Providing β-lactams a helping hand: targeting the AmpC β-lactamase induction pathway

Future Microbiol. (2011) 6(12), 1415–1427

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b‑lactams constitute over 50% of all antibi‑otics in clinical use [1], yet their therapeutic value is being steadily eroded by the increasing prevalence of bacterial resistance mechanisms [2]. A formidable mechanism of resistance to b‑lactams in Pseudomonas aeruginosa and many other nonfermenting Gram‑negative bacilli, as well as many Enterobacteriaceae, is the produc‑tion of AmpC b‑lactamase. AmpC is a group I [3], class C [4,5], chromosomal b‑lactamase that is responsible for deactivating a wide range of dif‑ferent b‑lactams, including penicillins, cephems and monobactams [6]. With a few exceptions, particularly Escherichia coli, the expression of this enzyme is inducible by b‑lactams. AmpC‑mediated resistance is of particular concern in P. aeruginosa, since this opportunistic pathogen is a common cause of antibiotic‑resistant noso‑comial infections of cancer patients and burn victims suffering from compromised immune systems [7]. P. aeruginosa is also a prevalent cause of chronic and often fatal respiratory infections in patients with cystic fibrosis [8–10].

Clinically available b‑lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) do not effectively inhibit, and are, therefore, gen‑erally ineffective against, AmpC b‑lactamases [3,11]. Furthermore, because they contain the b‑lactam core, they can actually potentiate resistance mechanisms present within bac‑teria. Clavulanic acid, for example, induces AmpC in P. aeruginosa and Enterobacteriaceae, thereby compromising the bactericidal effect

of its partnering b‑lactam antibiotic [12,13]. Accordingly, effort has been focused on both the development of next‑generation b‑lactam‑based b‑lactamase inhibitors with improved perfor‑mance against existing resistance mechanisms, and novel non‑b‑lactam inhibitors that have the potential to subvert b‑lactamase‑mediated resistance mechanisms that are stimulated by cellular processes that respond to the b‑lactam core [11,14,15]. The most promising candidate currently being evaluated in clinical trials (in combination with ceftazidime) is the non‑b‑lactam inhibitor AVE1330A (NXL‑104), a bridged bicyclico[3.2.1]diazabicyclo‑octanone that effectively inhibits Ambler class A and C b‑lactamases [16]. BAL30376 is a also a prom‑ising drug combination, containing a bridged monobactam (BAL29880) as an AmpC inhibi‑tor together with a siderophore‑monobactam (BAL19764) and clavulanate to provide broad‑spectrum efficacy against Enterobacteriaceae and nonfermenters [17]. Other strategies currently being evaluated in clinical trials include modi‑fied b‑lactam antibiotics that are designed to be stable against hydrolysis by AmpC enzymes such as CXA‑101 (FR264205), a new cephalosporin that is resistant to the activity of P. aeruginosa AmpC [18,19].

An alternative strategy that departs entirely from direct inhibition of b‑lactamases is cur‑rently being explored to address b‑lactam antibi‑otic resistance in Gram‑negative bacteria carry‑ing inducible chromosomal AmpC. Exposure of

146510.2217/FMB.11.128 © 2011 Future Medicine Ltd ISSN 1746-0913

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Providing b-lactams a helping hand: targeting the AmpC b-lactamase induction pathway

Brian L Mark*1, David J Vocadlo2 & Antonio Oliver3 1Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada2Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada3Servicio de Microbiología & Unidad de Investigación, Hospital Son Espases, Instituto Universitario de Investigación en Ciencias de la Salud, Palma de Mallorca, Spain *Author for correspondence: Tel.: +1 204 480 1430 n Fax: +1 204 474 7603 n [email protected]

A major cause of the clinical failure of broad-spectrum b-lactam antibiotics against Pseudomonas aeruginosa and many Enterobacteriaceae species are chromosomal mutations that lead to the hyperproduction of AmpC b-lactamase. These mutations typically affect proteins within the peptidoglycan (PG) recycling pathway, as well as proteins that are modulated by metabolic intermediates of this pathway. Blocking PG recycling and associated sensing mechanisms with small-molecule inhibitors holds promise as a strategy for overcoming AmpC-mediated resistance that results from the selection of mutations during b-lactam therapy, or from the direct acquisition of infections by AmpC-producing mutants. Here we report on the structural and functional biology of potential drug targets within the Gram-negative PG recycling pathway and the utility of blocking PG recycling as a means of attenuating AmpC-mediated resistance in P. aeruginosa.

