Auxiliary factors: a chink in the armor of MRSA resistance to β-lactam antibiotics

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Auxiliary factors: a chink in the armor of MRSA resistance to b-lactam antibiotics Terry Roemer1, Tanja Schneider2,3 and Mariana G Pinho4 Combination agents provide an important orthogonal approach to treat infectious diseases, particularly those caused by drug resistant pathogens. Indeed, applying a biologically ‘rational’ and systems-level paradigm to discover potent, selective, and synergistic agents would augment current (and arguably overly relied upon) empirical and serendipitous approaches to such discovery efforts. Here, we review the cellular mechanisms of b-lactam drug resistance and tolerance achieved amongst methicillin-resistant Staphylococcus aureus (MRSA) as well as their molecular targets and strategies to identify cognate inhibitors as potential combination agents to restore b-lactam efficacy against MRSA. Addresses 1 Infectious Disease Research, Merck Research Laboratories, Kenilworth, NJ 07033, USA 2 Institute of Medical Microbiology, Immunology and ParasitologyPharmaceutical Microbiology Section, University of Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany 3 German Centre for Infection Research (DZIF), Partner site BonnCologne, Bonn, Germany 4 Laboratory of Bacterial Cell Biology, Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Av. da Repu´blica, Oeiras 2780157, Portugal Corresponding authors: Roemer, Terry ([email protected]), Schneider, Tanja ([email protected]) and Pinho, Mariana G ([email protected])

Current Opinion in Microbiology 2013, 16:538–548 This review comes from a themed issue on Antimicrobials Edited by Robert EW Hancock and Hans-Georg Sahl For a complete overview see the Issue and the Editorial Available online 26th July 2013 1369-5274/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mib.2013.06.012

Introduction Staphylococcus aureus remains the leading cause of life threatening Gram-positive bacterial infections in hospital and community settings [1,2]. In large part, this is due to the inevitable selection of diverse S. aureus drug resistance mechanisms following use/overuse/misuse of antibiotic therapeutics. For example, methicillin resistant S. aureus (MRSA), which first appeared as short as one year after its introduction to the clinic and is highly cross resistant to nearly all b-lactam antibiotics, presently represents over 50% of all S. aureus clinical isolates in the USA [3]. Compounding this issue, S. aureus isolates resistant to important alternative standard of care Current Opinion in Microbiology 2013, 16:538–548

antibiotics, including vancomycin, linezolid, or daptomycin, continue to be reported [4,5] and the discovery of new antibiotics has met with little success [6]. Consequently, redoubling our efforts and applying novel strategies to address b-lactam resistance associated with this important pathogen is urgently needed [2]. Despite this alarming incidence of drug resistance, blactams starting with penicillin and through its stepwise improvements (from cephalosporins to present day carbapenems), remain the most clinically relevant class of antibiotics [7]. b-lactams act by inhibiting synthesis and chemical cross-linking of the bacterial-specific cell wall polymer, peptidoglycan (PG), resulting in weakening of the cell wall and eventual lysis of the cell [8]. Mechanistically, b-lactams act as pseudosubstrates and acylate the active site of penicillin-binding proteins (PBPs) to inhibit transpeptidation-dependent cross-linking of PG polymers [9]. The molecular basis of MRSA b-lactam resistance is also well understood [9,10], involving the lateral transfer of the mecA gene, possibly from the related organisms Staphylococcus sciuri [10,11] or Staphylococcus fleurettii [12], and which encodes an additional PBP (PBP2A) with markedly reduced affinity to b-lactams versus endogenous S. aureus PBP enzymes [13,14]. Consequently, MRSA synthesizes PG and is viable in otherwise lethal concentrations of b-lactam antibiotics, through the cooperative function of PBP2 and PBP2A, which provide the necessary transglycosylation and transpeptidation activity, respectively, to polymerize PG monomers and cross-link PG polymer chains [14,15,16,17]. Combatting resistance mechanisms of these ‘miracle drugs’ is essential to restoring their efficacy and in the case of b-lactam resistant Gram-negative bacteria, this has proven highly impactful [18]. Success has involved a combination therapy strategy [19], namely restoring blactam antibiotic activity by pairing the drug with a second agent (a b-lactamase inhibitor) which inactivates specific members of the b-lactamase enzyme family responsible for hydrolysis of b-lactams [18]. As such agents inactivate the function of the resistance mechanism, they effectively ‘reset’ the pathogen to antibiotic susceptible levels, thereby potentiating the effect of the b-lactam. A combination of such agents is synergistic against the drug resistant microbe, as demonstrated by using standard checkerboard serial dilution assays [20]. However, as MRSA evades the effects of b-lactams through PBP2A rather than b-lactamases, alternative www.sciencedirect.com

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strategies are required. For example, combining highly selective PBP2A agents in combination with b-lactams, or developing single agent b-lactams whose PBP inhibitory spectrum includes PBP2A, such as the fifth generation cephalosporin, ceftobiprole, is certainly a sensible strategy [21]. Alternatively, genetics-based approaches have identified a large number of auxiliary factors required for robust expression of the PBP2A-dependent MRSA b-lactam resistance [22]. Such targets are therefore analogous to b-lactamases in the context of their inactivation resetting MRSA susceptibility to b-lactams and may provide a wide assortment of opportunities to indirectly cripple or inactivate PBP2A activity. Here we review the cellular processes whose function impacts PBP2A-based MRSA b-lactam resistance, strategies to identify cognate inhibitors to such targets, and their potential as combination agents to restore b-lactam efficacy against MRSA.

