, Barber KE, Raut A, Aboutaleb M, Sakoulas G,Rybak MJ. 2015a. b-Lactam combinations with dapto-mycin provide synergy against vancomycin-resistant E.faecalis and E. faecium. J Antimicrob Chemother 70: 1738–1743.

Smith JR, Barber KE, Raut A, Rybak MJ. 2015b. b-Lactamsenhance daptomycin activity against vancomycin-resis-tant E. faecalis and E. faecium in in vitro pharmacokinet-ic/pharmacodynamic models. Antimicrob Agents Che-mother 59: 2842–2848.

Solapure S, Dinesh N, Shandil R, Ramachandran V, SharmaS, Bhattacharjee D, Ganguly S, Reddy J, Ahuja V, Pandu-ga V, et al. 2013. In vitro and in vivo efficacy of b-lac-tams against replicating and slowly growing/nonrepli-cating M. tuberculosis. Antimicrob Agents Chemother 57:2506–2510.

Sorg RA, Veening JW. 2015. Microscale insights into pneu-mococcal antibiotic mutant selection windows. Nat Com-mun 6: 8773.

Soroka D, Li de la Sierra-Gallay I, Dubee V, Triboulet S, vanTilbeurgh H, Compain F, Ballell L, Barros D, Mainardi JL,Hugonnet JE, et al. 2015. Hydrolysis of clavulanate byMycobacterium tuberculosis b-lactamase BlaC harboringa canonical SDN motif. Antimicrob Agents Chemother 59:5714–5720.

Spratt BG. 2012. The 2011 Garrod lecture: From penicillin-binding proteins to molecular epidemiology. J Antimi-crob Chem 67: 1578–1588.

Starikova I, Al-Haroni M, Werner G, Roberts AP, Sørum V,Nielsen KM, Johnsen PJ. 2013. Fitness costs of variousmobile genetic elements in E. faecium and E. faecalis. JAntimicrob Chem 68: 2755–2765.

Staude MW, Frederick TE, Natarajan SV, Wilson BD, TannerCE, Ruggiero ST, Mobashery S, Peng JW. 2015. Investiga-tion of signal transduction routes within the sensor/transducer protein BlaR1 of S. aureus. Biochemistry 54:1600–1610.

Stryjewski ME, Corey GR. 2014. Methicillin-resistant S. au-reus: An evolving pathogen. Clin Infect Dis 58: S10–S19.

Tan CM, Therien AG, Lu J, Lee SH, Caron A, Gill CJ, Le-beau-Jacob C, Benton-Perdomo L, Monteiro JM, PereiraPM, et al. 2012. Restoring methicillin-resistant S. aureussusceptibility to b-lactam antibiotics. Sci Transl Med 4:126ra35.

Todorova K, Maurer P, Rieger M, Becker T, Bui NK, Gray J,Vollmer W, Hakenbeck R. 2015. Transfer of penicillin re-sistance from S. oralis to S. pneumoniae identifies murE asresistance determinant. Mol Microbiol 97: 866–880.

Tomasz A. 1979. The mechanism of the irreversible antimi-crobial effects of penicillins: How the b-lactams kill andlyse bacteria. Annu Rev Microbiol 33: 113–137.

Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VGJr. 2015. S. aureus infections: Epidemiology, pathophys-iology, clinical manifestations, and management. ClinMicrobiol Rev 28: 603–661.

Tremblay LW, Hugonnet JE, Blanchard JS. 2008. Structure ofthe covalent adduct formed between M. tuberculosis b-lactamase and clavulanate. Biochemistry 47: 5312–5316.

Tremblay LW, Fan F, Blanchard JS. 2010. Biochemical andstructural characterization of M. tuberculosisb-lactamasewith the carbapenems ertapenem and doripenem. Bio-chemistry 49: 3766–3773.

Triboulet S, Arthur M, Mainardi JL, Veckerle C, Dubee V,Nguekam-Moumi A, Gutmann L, Rice LB, Hugonnet JE.2011. Inactivation kinetics of a new target of b-lactamantibiotics. J Biol Chem 286: 22777–22784.

Triboulet S, Dubee V, Lecoq L, Bougault C, Mainardi JL,Rice LB, Etheve-Quelquejeu M, Gutmann L, Marie A,Dubost L, et al. 2013. Kinetic features of L,D-transpepti-dase inactivation critical for b-lactam antibacterial activ-ity. PLoS ONE 8: e67831.