Keywords

n AmpC n AmpG n AmpR n antibiotic resistance n b-lactam n b-lactamase n cystic fibrosis n FtsZ n NagZ n Pseudomonas aeruginosa

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Future Microbiol. (2011) 6(12)1466 future science group

MurQ

ATP,Mg2+

(+) Induce

Repress (-)

NagZ

AmpD

Biosynthesis (multiple steps)

Peptidoglycan (PG)

AmpR

ampR ampC

Mpl

AmpG

GlcNAc

anhMurNAc

GlcNAc

anhMurNAcIncreased PG fragmentation

AmpC

Exp

orte

d

PorinLPS

OR ?

UDP-MurNAc

MurF

AmpC inducere.g., Imipenem (strong)Ceftazidime (weak)

(R2 predominates when PBPs are inhibited by β-lactams)

OR

Mpl

PBP4

AnmKMurNAc -6-P

GlcNAc-6-PD-lactate

R1 =

R2 =

Special Report Mark, Vocadlo & Oliver

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these bacteria to b‑lactams leads to the induction of AmpC expression, a process that is intricately controlled by metabolites of the Gram‑negative peptidoglycan (PG) recycling pathway (Figure 1) [20]. PG recycling pathways are highly conserved in Gram‑negative bacteria and involve a num‑ber of proteins that are potential targets for small‑molecule inhibitors; the idea being that co‑administration of such inhibitors with b‑lac‑tams should block PG recycling and suppress AmpC induction during therapy. Importantly, therapeutic use of b‑lactams against P. aerugi-nosa and enterobacteria often selects for mutants with chromosomal mutations that cause con‑stitutive, high‑level AmpC hyperproduction with consequent failure of the treatment [6,7,21]. Nevertheless, recent data suggest that inhibit‑ing PG recycling can also suppress mutational AmpC hyperproduction [22–24], offering a route to attenuate this problematic emergence of b‑lactam resistance. Here we report our current understanding of the structural and functional biology of these potential drug targets and the application of this knowledge towards the devel‑opment of small‑molecule‑based approaches to suppress AmpC‑mediated resistance in P. aeruginosa.

How does PG recycling regulate AmpC production?

PG is a mesh‑like heteropolymer of glycan strands (comprised of alternating b‑1,4‑linked N‑acetylglucosamine [GlcNAc] and N‑acetylmuramic acid [MurNAc] sugars) that are crosslinked by short‑stem peptides [25]. The PG is a key component of the cell wall structure, encasing the bacterium to maintain its overall shape and protect it from osmotic lysis [26]. The PG recycling pathway enables the bacterium to efficiently remodel the PG cell wall during growth and division, but its activity must be carefully regulated so as to not compromise the integrity of the cell wall [20].

The inhibition of penicillin‑binding proteins by b‑lactams results in the continuing activity of PG hydrolases leading to cell lysis and death [27]. For this reason the production of b‑lactamases, which protect penicillin‑binding proteins from

inactivation, have consequently evolved within bacteria to maintain appropriate PG metabo‑lism and so protect the integrity of the cell wall. P. aeruginosa is proposed to sense cell wall dis‑ruption and induce expression of AmpC via the ampC gene induction pathway to rebalance PG homeostasis by hydrolytically deactivating the antibiotic, which in turn returns AmpC production to basal levels (Figure 1) [6,28,29].

During normal growth and division, frag‑ments of the PG cell wall are excised by auto‑lysins [30] to produce a series of periplasmic GlcNAc‑1,6‑anhydroMurNAc‑peptides (tri‑, tetra‑ and penta‑peptide species) [20]. These muropeptides are transported into the cytosol through the inner membrane permease AmpG [31,32]. After arriving in the cytosol, the GlcNAc sugar is removed from the muropeptides by the glycoside hydrolase NagZ [33,34]. The resulting pool of 1,6‑anhydroMurNAc‑tri‑ and penta‑peptide molecules [20] are subsequently recycled to form UDP‑MurNAc‑pentapeptide, a PG precursor that becomes reincorporated into the PG cell wall (Figure 1) [20]. From the pool of 1,6‑anhydroMurNAc‑peptide catabolites, the tripeptide [28] or pentapeptide species [35] have both been proposed to be the signaling molecule that induces ampC transcription, whereas the anabolic product UDP‑MurNAc pentapeptide is thought to repress ampC transcription (Figure 1)

[28]. The 1,6‑anhydro‑MurNAc‑tetrapeptide species does not accumulate in the cytosol [29], since it is converted into 1,6‑anhydro‑MurNAc‑tripeptide by the cytosolic l,d‑carboxypeptid‑ease, LdcA [36]. There is no evidence in the lit‑erature to suggest that the tetrapeptide species is involved in AmpR activation.