S. aureus cell wall biogenesis In S. aureus, the mode of action of b-lactams, as well as the molecular basis of their acquired resistance, is intimately related to PG biosynthesis and cell wall biogenesis [9,17]. PG is an essential component of the cell wall, providing strength to resist the high internal osmotic pressure and maintaining cell shape. The PG polymer is composed of glycan strands made of a repeating disaccharide unit comprising N-acetyl-glucosamine (GlcNAc) and Nacetyl-muramic acid (MurNAc) that are cross-linked by short peptides [23]. The PG monomer is synthesized within the cytoplasm in a multistep process (Figure 1). GlmS, GlmM, and GlmU enzymes sequentially convert fructose-6-P to UDP-GlcNAc, which is later converted to UDP-MurNAc through the activities of MurA, MurZ, and MurB. A series of PG ligases (MurC-F) then sequentially synthesize a pentapeptide (L-Ala-D-Glu-L-Lys-D-Ala-DAla) extension from UDP-MurNAc, resulting in the soluble PG precursor, UDP-MurNAc-pentapeptide (also referred to as Park’s nucleotide) [23]. To complete PG monomer synthesis and facilitate export to the cell surface, UDP-MurNAc-pentapeptide is linked to a lipid carrier (undecaprenyl-phosphate; C55-P) by the membrane protein MraY (resulting in undecaprenyl-P-P-MurNAc-pentapeptide, referred to as lipid I) and finally MurG utilizes UDP-GlcNAc substrate to build the final PG precursor, undecaprenyl-P-P-(GlcNAc)MurNAcpentapeptide, or lipid II [23,24]. S. aureus lipid II is further modified by a family of peptidyltransferases (FemX, FemA, and FemB) which complete a pentaglycine bridge peptide extending from the pentapeptide LLys residue, which ultimately allows for the efficient cross-linking of PG in the cell wall [25,26]. In addition, GatD and MurT enzymes amidate D-Glu in the pentapeptide to form D-glutamine [27,28], an essential modification required for PBP-catalyzed polymerization [29]. Export of the fully modified PG lipid II precursor is facilitated by a transporter(s), likely FtsW and/or RodA www.sciencedirect.com

[30] with a possible supporting role by MurJ/SAV1754 [31,32], which flip the membrane bound lipid II from the inner to outer leaflet of the plasma membrane, exposing the substrate to PBP family proteins, PBP1-4, as well as PBP2A in the case of MRSA. Depending on their specific enzymatic activity, PBPs then polymerize linear chains of the PG polymer via transglycosylation of disaccharide units to form glycan strands and cross-link adjacent PG polymers by transpeptidation of the pentaglycine bridge of one unit in a glycan strand to the penultimate D-Ala residue of another pentapeptide unit in a second glycan strand [17,23,24]. A convergence in our identification of the S. aureus genes involved in PG biosynthesis and of the genetic factors that are required, together with mecA, for S. aureus to resist the effects of b-lactams, originated in the 1980s from the pioneering work from the Berger-Bachi and Tomasz labs, who screened Tn551 mutants of MRSA for reduced levels of methicillin resistance [22,33,34]. In addition to mecA, several other Tn551 insertional inactivation mutants were identified, corresponding to several accessory factors which were named fem genes (for factor essential for methicillin resistance) or aux genes (for auxiliary genes) (Table 1) [22,34]. Importantly, these fem mutations were unlinked to mecA, and PBP2A expression was unaltered despite dramatic hypersensitivity of these mutants to methicillin. Thus, b-lactam resistance of MRSA is multifactorial and PBP2A expression is necessary but not sufficient. Consistent with this view, no correlation exists between PBP2A expression levels and the level of MRSA resistance to methicillin [34]. Indeed, subsequent Tn551-based screens have revealed many additional genes necessary for MRSA resistance (Table 1). A large fraction of these accessory factors contribute to MRSA b-lactam resistance by providing normal levels of structurally wild type PG precursors for PBPs to complete PG synthesis and crosslinking. Changes in the PBP substrate can have deleterious effects in enzyme efficiency to complete sufficient PG synthesis necessary to grow and divide in otherwise bacteriocidal concentrations of a b-lactam [34]. However, such forward genetic strategies may not provide a complete annotation of b-lactam susceptibility determinants, since PG biosynthesis is essential for cell viability, as might be other potential processes contributing to MRSA resistance, and such approaches bias against identifying viable loss of function mutants. To address this concern, a reverse genetic strategy was recently performed, whereby a library of 245 plasmids, each maintaining an inducible antisense interference fragment targeting a specific S. aureus essential gene transcript [35], was introduced into the hospital-acquired MRSA strain COL and communityacquired MRSA isolate USA300 [36]. The resulting strain libraries were then screened under partially inducing conditions to identify additional genes required for b-lactam resistance of MRSA [36]. In so doing, Current Opinion in Microbiology 2013, 16:538–548