Triboulet S, Bougault CM, Laguri C, Hugonnet JE, ArthurM, Simorre J-P. 2015. Acyl acceptor recognition by En-terococcus faecium L,D-transpeptidase Ldtfm. Mol Micro-biol 98: 90–100.

Tsubakishita S, Kuwahara-Arai K, Sasaki T, Hiramatsu K.2010. Origin and molecular evolution of the determinantof methicillin resistance in Staphylococci. AntimicrobAgents Chemother 54: 4352–4359.

Tsui HCT, Boersma MJ, Vella SA, Kocaoglu O, Kuru E, Pe-ceny JK, Carlson EE, VanNieuwenhze MS, Brun YV, ShawSL, et al. 2014. Pbp2x localizes separately from Pbp2b andother peptidoglycan synthesis proteins during later stagesof cell division of S. pneumoniae D39. Mol Microbiol 94:21–40.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Turner RD, Ratcliffe EC, Wheeler R, Golestanian R, HobbsJK, Foster SJ. 2010. Peptidoglycan architecture can spec-ify division planes in S. aureus. Nat Commun 1: 1025.

Turner RD, Vollmer W, Foster SJ. 2014. Different walls forrods and balls: The diversity of peptidoglycan. Mol Mi-crobiol 91: 862–874.

van Tonder AJ, Bray JE, Roalfe L, White R, Zancolli M, QuirkSJ, Haraldsson G, Jolley KA, Maiden MCJ, Bentley SD,et al. 2015. Genomics reveals the worldwide distributionof multidrug-resistant serotype 6E pneumococci. J ClinMicrobiol 53: 2271–2285.

Veziris N, Truffot C, Mainardi JL, Jarlier V. 2011. Activity ofcarbapenems combined with clavulanate against murinetuberculosis. Antimicrob Agents Chemother 55: 2597–2600.

Vollmer W, Blanot D, de Pedro MA. 2008. Peptidoglycanstructure and architecture. FEMS Microbiol Rev 32:149–167.

Wang H, Gill CJ, Lee SH, Mann P, Zuck P, Meredith TC,Murgolo N, She X, Kales S, Liang L, et al. 2013. Discoveryof wall teichoic acid inhibitors as potential anti-MRSAb-lactam combination agents. Chem Biol 20: 272–284.

Waxman DJ, Strominger JL. 1983. PBPs and the mechanismof action of b-lactam antibiotics. Annu Rev Biochem 52:825–869.

Werner G, Coque TM, Franz CMA, Grohmann E, HegstadK, Jensen L, van Schaik W, Weaver K. 2013. Antibioticresistant Enterococci—Tales of a drug resistance gene traf-ficker. Int J Med Microbiol 303: 360–379.

Werth BJ, Barber KE, Tran KNT, Nonejuie P, Sakoulas G,Pogliano J, Rybak MJ. 2015. Ceftobiprole and ampicillinincrease daptomycin susceptibility of daptomycin-sus-ceptible and -resistant VRE. J Antimicrob Chemother 70:489–493.

Wheeler R, Mesnage S, Boneca IG, Hobbs JK, Foster SJ.2011. Super-resolution microscopy reveals cell wall dy-namics and peptidoglycan architecture in ovococcal bac-teria. Mol Microbiol 82: 1096–1109.

Winstel V, Xia G, Peschel A. 2014. Pathways and roles of wallteichoic acid glycosylation in S. aureus. Int J Med Micro-biol 304: 215–221.

Wivagg CN, Bhattacharyya RP, Hung DT. 2014. Mechanismsof b-lactam killing and resistance in the context ofM. tuberculosis. J Antibiot 67: 645–654.

Wivagg CN, Wellington S, Gomez JE, Hung DT. 2016. Lossof a class A penicillin-binding protein alters b-lactamsusceptibilities in M. tuberculosis. ACS Infect Dis 2:104–110.

Xu H, Hazra S, Blanchard JS. 2012. NXL104 irreversiblyinhibits the b-lactamase from M. tuberculosis. Biochem-istry 51: 4551–4557.

Yamada M, Watanabe T, Baba N, Takeuchi Y, Ohsawa F,Gomi S. 2008. Crystal structures of biapenem and tebi-penem complexed with PBPs 2X and 1A from S. pneumo-niae. Antimicrob Agents Chemother 52: 2053–2060.