The relative levels of the catabolic and ana‑bolic PG metabolites govern whether ampC is transcribed. This is achieved through what is believed to be competitive binding of the mol‑ecules to the LysR‑type transcriptional regulator AmpR [28]. Together, the ampR and ampC genes form a divergent operon with overlapping pro‑moters to which AmpR binds and regulates the transcription of both genes [37,38]. In the absence of b‑lactams, the cytosolic concentration of 1,6‑anhydro‑MurNAc‑peptide is controlled by

Figure 1. Gram-negative peptidoglycan recycling pathway regulates AmpC b-lactamase induction. NagZ removes GlcNAc from internalized muropeptides to form 1,6-anhydro-MurNAc-tripeptide or pentapeptide species (boxed right), one or both of which convert AmpR into an activator of ampC transcription. AmpD is essential for breaking down these species by removing the stem peptide from 1,6-anhydroMurNAc (anhMurNAc). The anhMurNAc sugar is converted to MurNAc-6-P by the anhMurNAc kinase AnmK and subsequently processed into GlcNAc-6-P by the etherase MurQ [88–90]. The AmpR corepressor, UDP-MurNAc pentapeptide, is shown boxed on the left. It is assembled from UDP-MurNAc and stem peptides via the activities of the murein peptide ligase Mpl and murine synthase MurF. Loss-of-function mutations in PBP4 (dacB null mutations) and AmpD (ampD null mutations) lead to constitutive AmpC hyperproduction that is mediated by the activities of NagZ and AmpG.

Providing b‑lactams a helping hand: targeting the AmpC b‑lactamase induction pathway Special Report


the activity of AmpD, an N-acetyl‑muramyl‑l-alanine amidase that removes the stem peptide from the 1,6‑anhydroMurNAc sugar (Figure 1) [39,40]. UDP‑MurNAc‑pentapeptide therefore predominates and binds AmpR, so favoring the repression of ampC transcription. Exposure to b‑lactams, however, elevates PG fragmenta‑tion, allowing 1,6‑anhydro‑MurNAc‑peptides to accumulate, enabling most likely either the tripeptide [35] or pentapeptide species [28] to dis‑place UDP‑MurNAc‑pentapeptide from AmpR and favor transcriptional activation of ampC [28]. The resulting AmpC enzyme deactivates b‑lactams that are present and so re‑establishes proper PG homeostasis, which in turn brings AmpC production back to basal levels.

Constitutive AmpC hyperproductionb‑lactams vary significantly in their capacity to induce ampC gene expression. Although sus‑ceptible to hydrolysis by AmpC, some penicil‑lins (such as piperacillin), cephalosporins (such as cefepime or ceftazidime) and monobactams (aztreonam) exhibit antipseudomonal activity because they are weak inducers of ampC expres‑sion [21]. Clinical use, however, frequently selects for chromosomal mutations in P. aeruginosa that cause high‑level constitutive AmpC expres‑sion and subsequent therapeutic failure of these b‑lactams [6]. Development of antipseudomonal b‑lactam resistance through mutational overex‑pression of AmpC is a frequent outcome of the treatment of nosocomial infections [41] and also in chronic lung infections of cystic fibrosis patients [42], where this problem is further amplified by the high prevalence of hypermutable (mutator) strains that show dramatically increased sponta‑neous mutation rates due to defects in the DNA mismatch repair system [43]. Moreover, AmpC hyperproduction is a nearly universal compo‑nent of multidrug‑resistant strains in the cys‑tic fibrosis setting [44] and documented in over 20% of P. aeruginosa isolates that produce severe bloodstream nosocomial infections [45,46].

Constitutive AmpC hyperproduction com‑monly arises from the selection of ampD null mutations (Figure 1) [47–50]. Loss of AmpD func‑tion significantly increases the concentration of cytosolic 1,6‑anhydro‑MurNAc‑peptides, which leads to AmpC hyperproduction and fail‑ure of the antibiotic [39,51]. Interestingly, unlike Enterobacteriaceae, P. aeruginosa encodes three ampD genes: ampD, ampDh2 and ampDh3 [51]. The enzymes encoded by these genes work in con‑cert to repress ampC induction, since step‑wise inactivation of their genes results in progressive

upregulation of ampC expression. Multiple ampD homologss may allow P. aeruginosa to easily acquire moderate b‑lactam resistance by partially derepressing ampC expression via loss of AmpD function, while sustaining PG recycling via the actions of AmpDh2 and AmpDh3. Moreover, partial derepression appears to avoid compromis‑ing fitness and virulence that is associated with high‑level constitutive AmpC hyperproduction [52]. Indeed, while ampD mutations are frequently found among clinical isolates, full ampC dere‑pression due to the additional inactivation of ampDh2 and ampDh3 is not [53].