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Figure 1

peptidoglycan 2

40

TG targocil L275 L555 L524

krisynomycin M131

tunicamycin ticlopidine

SpsB LCP 2

40

2

moenomycin β-lactams

TP

hBD3

PBP

DMPI

Atl

PknB

40

FtsQ LtaA

FtsW

FtsL RodA

TarH

DltD 40

DltA DltC

MurJ

MraY

TarO

TarG

DltB

LtaS

TarS↑

TarB-L

MurG

TarA

2

↑GlmU ↑GlmM

NVA-FMDP

FtsB FemB FtsZ

Mur A-F

↑TarM

FemX FemA

GlyS

L337

cyslabdan

FtsA

MurT ZapA

GatD

fosfomycin D-cycloserin

PC190723

↑GlmS

Fruc-6-P

GlcNAc

ManNAc

MurNAc-pp

GroP

C55-P Gly5-bridge

PG

RtoP

Ala

Glc2

Gln

DAG Current Opinion in Microbiology

Schematic representation of cell envelope biosynthesis and modification reactions. Compounds exhibiting synergistic antimicrobial activities with blactams are depicted in red. GlcNAc, N-acetyl-glucosamine; MurNAc-pp, N-acetyl-muramic acid pentapeptide; C55-P, undecapreyl-phosphate; Gly5bridge, pentaglycin interpeptide bridge; ManNAc, N-acetylmannosamine; GroP, glycerolphosphate, RtoP, ribitolphosphate; Glc, glucose; DAG, diacylglycerol; PG, phosphatidylglycerol; Ala, alanine; Gln, glutamine; TP, transpeptidase; TG, transglycosylase.

essentially all known PG biosynthesis genes as well as novel ones implicated in lipid II amidation (gatD and murT, [27,28] and translocation (SAV1754/murJ; [31] were identified as uniquely impacting MRSA susceptibility to a broad assortment of penicillins, cephalosporins, and carbapenems (Table 1). Interestingly, other cellular processes were also identified to contribute to MRSA blactam resistance, including essential aspects of cell division ( ftsZ and ftsA) and protein secretion (spsB), as well as wall teichoic acid biosynthesis (tarL) and signal transduction systems (SAV1220/pknB) [36]. Therefore, although PBP2A is the fundamental drug resistance determinant of MRSA, the coordinated activity of a broad network of interdependent processes contributes greatly to its functional activity in providing b-lactam resistance. Further, the above genetic analyses predict that cognate small molecule inhibitors of these targets would mimic the often massively restored b-lactam susceptibility phenotypes of such mutants. Early demonstration that target-specific inhibitors to auxiliary factors could pharmacologically resensitize MRSA to b-lactams was achieved by combining methicillin with sub-MIC levels of fosfomycin, a clinically relevant inhibitor of MurA and its paralog, MurZ; [37] or D-cycloserine, which inhibits D-Ala-D-Ala incorporation into the PG pentapeptide [38,39]. Various other Current Opinion in Microbiology 2013, 16:538–548

examples followed. Cefoxitin, a clinically used b-lactam with high affinity for PBP4, synergizes with various other b-lactams against community-acquired MRSA strains [40,41]. However, this effect is not observed in tested hospital-acquired MRSA strains which, contrary to community acquired strains, do not require PBP4 for expression of b-lactam resistance. Moenomycin, a non-clinically used inhibitor of transglycosylation, also acts synergistically with b-lactams by inactivating the PBP2 transglycosylase domain that is required, together with the transpeptidase domain of PBP2A, for peptidoglycan synthesis in the presence of b-lactams [15]. Similarly, cognate inhibitors of GlmS (NVA-FMDP) significantly synergize imipenem activity against MRSA and recapitulate genetic findings [36]. Recently, the discovery and mechanistic characterization of the natural product, cyslabdan, which markedly potentiates imipenem activity against MRSA, has been demonstrated to inhibit pentaglycine interpeptide bridge synthesis by inhibiting FemA [42] and ultimately crippling PG cross-linking. DMPI, an inhibitor of SAV1754 [31] and likely FtsW as well [36] is also synergistic in combination with b-lactams [31], presumably by interfering with lipid II translocation and extracellular localization of PBP substrate. Finally, host defense peptides are broadly demonstrated to bind lipid II [43] and representative peptides, hBD3 and CAP18 strongly synergize with methicillin against diverse MRSA www.sciencedirect.com

