Exploring the potential of boronic acids as inhibitors of OXA‐24/40 β‐lactamase

β‐lactam antibiotics are crucial to the management of bacterial infections in the medical community. Due to overuse and misuse, clinically significant bacteria are now resistant to many commercially available antibiotics. The most widespread resistance mechanism to β‐lactams is the expression of β‐lactamase enzymes. To overcome β‐lactamase mediated resistance, inhibitors were designed to inactivate these enzymes. However, current inhibitors (clavulanic acid, tazobactam, and sulbactam) for β‐lactamases also contain the characteristic β‐lactam ring, making them susceptible to resistance mechanisms employed by bacteria. This presents a critical need for novel, non‐β‐lactam inhibitors that can circumvent these resistance mechanisms. The carbapenem‐hydrolyzing class D β‐lactamases (CHDLs) are of particular concern, given that they efficiently hydrolyze potent carbapenem antibiotics. Unfortunately, these enzymes are not inhibited by clinically available β‐lactamase inhibitors, nor are they effectively inhibited by the newest, non‐β‐lactam inhibitor, avibactam. Boronic acids are known transition state analog inhibitors of class A and C β‐lactamases, and are not extensively characterized as inhibitors of class D β‐lactamases. Importantly, boronic acids provide a novel way to potentially inhibit class D β‐lactamases. Sixteen boronic acids were selected and tested for inhibition of the CHDL OXA‐24/40. Several compounds were identified as effective inhibitors of OXA‐24/40, with K_i values as low as 5 μM. The X‐ray crystal structures of OXA‐24/40 in complex with BA3, BA4, BA8, and BA16 were determined and revealed the importance of interactions with hydrophobic residues Tyr112 and Trp115. These boronic acids serve as progenitors in optimization efforts of a novel series of inhibitors for class D β‐lactamases.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

The global regulator Crc modulates metabolism,susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross‐talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high‐throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing‐regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta‐lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.     

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

One ring to rule them all: Current trends in combating bacterial resistance to the β‐lactams

From humble beginnings of a contaminated petri dish, β‐lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an “arms race” with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever‐dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in β‐lactam research. Here the current state of β‐lactam antibiotics from a structural perspective was reviewed.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Solution structure of protein synthesis initiation factor 1 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa‐IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa‐IF1. Pa‐IF1 consists of a five‐stranded β‐sheet with an unusual extended β‐strand at the C‐terminus and one short α‐helix arranged in the sequential order β1‐β2‐β3‐α1‐β4‐β5. The structure adopts a typical β‐barrel fold and contains an oligomer‐binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α‐helix may compose the binding interface with the 30S subunit.     

A novel pathway for outer membrane protein biogenesis in Gram‐negative bacteria

The understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM.     

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Elucidating the role of Trp105 in the KPC-2 ��-lactamase

The molecular basis of resistance to β‐lactams and β‐lactam‐β‐lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β‐lactam therapy. Recent crystal structures of KPC‐2 and other class A β‐lactamases suggest that Ambler position Trp105 may be of importance in binding β‐lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC‐2 by conducting site‐saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, ‐Tyr, ‐Asn, and ‐His KPC‐2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC‐2. Interestingly, most of the variants showed increased MICs to ampicillin‐clavulanic acid but not to ampicillin‐sulbactam or piperacillin‐tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, ‐Asn, ‐Leu, and ‐Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β‐lactamase displayed improved catalytic efficiencies, k_cat/K_m, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k_cat/K_m toward cefotaxime and imipenem when compared to WT β‐lactamase. The Trp105Asn variant exhibited increased K_ms for all substrates. In contrast, the Trp105Leu and ‐Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k_cat/K_m) for all substrates. With respect to clavulanic acid, the K_is and partition ratios were increased for the Trp105Phe, ‐Asn, and ‐Val variants. We conclude that interactions between Trp105 of KPC‐2 and the β‐lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β‐lactam and β‐lactamase inhibitor discrimination.     

Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.     

Crystal structures of OprN and OprJ,outer membrane factors of multidrug tripartite efflux pumps of Pseudomonas aeruginosa

The genome of Pseudomonas aeruginosa encodes tripartite efflux pumps that extrude functionally and structurally dissimilar antibiotics from the bacterial cell. MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM are the main tripartite efflux pumps responsible for multidrug resistance in P. aeruginosa. The outer membrane factors OprN, OprJ, and OprM are essential components of functional tripartite efflux pumps. To elucidate the structural basis of multidrug resistance, we determined the crystal structures of OprN and OprJ. These structures revealed several features, including tri‐acylation of the N‐terminal cysteine, a small pore in the β‐barrel domain, and a tightly sealed gate in the α‐barrel domain. Despite the overall similarity of OprN, OprJ, and OprM, a comparison of their structures and electrostatic distributions revealed subtle differences at the periplasmic end of the α‐barrel domain. These results suggested that the overall structures of these outer membrane factors are specifically optimized for particular tripartite efflux pumps. Proteins 2016; 84:759–769. © 2016 Wiley Periodicals, Inc.     