Spontaneous inactivation of dacB, which encodes a nonessential penicillin‑binding protein (PBP4), has also been found to cause clinically relevant antipseudomonal b‑lactam resistance in P. aeruginosa via hyperproduction of AmpC at levels exceeding those produced by ampD null mutants [54]. Inactivation of this PBP results in a highly efficient and complex b‑lactam resistance response, triggering overproduction of chromo‑somal AmpC and the specific activation of the CreBC (BlrAB) two‑component regulator, which in turn plays a role in resistance through a still unknown mechanism. Surveys of b‑lactam‑resis‑tant isolates of P. aeruginosa show that ampD and dacB null mutations (frequently combined) are a frequent cause of clinical resistance to antipseu‑domonal b‑lactams [48,54]. Finally, while specific point mutations in AmpR have been reported to cause constitutive AmpC hyperproduction [48,55,56], they appear to be rare compared with ampD and dacB null mutations [48,54]. Their lower prevalence may ref lect the small pro‑portion of such viable point mutations within the entire pool of mutations leading to AmpC hyperproduction, which is likely dominated by thousands of ampD or dacB inactivating muta‑tions that have established fitness. Indeed, AmpR acts as a transcriptional regulator of many genes within P. aeruginosa [57], and altering its function may adversely affect the expression of genes that are essential to fitness or virulence. Nevertheless, wider studies investigating the prevalence and role of ampR mutations in the clinical setting are still required.

Unlike antipseudomonal b‑lactams, carbap‑enems are strong inducers of AmpC, yet they remain effective against P. aeruginosa because AmpC hydrolyzes them only very slowly [6]. Remarkably, in the case of P. aeruginosa, this poor activity against carbapenems becomes clinically significant within spontaneous mutants having reduced levels of the outer membrane porin, OprD [58–60]. In this case, the lower OprD levels



leading to reduced membrane permeability acts synergistically with AmpC production to confer clinical resistance to carbapenems [59]. Repression or inactivation of porins may also result in low‑level carbapenem resistance in Enterobacteriaceae isolates overexpressing chromosomal AmpC b‑lactamases [6]. It is noteworthy that the most frequently selected mutations that cause clini‑cal failure of penicillins, cephalosporins and monobactams (ampD and dacB mutations) and carbapenems (particularly repression or inac‑tivation of OprD) against P. aeruginosa (and Enterobacteriaceae) all depend upon the induc‑tion of AmpC by metabolites of the PG recycling pathway. Considering this common dependence on PG recycling, pharmacological strategies aimed at blocking PG recycling not only hold promise for suppressing intrinsic AmpC pro‑duction, but also for blocking the clinically rel‑evant AmpC hyperproduction that results from the selection of chromosomal mutations during b‑lactam therapy, or from the direct acquisition of infections by AmpC‑hyperproducing strains.

PG recycling proteins as targets for attenuating AmpC‑mediated b‑lactam resistance

Our advancing knowledge of the structure and function of proteins comprising the Gram‑negative PG recycling pathway is providing

insight into the possible development of small molecules to block AmpC induction during b‑lactam therapy. Table 1 lists proteins from the PG recycling pathway that are potential targets for the development of such a strategy. Below we summarize recent findings that support the exploitation of these proteins as small‑molecule targets for repressing inducible AmpC‑mediated b‑lactam resistance.

NagZGenetic inactivation of nagZ reverses & prevents b‑lactam resistance in P. aeruginosaGiven that NagZ catalyzes the formation of 1,6‑anhydro‑MurNAc‑peptide species [33,34], and is necessary for AmpC overproduction [22,34], blocking its activity is being explored as a strategy to repress ampC expression. Genetic inactivation of nagZ in the P. aeruginosa ref‑erence strain PAO1, and in an isogenic triple ampD null mutant (ampD-, ampDh2- and ampDh3-) recently revealed that NagZ plays a central role in the high‑level b‑lactam resistance that is associated loss of with AmpD activity (Table 2) [22]. Loss of NagZ activity repressed intrinsic b‑lactam resistance up to four‑fold in PAO1, which is consistent with the work of Votsch and Templin, who found that a strain of Escherichia coli lacking NagZ produced a quarter

Table 1. Putative inhibitor targets of the AmpC b-lactamase induction pathway.