MRSA resistance to beta-lactam antibiotics Roemer, Schneider and Pinho 541

Table 1 Auxiliary factors required for expression of MRSA b-lactam resistance. Gene (alternative names) femX (fmhB) femA femB femC (glnR) glmS glmM (femD) murA murB murC-F (femF) glyS gatD murT SAV1754 (mviN, murJ) ftsW pbp1 (pbpA) pbp2 pbp4 fmtA ftsA ftsZ llm (tarO) tarA tarB tarD tarL tarI tarS ltaS pknB (stk1, aux2) spsB sigB vraSR

Function

Refs

Peptidyltransferase, addition of first glycine to pentaglycine bridge Peptidyltransferase, addition of 2nd and 3rd glycine to pentaglycine bridge Peptidyltransferase, addition of 4th and 5th glycine to pentaglycine bridge Glutamine synthase repressor, inactivation reduces amidation of stem peptide Glucosamine-6-phosphate synthase, converts fructose-6-phosphate to glucosamine-6-phosphate Glucosamine-1-phosphate mutase, converts glucosamine-6-phosphate to glucosamine-1-phosphate Transferase; converts UDP-GlcNAc to UDP-GlcNAc-enoylpyruvate Reductase, converts UDP-GlcNAc-enoylpyruvate to UDP-MurNAc Mur ligases adding L-Ala, D-Glu, L-Lys, and D-Ala-D-Ala respectively to form the UDP-MurNAc-pentapeptide Glycine tRNA synthetase; provides glycine substrate for pentaglycine bridge formation Glutamine amidotransferase, amidation of lipid II Mur ligase homolog, amidation of lipid II Proposed lipid II translocase Proposed lipid II translocase PBP with transpeptidation activity PBP with transglycosylation and transpeptidation activity PBP with transpeptidation activity Accessory PBP with transpeptidation activity Divisome component Bacterial ortholog of tubulin, Divisome component Phosphosugar transferase; forms first precursor in WTA synthesis (GlcNAc-pyrophosphate-undecaprenol) N-acetylmannosamine transferase; forms second intermediate in WTA synthesis (ManNAc-GlcNAc-pyrophosphate-undecaprenol) Glycerophosphate transferase; forms third intermediate in WTA synthesis (glycerol-3-phosphate-ManNAc-GlcNAc-pyrophosphate-undecaprenol) WTA synthesis, produces CDP-glycerol substrate for TarB Ribitolphosphotransferase, required for poly ribitol-phosphate extension of WTA WTA synthesis, produces CDP-ribitol substrate for TarL Glycosyltransferase required of b-O-GlcNAc addition to WTA Lipoteichoic acid synthase Eukaryotic-like serine/threonine kinase Signal peptidase I Alternate transcription factor Two component signal transduction sensor of cell wall stress

[93] [94] [95] [96] [36] [36,97] [36] [36] [33,36] [36] [27,28] [27,28] [31,36] [36] [36] [15,36] [41] [98] [36] [36,48] [50,51] [67,68]

clinical isolates [44]. Therefore, structural changes or reduced PBP substrate levels would impair PBPs from completing PG synthesis and crosslinking when dually challenged by the effects of a b-lactam. Consistent with the view that methicillin-potentiating effects of these agents against MRSA are due to their inhibitory effect on PG biosynthesis, antibiotics targeting other cellular processes failed to potentiate methicillin [39]. Auxiliary factors may also function to correctly localize PBP enzymes to the site of active cell wall synthesis and cell division, the septum. As S. aureus PBP functions are spatially and temporally controlled throughout the cell cycle [17,45,46], loss of any of these auxiliary factors could disrupt the cooperative function of PBP2 and PBP2A at the septum and sensitize MRSA to blactams. For example, the divisome is a complex multimolecular structure that drives cytokinesis, and its assembly is mediated by the prokaryotic tubulin homolog, FtsZ [47]. FtsZ polymerizes in a GTP-dependent manner to form a cytoskeletal scaffold referred to as the Z ring, which coordinates septal localization of cell wall and division proteins, including PBPs, to sites of active www.sciencedirect.com

[67] [67] [36] [67] [72] [77] [34,86] [36] [99] [81,83]

cell wall biogenesis [45,46,47]. Recently, we demonstrated that the FtsZ inhibitor, PC190723 markedly resensitizes MRSA to diverse b-lactams both in vitro and in an animal infection model [48]. Mechanistically, this is likely achieved by the profound delocalization of PBP2 from its site of function in PC190723treated cells [48]. Therefore, synergy between these agents results in a substantially reduced level of blactam being required to inhibit the residual PBP2 that is functionally active at the septum. Importantly, PBP localization is also dependent on binding to its membrane-bound substrate, lipid II, (PBP2, [46]) as well as wall teichoic acid (PBP4, [49]), emphasizing the importance of these cell wall biosynthetic products themselves in contributing to the buffering capacity of MRSA to b-lactams. Consistent with this view, we have recently identified a MurG-selective inhibitor (L337 or murgocil) that depletes lipid II synthesis, which specifically synergizes with b-lactams and disrupts PBP2 localization (manuscript in preparation). Therefore, impairing any of a variety of PBP localization mechanisms is expected to uncouple PBPs (most notably PBP2) from their substrate as well as the Current Opinion in Microbiology 2013, 16:538–548

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coordinated PG synthesis (and hydrolysis) with other structural components of the cell wall.