Young KD. 2006. The selective value of bacterial shape. Mi-crobiol Mol Biol Rev 70: 660–703.

Young KD. 2007. Bacterial morphology: Why have differentshapes? Curr Opin Microbiol 10: 596–600.

Zapun A, Contreras-Martel C, Vernet T. 2008. PPBs andb-lactam resistance. FEMS Microbiol Rev 32: 361–385.

Zerfass I, Hakenbeck R, Denapaite D. 2009. An importantsite in PBP2x of penicillin-resistant clinical isolates ofS. pneumoniae: Mutational analysis of Thr338. Antimi-crob Agents Chemother 53: 1107–1115.

Zhang X, Paganelli FL, Bierschenk D, Kuipers A, BontenMJM, Willems RJL, van Schaik W. 2012. Genome-wideidentification of ampicillin resistance determinants inE. faecium. PLoS Genet 8: e1002804.

Zhang D, Wang Y, Lu J, Pang Y. 2015. In vitro activity ofb-lactams in combination with b-lactamase inhibitorsagainst multidrug-resistant Mycobacterium tuberculosisisolates. Antimicrob Agents Chemother 60: 393–399.

Zhou Y, Antignac A, Wu SW, Tomasz A. 2008. PBPs and cellwall composition in b-lactam-sensitive and -resistantstrains of S. sciuri. J Bacteriol 190: 508–514.

Zorzi W, Zhou XY, Dardenne O, Lamotte J, Raze D, Pierre J,Gutmann L, Coyette J. 1996. Structure of the low-affinityPBP5fm in wild-type and highly penicillin-resistantstrains of E. faecium. J Bacteriol 178: 4948–4957.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



18, 20162016; doi: 10.1101/cshperspect.a025221 originally published online AprilCold Spring Harb Perspect Med

Jed F. Fisher and Shahriar Mobashery Mycobacterium tuberculosis-Lactam Resistance Mechanisms: Gram-Positive Bacteria and β

Subject Collection Antibiotics and Antibiotic Resistance

Fosfomycin: Mechanism and ResistanceLynn L. Silver Resistance

The Whys and Wherefores of Antibiotic

Cameron R. Strachan and Julian Davies

Mode of Action and Resistance−−Pleuromutilins: Potent Drugs for Resistant Bugs

Susanne Paukner and Rosemarie Riedl

-Lactamases: A Focus on Current ChallengesβRobert A. Bonomo

Appropriate Targets for Antibacterial DrugsLynn L. Silver Mechanism of Action and Resistance

Approved Glycopeptide Antibacterial Drugs:

et al.Daina Zeng, Dmitri Debabov, Theresa L. Hartsell,

of ResistancePleuromutilins: Mode of Action and Mechanisms Lincosamides, Streptogramins, Phenicols, and

et al.Stefan Schwarz, Jianzhong Shen, Kristina Kadlec,

Enterococci andStaphylococcus aureusDaptomycin in

Mechanism of Action and Resistance to

AriasWilliam R. Miller, Arnold S. Bayer and Cesar A.

Health PathogensResistance to Macrolide Antibiotics in Public

al.Corey Fyfe, Trudy H. Grossman, Kathy Kerstein, et

ResistancePolymyxin: Alternative Mechanisms of Action and

al.Michael J. Trimble, Patrik Mlynárcik, Milan Kolár, et

Antibiotic InhibitionBacterial Protein Synthesis as a Target for

Stefan Arenz and Daniel N. WilsonMechanisms of Action and ResistanceTopoisomerase Inhibitors: Fluoroquinolone

David C. Hooper and George A. Jacoby

through Resistance to the Next GenerationAntibacterial Antifolates: From Development

AndersonAlexavier Estrada, Dennis L. Wright and Amy C.

Overview-Lactamase Inhibitors: Anβ-Lactams and β

Karen Bush and Patricia A. Bradford

Lipopolysaccharide Biosynthetic Enzyme LpxCAntibacterial Drug Discovery Targeting the

Alice L. Erwin

Rifamycins, Alone and in CombinationDavid M. Rothstein

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved


http://perspectivesinmedicine.cshlp.org/cgi/collection/

http://perspectivesinmedicine.cshlp.org/cgi/collection/


b-lactam resistance mechanisms: gram-positive bacteria...

Documents