Protein 3D structure PDB entries Effect of inhibition

NagZ 1Y651TR92OXN3GSM3GS6

Block formation of the AmpR activator molecule 1,6-anhydro-MurNAc-tri(or penta)peptide

AmpG N/A Block entry of all PG fragments that activate AmpR-mediated ampc transcription

AmpR

(Effector binding domain only)

3KOT3KOS

Block AmpR from binding 1,6-anhydroMurNAc-tri (or penta) peptide activator molecule

FtsZ 1FSZ1RQ72VAW2VAM2R6R

Increase cytosolic amount of the AmpR corepressor UDP-MurNAc-pentapeptide

BlrAB N/A Inactivation of BlrAB (CreBC) two-component regulator mitigates b-lactam resistance driven by dacB (PBP4) mutation in Pseudomonas aeruginosa



of the amount of AmpC from a plasmid‑borne ampC–ampR operon compared with wild‑type E. coli harboring the same plasmid [34]. More striking, however, is the marked attenuation that nagZ inactivation has on the high antip‑seudomonal b‑lactam resistance associated with loss of AmpD activity; inactivation of the gene restored b‑lactam susceptibility in the ampD triple mutant, returning the MICs of ceftazi‑dime, piperacillin and aztreonam to below their respective pharmacological resistance break‑point concentrations (Table 2) [22]. Furthermore, loss of NagZ restored wild‑type b‑lactam MIC levels and ampC expression in a dacB- mutant of PAO1 and significantly reduced the impact of the dacB-ampD- double mutant (e.g., the MIC for ceftazidime against the dacB-ampD- double mutant was shown to drop from 96 to 4 µg/ml, and ampC expression from ca. 1000‑fold to ca. 50‑fold higher than that for PAO1) [23]. The mechanism by which dacB null mutations cause constitutive AmpC hyperproduction is not fully understood, but, it relies on NagZ. Intriguingly in this regard, loss of NagZ activity blocks the overexpression of creD (blrD), which is a marker

of the activation of the CreBC (BlrAB) two‑component regulator that is involved in the dacB resistance phenotype [54]. Although wild‑type MICs are restored when nagZ is inactivated in ampD or dacB mutants, wild‑type MICs are not fully restored when nagZ is inactivated in strains carrying multiple resistance mutations (e.g., null mutations in all three ampD genes [ampD, ampDh2 and ampDh3] or null muta‑tions in both ampD and dacB) (Table 2) [22,24]. The reason for the residual AmpC production in these mutants after removal of NagZ activity remains unclear.

In addition to repressing AmpC induction, nagZ inactivation has also been shown to impede the ability of P. aeruginosa PAO1 to develop cef‑tazidime resistance [23]. A population ana lysis revealed that spontaneous resistance mutants could not be obtained at concentrations exceed‑ing the susceptibility breakpoint of ceftazidime (8 µg/ml) for a nagZ null mutant, but were obtained for wild‑type PAO1. Therefore, NagZ is envisaged as a candidate drug target to both revert and possibly prevent AmpC‑mediated b‑lactam resistance in P. aeruginosa.

Table 2. Effect of inactivating or inhibiting key peptidoglycan recycling proteins on the MICs of b-lactams against resistant mutants of Pseudomonas aeruginosa PAO1.

Strain MIC (µg/ml)

CAZ CAZ + PUGNAc¶ PTZ PTZ + PUGNAc ATM ATM + PUGNAc IMP

Wild-type (PA)† 1 3 3 2

Resistance mutants

PADampD‡ 8 4 48 24 8 4 1PADampDampDh2ampDh3§ 48 >256 24 1.5PADdacB‡ 24 8 64 32 12 4 2PADdacBampD† 96 >256 48 2PADoprD† 1.5 2 2 >32

Effect of inactivating nagZ

PADnagZ† 1 3 3 1PADampDnagZ† 2 6 3 1PADampDampDh2ampDh3nagZ§ 12 24 4 1.5PADdacBnagZ† 1.5 4 3 1PADdacBampDnagZ† 4 24 6 1.5PADoprDnagZ† 1.5 2 2 6

Effect of inactivating ampG

PADampG† PADampDampG†

11

33

21.5

0.380.38

PADdacBampG† 1 2 1.5 0.38PADdacBampDampG† 0.75 1.5 1.5 0.38PADoprDampG† 1.5 2 2 0.5†Reproduced from [24].‡Reproduced from [23].§Reproduced from [22].¶[PUGNAc] = 0.5 mM.ATM: aztreonam; CAZ: Ceftazidime; IMP: Imipenem; PA: Pseudomonas aeruginosa; PTZ: piperacillin-tazobactam; PUGNAc: O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate.



Potential of NagZ inhibitorsPUGNAc (O ‑ (2‑acetamido‑2‑deoxy-d-glucopyranosylidene)amino‑N-phenylcarba‑mate) is a potent, nonselective inhibitor of N‑acetyl‑b-d-glucosaminidases [61], including NagZ (K

i = 48 nM) (Figure 2) [62]. While it does

not possess antimicrobial properties [22,62], co‑administration with ceftazidime, piperacillin or aztreonam enhances the efficacy of these anti‑biotics against ampD and dacB null mutants of P. aeruginosa [22,23]. Interestingly, repression of AmpC production is greater in the dacB mutant (reducing the ceftazidime MICs from 24 to 6 µg/ml) than the ampD mutant (reducing the MICs from 8 to 4 µg/ml) [23]. These effects using NagZ inhibitors are smaller than when nagZ is genetically deleted from the microbe, perhaps due to the limited permeability of the compounds through the outer membrane or from their limited potency.