Wall teichoic acid One of the first reported auxiliary factors is the previously uncharacterized gene, llm [50]. Although its molecular function was unknown at the time, IIm mutants demonstrated a profoundly restored b-lactam susceptibility in a broad set of MRSA clinical isolates examined [50]. llm has led a largely silent existence until recently, when it was recognized to be the same gene as tarO [51], encoding the first enzymatic step in wall teichoic acid (WTA) biosynthesis [52]. WTA is a glycophosphate-rich cell wall polymer common to Gram-positive bacteria [52,53]. S. aureus WTA is composed of a GlcNAc b-(1–3) ManNAc disaccharide to which a short glycerol-3-phosphate repeat unit and extensive ribitol-3-phosphate terminal chain is added [52]. Unlike PG, WTA is not required for cell viability per se [54,55] (see below) but plays important roles in growth, division and morphology of the cell [49,51,52,56,57]. Defects in WTA synthesis also produce strong avirulence phenotypes in multiple S. aureus animal infection models [53,54]. Like PG, WTA polymers are sequentially synthesized on the central undecaprenyl phosphate carrier lipid C55-P by a series of Tar enzymes localized on the inner face of the cytoplasmic membrane, before being exported to the cell surface by a two component ATP-binding cassette (ABC) transporter system and cross-linked to PG by the family of LCP proteins [52,57,58] Recently, it has been demonstrated that tunicamycin is a highly potent inhibitor of TarOdependent WTA synthesis at sub-MIC concentrations [51], thereby providing a molecular basis for the initial finding that tunicamcyin is a potent synergist of methicillin against MRSA [38] and verifying that tunicamycintreated MRSA and MRSA llm/tarO mutants share common b-lactam susceptibility phenotypes [51]. Mechanistically, potentiation between these agents is likely complex, involving the disruption of both WTA polymer –dependent septum localization of proteins such as PBP4, which mediates PG cross-linking [49], as well as impairing normal WTA-mediated exclusion of the major autolysin, Atl, from the old, peripheral cell wall [59]. For these reasons, we and others have invested greatly in developing phenotypic screens to identify novel inhibitors of WTA biosynthesis. Pivotal to these efforts is the observation that late steps in WTA biosynthesis, in either S. aureus or Bacillus subtilis, are essential for cell viability whereas early steps (encoded by tarO and tarA, respectively) are not [54,55,60,61,62]. Further, late stage WTA genes are conditionally essential since they are dispensable in either a tarO or tarA deletion background, presumably because late stage mutants accumulate toxic WTA intermediate polymers and/or sequestering WTA intermediates effectively dilutes undecaprenyl lipid carrier levels to the point where PG synthesis is dramatically Current Opinion in Microbiology 2013, 16:538–548

impacted [55,61]. This gene dispensability phenomenon, often referred to as the ‘essential gene paradox’ or EGP [55,60,61], provides a powerful strategy for identifying WTA inhibitors [63]. Walker and colleagues have elegantly exploited EGP by screening for late stage WTA inhibitors that phenocopy the genetic characterization of the pathway [63]. Such compounds should display intrinsic bioactivity against wild type S. aureus but lack activity against S. aureus strains in which flux into the WTA pathway is abolished, either by genetic (e.g. tarO deletion) or pharmacological (e.g. tunicamycin) means [63]. One compound originally identified in this manner and subsequently optimized for potency is targocil [64,65]. Drug resistance mutant isolation revealed that targocil inhibits TarG, an essential subunit of the WTA ABC transporter [63,66]. As expected, resistance to targocil is also achieved by lossof-function mutations in tarO or tarA, which effectively bypass the mechanism of action of targocil [63]. Although the frequency of resistance (FOR) to targocil is high [64,67], the contribution of DtarO and DtarA mutants to targocil resistance is eliminated in the presence of oxacillin [68]. Further, such by-pass mutants are profoundly attenuated in virulence in several models of S. aureus infection [53,54,67], thus likely mitigating such issues from a b-lactam combination agent context. Wang et al. [67] have performed an analogous high throughput screen against the Merck internal synthetic library and identified several additional structurally distinct WTA inhibitors, all targeting TarG, which also potentiate the effect of imipenem in a murine infection model of MRSA [67]. Therefore, TarG appears to be a highly druggable target versus other late stage WTA enzymes. Finally, genetic validation that defects in early or late stage WTA biogenesis restore imipenem efficacy, even greater than achieved by co-administering TarG inhibitors and imipenem in the same animal MRSA infection model, underscores the therapeutic potential pharmacologically superior WTA inhibitors may possess [67]. Inhibitors of cell wall synthesis, cell division, and late stage WTA synthesis are intrinsically bioactive and therefore provide dual opportunities, as single standalone therapeutic agents, as well as b-lactam potentiation agents. Contrarily, early stage WTA inhibitors would lack antibacterial activity and solely serve as b-lactam combination agents. In this respect, such targets are completely analogous to b-lactamases and provide development opportunities for combination agents most closely related to those successfully applied to treat Gram-negative blactam resistant pathogens. Tunicamycin is however unlikely to serve such a role, as it is a promiscuous inhibitor of multiple lipid carrier-based reactions both in prokaryotes (e.g. TarO/WTA, MraY/PG [63]) and eukaryotes (Alg7p/N-linked glycosylation [69]) and is consequently highly cytotoxic to mammals. Perhaps natural products www.sciencedirect.com