Endopeptidase-Mediated Beta Lactam Tolerance

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate - sphere formation vs. lysis – after treatment with antibiotics that target cell wall synthesis.     

V‐antigen homologs in pathogenic gram‐negative bacteria

Gram‐negative bacteria cause many types of infections in animals from fish and shrimps to humans. Bacteria use Type III secretion systems (TTSSs) to translocate their toxins directly into eukaryotic cells. The V‐antigen is a multifunctional protein required for the TTSS in Yersinia and Pseudomonas aeruginosa. V‐antigen vaccines and anti‐V‐antigen antisera confer protection against Yersinia or P. aeruginosa infections in animal models. The V‐antigen forms a pentameric cap structure at the tip of the Type III secretory needle; this structure, which has evolved from the bacterial flagellar cap structure, is indispensable for toxin translocation. Various pathogenic gram‐negative bacteria such as Photorhabdus luminescens, Vibrio spp., and Aeromonas spp. encode homologs of the V‐antigen. Because the V‐antigens of pathogenic gram‐negative bacteria play a key role in toxin translocation, they are potential therapeutic targets for combatting bacterial virulence. In the USA and Europe, these vaccines and specific antibodies against V‐antigens are in clinical trials investigating the treatment of Yersinia or P. aeruginosa infections. Pathogenic gram‐negative bacteria are of great interest because of their ability to infect fish and shrimp farms, their potential for exploitation in biological terrorism attacks, and their ability to cause opportunistic infections in humans. Thus, elucidation of the roles of the V‐antigen in the TTSS and mechanisms by which these functions can be blocked is critical to facilitating the development of improved anti‐V‐antigen strategies.     

Bacterial outer membrane constriction

The outer membrane of Gram‐negative bacteria is a crucial permeability barrier allowing the cells to survive a myriad of toxic compounds, including many antibiotics. This innate form of antibiotic resistance is compounded by the evolution of more active mechanisms of resistance such as efflux pumps, reducing the already limited number of clinically relevant treatments for Gram‐negative pathogens. During cell division Gram‐negative bacteria must coordinate constriction of the outer membrane in conjunction with other crucial layers of the cell envelope, the peptidoglycan cell wall and the inner membrane. Coordination is crucial in maintaining structural integrity of the envelope, and represents a highly vulnerable time for the cell as any failure can be fatal, if not least disadvantageous. However, the molecular mechanisms of cell division and how the biogenesis of the three layers is synchronised during constriction remain largely unknown. Perturbations of the outer membrane have been shown to increase the effectiveness of antibiotics in vitro, and so with improved understanding of this process we may be able to exploit this vulnerability and improve the effectiveness of antibiotic treatments. In this review the current knowledge of how Gram‐negative bacteria facilitate constriction of their outer membranes during cell division will be discussed.     

Comparison of separation performances of amylose‐ and cellulose‐based stationary phases in the high‐performance liquid chromatographic enantioseparation of stereoisomers of β‐lactams

High‐performance liquid chromatographic methods were developed for the separation of the enantiomers of 19 β‐lactams. The direct separations were performed on chiral stationary phases containing either amylose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® AmyCoat? column) or cellulose‐tris‐3,5‐dimethylphenyl carbamate, (Kromasil® CelluCoat? column) as chiral selector. The different methods were compared in systematic chromatographic examinations. The separations were carried out with good selectivity and resolution. The AmyCoat? and CelluCoat? columns appear to be highly complementary. The best separations of bi‐ and tricyclic β‐lactam stereoisomers were obtained with the AmyCoat? column, whereas the 4‐aryl‐substituted β‐lactams were better separated on the CelluCoat? column. The elution sequence was determined in all cases; no general rule could be established. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity

New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β‐hairpin antimicrobial peptide arenicin‐1 as a template for design of more potent antimicrobials. In particular, six shortened 17‐residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin‐1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two‐fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram‐positive and Gram‐negative bacteria was significantly increased compared with that of arenicin‐1 while the mechanism of action remained the same. Like arenicin‐1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug‐resistant Gram‐negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin‐1 in water solution, but significantly differed in membrane‐mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β‐hairpin antimicrobial peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

c‐di‐AMP modulates Listeria monocytogenes central metabolism to regulate growth,antibiotic resistance and osmoregulation

Cyclic diadenosine monophosphate (c‐di‐AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall‐active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β‐lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl‐CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100‐fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c‐di‐AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down‐stream enzymes suppressed ΔdacA phenotypes. These data suggested that c‐di‐AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild‐type bacteria.     

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

A large number of β‐lactamases have emerged that are capable of conferring bacterial resistance to β‐lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM‐1 β‐lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A β‐lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the β‐lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM‐1 β‐lactamase to positions 220, 272, and 276 by site‐directed mutagenesis. Kinetic analysis of the engineered β‐lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A β‐lactamases.     

A multifaceted cellular damage repair and prevention pathway promotes high‐level tolerance to β‐lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two‐component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high‐level β‐lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β‐lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β‐lactams. We propose that VxrAB reduces antibiotic‐induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β‐lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism''s ability to counteract diverse antibiotic‐induced stresses promotes high‐level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.