In addition to permeability and potency, another challenge facing the use of PUGNAc as an inhibitor of NagZ is that it also potently inhibits the functionally related human enzymes O‑GlcNAcase and b‑hexosaminidase. NagZ removes GlcNAc from PG recycling

intermediates using a catalytic mechanism involving a covalent glycosyl enzyme intermedi‑ate [63–65]. By contrast, human O‑GlcNAcase and b‑hexosaminidase remove GlcNAc from glyco‑conjugates using a neighboring‑group catalytic mechanism that proceeds through an oxazoline intermediate [66–70]. By exploiting these mecha‑nistic differences, we recently developed 2‑N‑acyl derivatives of PUGNAc that exhibit 100‑fold selectivity for NagZ over the functionally related human enzymes [62,71]. Selectivity arises from structural differences in the active site of NagZ versus O‑GlcNAcase or b‑hexosaminidase [71]. The 2‑N‑acyl binding site with the NagZ active site is solvent accessible, allowing bulky aliphatic extensions to be added to the moiety without sub‑stantially reducing binding affinity (Figure 2). By contrast, O‑GlcNAcase and b‑hexosaminidase must position the 2‑N‑acyl group of PUGNAc within a constrained pocket at the base of their active sites, which cannot accommodate sig‑nificant bulk at this position on the inhibitor. Consistent with results obtained for PUGNAc, the derivative EtBuPUG (Figure 2) has recently been shown to repress AmpC production in an ampD null mutant of P. aeruginosa [22].

OHO

HO

HO

HN ON O

O

HN

OHO

HO

HO

NHN O

O

HN

CH3H3CO

0.048 µM 0.046 µM0.036 µM

PUGNAc inhibition constants (Ki)

NagZO-GlcNAcaseβ-hexosaminidase

3.5 µM

337 µM>3000 µM

EtBuPUG inhibition constants (Ki)

NagZ

O-GlcNAcaseβ-hexosaminidase

Figure 2. Small-molecule inhibitors of NagZ. (A) Crystal structure of NagZ from Vibrio cholerae bound to PUGNAc. (B) The 2-N-acyl binding site of PUGNAc (circled yellow) within the NagZ active site is open and solvent accessible, allowing bulky aliphatic extensions to be added to the moiety without substantially reducing binding affinity. These 2-N-acyl derivatives are selective for NagZ over the functionally related human enzymes O-GlcNAcase and b-hexosaminidase. (C) An example of a NagZ selective PUGNAc derivative with a branched extension appended to the N-acyl group. Inhibition constants for PUGNAc and EtBuPUG are reproduced from [62].



Small molecule‑based inhibition of NagZ could potentiate the efficacy of a broad spectrum of b‑lactams and possibly prevent the sponta‑neous occurrence of highly resistant mutants. Further, selection of NagZ mutations that decrease binding affinity of sugar‑based inhibi‑tors such as 2‑N‑acyl derivatives of PUGNAc, could well compromise the catalytic efficiency of the enzyme, thereby reducing production of the inducing anhydomuropeptide and ensur‑ing continued repression of AmpC produc‑tion. Collectively, these observations suggest improved NagZ inhibitors could eventually prove useful to combat P. aeruginosa infections.

AmpGGenetic inactivation of ampG reverses broad‑spectrum b‑lactam resistance in P. aeruginosaThe inner membrane permease, AmpG, plays an essential role in AmpC induction [72–75] by importing excised PG fragments into the bacte‑rial cytosol [31]. Genetic inactivation of AmpG not only fully restores penicillin, cephem and monobactam susceptibility in ampD [24,74] and dacB [24] null mutants of P. aeruginosa, it also reverses imipenem resistance that arises from loss of OprD function by completely inhibit‑ing AmpC induction [24]. The MIC for imipe‑nem against an OprD null mutant of PAO1, for instance, can be reduced from >32 to 0.5 µg/ml when ampG is inactivated (Table 2). In a com‑parative assessment of ampG and nagZ inac‑tivation in three clinical isolates known to be pan‑b-lactam resistant due to AmpC hyperpro‑duction, OprD inactivation and overexpression of several efflux pumps, a marked increase in sus‑ceptibility to ceftazidime and piperacillin‑tazo‑bactam was observed in both cases. Only ampG inactivation, however, fully restored wild‑type imipenem susceptibility [24]. Moreover, as for NagZ, loss of AmpG function also impairs the emergence of one‑step ceftazidime resistance in P. aeruginosa PAO1, suggesting that inhibition of AmpG could both reverse and prevent emer‑gence of broad‑spectrum b‑lactam resistance in P. aeruginosa and other Gram‑negative bacteria carrying the ampR–ampC operon.