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structurally related to tunicamycins (e.g. streptovirudins, septacidins, corynetoxins, or other nucleoside antibiotics [70]) may exhibit reduced toxicity and superior TarO selectivity. Alternatively, a b-lactam potentiation screen by Brown and colleagues [71] using a focused library of previously-approved drugs has successfully identified a novel TarO inhibitor (ticlopidine) which, accordingly, is unlikely to share similar toxicity issues. Farha et al. [71] demonstrate that ticlopidine, a clinically used antiplatelet drug lacking antibacterial activity, strongly synergizes with PBP2 and PBP4-selective b-lactams and that their synergy is suppressed in MRSA DtarO strain backgrounds. Further, ticlopidine-treated cells partially phenocopy the effects of DtarO cells, exhibiting reduced WTA levels, suppression of targocil bioactivity, and modest inhibition of TarO in vitro activity [71], albeit at extremely high (>100 mg/ml) and pharmacologically unachievable drug concentrations. Similar b-lactam potentiation, whole cell WTA pathway, and targetspecific biochemical screens are likely to uncover novel TarO and TarA-specific inhibitors potentially more suitable as b-lactam combination agents. Lastly, tarS, a third non-essential WTA pathway gene encoding a glycosyltransferase responsible for b-O-GlcNAc addition to the WTA polymer has been recently identified [72]. DtarS mutants prominently display restored b-lactam hypersusceptibility against MRSA, which is not the case for other enzymes that provide a-O-GlcNAc (TarM, [73]) and Dalanylation (DltA-D, [74]) tailoring modifications to WTA [72]. Unlike DtarO and DtarA mutants, however, DtarS strains still produce WTA polymer, implying that the bO-GlcNAc modification itself may serve a scaffolding function and perhaps assist PBP2A localization [72]. Thus a growing number of suitable non-essential b-lactam potentiation targets exist for identifying cognate inhibitors lacking intrinsic bioactivity and strong b-lactam potentiation activity.

Additional cell wall contributors to MRSA blactam resistance Considering the coordinated synthesis and maintenance of the S. aureus cell wall and its importance as a protective barrier between the bacterium and its external environment [17], it is perhaps not surprising that other structural constituents of its assembly and regulation are also important MRSA auxiliary factors. Lipoteichoic acid (LTA) represents a third important component of the cell wall of Gram-positive bacteria, in addition to PG and WTA [74]. In S. aureus, LTA is essential for growth and cell division and consists of a glycerol-phosphate polymer extended from a diacyl glycerol gentiobiose unit [75,76]. Depletion of LTA by transposon insertion into the 50 region of the LTA synthase gene, ltaS, strongly resensitizes MRSA to b-lactams in vitro [77] and, accordingly, imipenem is highly efficacious against such mutants in animal infection models (data not shown). Interestingly, LtaS is post-translationally regulated by the signal www.sciencedirect.com

peptidase enzyme, SpsB, via proteolytic cleavage [78] and either genetic [36] or pharmacological inhibition of SpsB [79] dramatically resensitizes MRSA to b-lactams. Curiously, ltaS mutations also confer resistance to SpsB inhibitors, krisynomycin and the actinocarbasin synthetic analog, M131 [77]. Cell wall two-component signal transduction systems (TCS) sense cell surface damage and trigger protective stress responses [80]. The sentinel S. aureus cell wall TCS is encoded by vraSR, which induces a complex cell wall stress stimulon (CWSS) comprising nearly 50 genes, including its own locus as well as pbp2, murZ, fmtA, tarA and sa0908 (encoding one of the WTA LCP proteins, [57]) specifically in response to cell wall acting antibiotics [81,82] or cell wall mutations [83,84]. Accordingly, mutants in vraSR are unable to mobilize a cell wall stress response [81] and are also hypersusceptible to vraSRinducing agents, including b-lactams [81,83]. Jo et al. [85] extended these results by demonstrating the restored efficacy of oxacillin in animal infection models against the community acquired MRSA strain USA300, if deleted of vraSR. Originally identified and annotated as aux2 by Tomasz and colleagues [34], a S. aureus eukaryote-like serine/ threonine kinase (renamed pknB or stk1) and phosphatase (aux1 or stp1) regulatory circuit has also gained significant interest of late, both in terms of its role as a b-lactam susceptibility determinant of MRSA [86,87] and its possible role in sensing cell wall stress [88,89]. The PknB kinase is composed of an N-terminal cytosolic kinase domain, a central transmembrane domain, and three Cterminal extracellular PASTA (penicillin-binding protein and serine/threonine kinase associated) domains, the latter of which is thought to bind b-lactams and PG [90]. Unfortunately, conflicting conclusions with regards to DpknB strains displaying attenuated virulence or hypervirulent phenotypes casts confusion on the suitability of PknB as a validated b-lactam potentiation target [87,91].

Small molecule synergists: early lessons learned The triumvirate of genetics, biochemistry and cell biology has revealed a systems level understanding of the targets and pathways required for MRSA b-lactam resistance. However, the discovery of small molecules inhibitors to several of these targets now sheds an even deeper knowledge of the benefits and obstacles of exploiting chemical synergy in developing b-lactam combination agents. Perhaps not unexpectedly, provided both inhibitors are potent bioactive agents targeting distinct essential proteins, their combination effectively reduces the FOR otherwise associated with each individual agent. Such conclusions are repeatedly drawn when examining the intrinsic FOR of FtsZ inhibitor PC190723, targocil and other TarG or SpsB inhibitors versus their reduced Current Opinion in Microbiology 2013, 16:538–548

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FOR when partnered with sub-MIC levels of imipenem [48,68,79,67]. In some instances, such as the SpsB inhibitor M131 and imipenem, this may manifest by the synergistic pair conferring a cidal effect whereas the individual agents are static [79]. In other instances, reduced FOR is achieved by the phenotypes of the resistant mutants themselves. For example, PC190723 resistance mutations solely map to FtsZ and many of these PC190723-resistant mutations dramatically resensitize MRSA to b-lactams [48], recapitulating the original antisense interference-based b-lactam hypersensitivity phenotype that identified the target [36]. Thus a strong counterselection against such drug resistant mutants would exist in the presence of a b-lactam and each agent selects against resistance to its companion antibiotic. Similarly, TarG inhibitors share this benefit, albeit through the accumulation of compensatory loss-offunction by-pass mutations in multiple earlier steps of WTA synthesis [63,67] which reset MRSA susceptibility to b-lactams [67,68]. Furthermore, S. aureus drug resistant by-pass mutants of TarG inhibitors are also profoundly attenuated in virulence as demonstrated in several distinct animal infection models [53,54,62,67]. Therefore, in multiple ways, the FOR associated with such compounds are substantially reduced in the context of a b-lactam combination agent in an animal infection setting of MRSA. It is important to note that small molecules predicted to synergize with b-lactams on the basis of well characterized genetic interactions between their cognate targets do not always manifest the anticipated degree of synergy when measured in in vitro assays [67,68]. Whether this reflects limitations to how well the checkerboard assay scores synergy amongst intrinsically bioactive versus nonbioactive agents should be investigated. For example, synergy between two potent bioactive agents interdicting two interdependent essential processes may be best observed over a period of several cell divisions using vital dyes, rather than optical density reading over hours or overnight growth. We also note that in vivo synergistic effects are reported that appear stronger than quantified in vitro using the checkerboard assay [48,67]. Considering the unique challenges MRSA faces in a host environment, synergistic effects between compounds in the context of an infection setting are important and should be studied further. For example, host defensins or other innate immunity factors may play important roles in facilitating synergy between these agents, particularly under conditions in which combination agents compromise the MRSA cell wall. It is also important to consider natural products (NP) in this pursuit. NP-derived inhibitors like krisynomycin [79], actinocarbasin [79], and cyslabdan [42] have desirable drug-like qualities that are often lacking in synthetic small molecules and continued screening of NP libraries for b-lactam potentiation should be encouraged. Considering that penicillins Current Opinion in Microbiology 2013, 16:538–548

themselves are NPs, the possibility that microbes themselves have evolved chemical-chemical potentiation strategies should not be ignored.

Conclusions Notwithstanding the unique pharmaceutical challenges of a combination agent development strategy, including co-dosing or co-formulation, route of drug administration, and unique clinical and regulatory issues, significant benefits are expected and encourage such an approach [19,92]. First, due to the unique chemical synergy achieved by pairing such agents, reduced drug levels of each individual component are required to achieve efficacy, therefore limiting potential toxicity and/or drugdrug interactions. Such an approach also dramatically expands the number of targets, well beyond PBP2A, that are suitable for drug interdiction and restoring b-lactam efficacy against MRSA. However, not all b-lactam potentiation targets are ‘created equal’ due to the extent or spectrum of their b-lactam susceptibility phenotype when deleted or depleted from the cell, or their simplicity for screening, intrinsic druggability, propensity for bypass suppression, cidal, static, or viable terminal phenotypes, intracellular or extracellular localization, or conservation across Staphylococci. Therefore a rich selection of targets, which has been illustrated in this review, is critically important. There is of course also the issue of identifying drug-like molecules to these targets, a historically difficult challenge. Considering their therapeutic potential however, many in the field remain dedicated to discovering such molecules. More broadly, applying a systems level approach to understanding the functional interdependencies between cellular processes, as applied in MRSA, also serves as a general model to rationally pursue combination agent small molecule discovery strategies against other drug resistant microbes to which novel therapeutics are urgently needed.