Due to its essential role in ampC expression and high conservation in Gram‑negative bacte‑ria, AmpG appears to be a promising drug target to supress AmpC induction. Realizing this goal will, however, require the development of new functional assays in order to identify candidate inhibitors. In addition to AmpG as a periplasmic target for suppressing AmpC induction during

b‑lactam therapy, inhibition of lytic transglyco‑sylases, a family of enzymes involved in the exci‑sion of PG fragments from the cell wall [76], may also provide a therapeutic mechanism to suppress AmpC production during b‑lactam therapy, since genetic inactivation of these enzymes has also been found to attenuate b‑lactam‑induced expression of AmpC [77].

AmpRLoss of AmpR function renders ampC noninducibleThe AmpC induction pathway ultimately con‑verges on the transcriptional regulator AmpR. Not surprisingly, ampR inactivation renders ampC noninducible [38]. Since AmpR regulates numerous genes in addition to ampC [57], potent small molecule inhibitors of AmpR based on the chemical motifs present in its small molecule corepressor, UDP‑MurNAc‑pentapeptide, could have a broad negative effect on the transcrip‑tional regulation of the genomes of opportunistic pathogens such as P. aeruginosa, in addition to suppressing the induction of AmpC. Modeling studies in conjunction with a crystal structure of the muropeptide‑binding domain of AmpR has recently provided some molecular insight into how the protein regulates ampC expression and could assist in the rational design of future AmpR inhibitors [78]. Further structural studies of the interactions of AmpR with its likely core‑pressor, UDP‑MurNAc‑pentapeptide, will be needed to enable future efforts to target AmpR.

Additional potential targets to block AmpC regulation

FtsZFtsZ is a highly conserved multidomain protein that is recruited to the site of cell division within bacteria. This essential protein forms the septal ring that is essential for bacterial cell division. FtsZ has been the focus of extensive studies (for review see [79–81]) and investigated as a potential antimicrobial drug target, for which some inhib‑itors have been described [82,83]. Interestingly, it has been found that when an E. coli temper‑ature‑sensitive ftsZ mutant is grown at a non‑permissive temperature, it not only exhibits impaired cell division, it becomes incapable of inducing ampC expression from a plasmid‑borne ampR–ampC operon when challenged with a strong inducer of AmpC (cefoxitin) [84,85]. The inability to induce AmpC results from a greatly increased amount UDP‑MurNAc‑pentapeptide in the ftsZ mutant, which the ampC activating muropeptides appear unable to competitively



Financial & competing interests disclosureResearch in the authors laboratories are supported by the Canadian Institutes of Health Research (BL Mark, DJ Vocadlo), Cystic Fibrosis Canada (BL Mark, DJ Vocadlo), The Manitoba Health Research Council (BL Mark) and Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, co-financed by European Development Regional Fund “A way to achieve Europe” ERDF, Spanish Network for Research in Infectious Diseases (REIPI RD06/0008) and grant PS09/00033 (A Oliver). BL Mark and DJ Vocadlo hold a Manitoba Research Chair and Canada Research chair, respec-tively. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

displace from AmpR to initiate transcription [85]. While the precise basis for the effects of FtsZ inhibition on UDP‑MurNAc‑pentapeptide levels remains unclear, testing FtsZ inhibitors, such as the broad‑spectrum antimicrobial san‑guinarine [86], may reveal that, in addition to their antimicrobial properties, FtsZ inhibitors may also be able to suppress b‑lactam resis‑tance in Gram‑negative pathogens containing inducible AmpC b‑lactamase. Accordingly, combining future FtsZ inhibitors with existing b‑lactams could provide potent broad‑spectrum antimicrobial therapies.

BlrAB (CreBC) two‑component regulatorFinally, another plausible target to minimize the impact of AmpC‑mediated resistance in P. aeru-ginosa is the BlrAB (CreBC) two‑component regulator. BlrAB is well known to regulate the expression of several b‑lactamases in Aeromonas spp. [87]. Interestingly, this two‑component regu‑lator has recently been found to play an important role in the b‑lactam resistance response driven by the inactivation of dacB in P. aeruginosa [54]. Noteworthy, the inactivation of BlrAB in a dacB mutant of P. aeruginosa significantly mitigated b‑lactam resistance, reducing ceftazidime MICs from 32 to 4 µg/ml [54]. Moreover, the inactiva‑tion of BlrAB in a wild‑type strain dramatically reduced its capacity to develop high‑level cef‑tazidime resistance through spontaneous muta‑tions. While high‑level ceftazidime‑resistant mutants were readily selected the from PAO1 wild‑type strain (mutation rate to ceftazidime [at breakpoint concentration, 16 µg/ml] resistance of 3 × 10‑8 mutants per cell division), mutation rates were below the detection limit (<1 × 10‑11) for the BlrAB knockout mutant [54].