Acknowledgements We thank Brigitte Berger-Ba¨chi, Alexander Tomasz, and colleagues in the field for their contributions in our understanding of auxiliary factors required for the expression of b-lactam resistance in MRSA. TS is supported by the German Research Foundation (DFG, SCHN1284/1-2) and the German Center for Infection Research (DZIF). MGP is funded by ERC-2012-StG-310987 from the European Research Council.

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Study examines the principle of applying chemical genetic interaction networks to predict chemical synergy between the FtsZ inhibitor, PC190723, and b-lactams against MRSA. Study also demonstrates therapeutic efficacy achieved by pairing a b-lactam potentiation agent with a b-lactam in a murine model of MRSA infection. Interestingly, multiple independently derived PC190723 drug resistant mutations mapping to FtsZ also restores MRSA susceptibility to b-lactams, highlighting how each of the paired agents can suppress resistance to the companion agent. 49. Atilano ML, Pereira PM, Yates J, Reed P, Veiga H, Pinho MG,  Filipe SR: Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus. Proc Natl Acad Sci USA 2010, 107:18991-18996. Shows that wall teichoic acids act as temporal and spatial regulators of peptidoglycan metabolism, controlling the level of cross-linking by regulating PBP4 localization. PBP4 normally localizes at the division septum, but in the absence of wall teichoic acids synthesis, it becomes delocalized. As a consequence, the peptidoglycan of DtarO mutants, impaired in wall teichoic acid biosynthesis, has a decreased degree of cross-linking and likely contributes to the restored susceptibility of MRSA DtarO mutants to b-lactams. 50. Maki H, Yamaguchi T, Murakami K: Cloning and characterization of a gene affecting the methicillin resistance level and the  autolysis rate in Staphylococcus aureus. J Bacteriol 1994, 176:4993-5000. Interesting study which was well ahead of its time, both in elucidating llm (tarO) as a key accessory factor to the expression of MRSA b-lactam resistance as well as providing a broad genetic demonstration of this phenotype across an expansive set of MRSA clinical isolates. See comments for [51]. 51. Campbell J, Singh AK, Santa Maria JP, Kim Y, Brown S,  Swoboda JG, Mylonakis E, Wilkinson B, Walker S: Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus. ACS Chem Biol 2011, 6:106-116. Genetic and pharmacological approaches are used to demonstrate that inactivation of TarO and blocking wall teichoic acid specifically sensitizes MRSA strains to b-lactams. 52. Swoboda JG, Campbell J, Meredith TC, Walker S: Wall teichoic acid function, biosynthesis, and inhibition. Chembiochem 2010, 11:35-45. 53. Weidenmaier C, Peschel A: Teichoic acids and related cell-wall glycopolymers in Gram-positive physiology and host interactions. Nat Rev Microbiol 2008, 6:276-287. 54. Weidenmaier C, Kokai-Kun JF, Kristian SA, Chanturiya T, Kalbacher H, Gross M, Nicholson G, Neumeister B, Mond JJ,  Peschel A: Role of teichoic acids in Staphylococcus aureus nasal colonization, a major risk factor in nosocomial infections. Nat Med 2004, 10:243-245. Study concludes that wall teichoic acids are not essential for S. aureus viability but that they serve as an important virulence determinant of pathogenesis. 55. D’Elia MA, Pereira MP, Brown ED: Are essential genes really essential? Trends Microbiol 2009, 17:433-438. 56. Schirner K, Marles-Wright J, Lewis RJ, Errington J: Distinct and essential morphogenic functions for wall- and lipo-teichoic acids in Bacillus subtilis. EMBO 2009, 28:830-842. 57. Dengler V, Meier PS, Heusser R, Kupferschmied P, Fazekas J, Friebe S, Staufer SB, Majcherczyk PA, Moreillon P, Berger-Ba¨chi B et al.: Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response. FEMS Microbiol Lett 2012, 333:109-120. 58. Brown S, Zhang YH, Walker S: A revised pathway proposed for Staphylococcus aureus wall teichoic acid biosynthesis based on in vitro reconstitution of the intracellular steps. Chem Biol 2008, 15:12-21. 59. Schlag M, Biswas R, Krismer B, Kohler T, Zoll S, Yu W, Schwarz H, Peschel A, Go¨tz F: Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl. Mol Microbiol 2010, 75:864-873. 60. D’Elia MA, Pereira MP, Chung YS, Zhao W, Chau A, Kenney TJ,  Sulavik MC, Black TA, Brown ED: Lesions in teichoic acid www.sciencedirect.com

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