Future perspectiveUnambiguous identification of the PG recycling metabolite(s) that converts AmpR into an acti‑vator of ampC transcription is yet to be achieved. Further studies will be needed to resolve this issue and the results will provide important insight into how the inactivation of nagZ can reverse b‑lactam resistance without fully repress‑ing ampC transcription, whereas inactivation of ampG can achieve both. Such data should also facilitate identification of AmpR antagonists. Given the data obtained using NagZ‑deficient strains, it appears that improvements to the potency and bioavailability of selective NagZ inhibitors are possible. Inhibitors of AmpG per‑mease also hold promise and we expect these could be used to reverse b‑lactam resistance that

would overcome all known chromosomal resis‑tance mutations. Several FtsZ inhibitors have been developed and there is clear evidence that loss of FtsZ function represses AmpC induction. These inhibitors should be examined for their ability to attenuate AmpC induction during challenge by b‑lactams.

Finally, while recent developments in b‑lac‑tamase inhibitor design show considerable prom‑ise at delivering therapeutically useful drugs that inhibit AmpC, it is possible that consti‑tutive AmpC hyperproducing mutants could overcome some of these next‑generation inhibi‑tors. Thus, combining b‑lactamase inhibitors with PG recycling inhibitors should be investi‑gated to ascertain whether they can synergisti‑cally alleviate b‑lactam resistance mediated by inducible AmpC.

ConclusionThe AmpC induction pathway includes a num‑ber of proteins that can be targeted with small molecule inhibitors to suppress AmpC induction and boost the efficacy of a broad spectrum of b‑lactam antibiotics. The recent development of a rationally designed inhibitor of NagZ that sup‑presses AmpC‑mediated resistance demonstrates the feasibility of these approaches. As research continues to develop in this area, it is likely that additional targets may ultimately yield potent PG recycling inhibitors that block resistance to a broad‑spectrum of b‑lactams, including car‑bapenems. The structural insights gained thus far will likely prove key in these endeavors and future structural studies are expected to also facilitate these efforts.



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Executive summary

AmpC induction pathwaynAmpC b-lactamases deactivate a wide range of b-lactams. The opportunistic pathogen Pseudomonas aeruginosa, as well as many

other Enterobacteriaceae carry a chromosomal AmpC gene that is induced by b-lactams, a process controlled by metabolites of the Gram-negative peptidoglycan (PG) recycling pathway. While natural AmpC induction accounts for intrinsic resistance to narrow-spectrum b-lactams (such as ampicillin), chromosomal mutations causing constitutive AmpC hyperproduction or reduced membrane permeability often lead to AmpC-mediated failure of broad-spectrum b-lactam therapy, including antipseudomonal penicillins, cephalosporins and carbapenems.

Blocking the PG recycling pathway to suppress AmpC-mediated resistancenPG recycling involves a number of proteins that are potential targets for small-molecule inhibitors. These inhibitors could be used to

block the production of PG recycling metabolites that induce AmpC production, thereby reducing AmpC levels and boosting the efficacy of coadministered b-lactams.

Evidence to support PG recycling targetsnLoss-of-function of the following PG-recycling proteins has been shown to reverse AmpC-mediated resistance in P. aeruginosa:

– The inner membrane permease, AmpG

– The glycoside hydrolase, NagZ

– The transcriptional regulator, AmpR

nSmall-molecule inhibitors of NagZ have been developed that demonstrate the potential of inhibiting PG recycling to suppress AmpC-mediated resistance in P. aeruginosa. Additional targets that indirectly suppress PG recycling, including the cell division protein FtsZ and the CreBC two-component regulator, are interesting targets to consider.

Future perspectivenImprovements to the potency and bioavailability of NagZ inhibitors are possible. Inhibitors of AmpG permease also hold promise to

reverse b-lactam resistance that would overcome all known chromosomal resistance mutations. Since loss of FtsZ function suppresses AmpC induction, available FtsZ inhibitors should be examined for their ability to attenuate AmpC induction during b-lactam therapy. Combining next-generation AmpC b-lactamase inhibitors with PG-recycling inhibitors should be investigated to ascertain whether they could synergistically alleviate b-lactam resistance mediated by inducible AmpC.



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Providing β-lactams a helping hand: targeting the AmpC β-lactamase induction pathway

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