Rational Design of Benzobisheterocycle Metallo-ß-Lactamase Inhibitors: A Tricyclic Scaffold Enhances Potency against Target Enzymes.

Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor taniborbactam against metallo-ß-lactamases.

Taniborbactam (TAN) is a novel broad-spectrum ß-lactamase inhibitor with significant activity against subclass B1 metallo-ß-lactamases (MBLs). Here, we showed that TAN exhibited an overall excellent activity against B1 MBLs including most NDM- and VIM-like as well as SPM-1, GIM-1, and DIM-1 enzymes, but not against SIM-1. Noteworthy, VIM-1-like enzymes (particularly VIM-83) were less inhibited by TAN than VIM-2-like. Like NDM-9, NDM-30 (also differing from NDM-1 by a single amino acid substitution) was resistant to TAN.

Structural basis of metallo-ß-lactamase resistance to taniborbactam.

The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.

Correction: 2-Mercaptomethyl-thiazolidines use conserved aromatic-S interactions to achieve broad-range inhibition of metallo-ß-lactamases.

[This corrects the article DOI: 10.1039/D0SC05172A.].

In-cell kinetic stability is an essential trait in metallo-ß-lactamase evolution.

Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-ß-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. The nonmetalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. Zn(II) binding renders the protein refractory to degradation by quenching the flexibility of this region. Membrane anchoring makes apo-NDM-1 less accessible to Prc and protects it from DegP, a cellular protease degrading misfolded, nonmetalated NDM-1 precursors. NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis. These observations link MBL-mediated resistance with the essential periplasmic metabolism, highlighting the importance of the cellular protein homeostasis.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Gating interactions steer loop conformational changes in the active site of the L1 metallo-ß-lactamase.

ß-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of ß-lactamases among pathogenic bacteria limits the efficacy of ß-lactam antibiotics. This has created a major public health crisis. The use of ß-lactamase inhibitors has proven useful in restoring the activity of ß-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-ß-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the ß-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-ß7 and ß12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-ß7 and ß12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

Characterization of a Novel Pathogen in Immunocompromised Patients: Elizabethkingia anophelis-Exploring the Scope of Resistance to Contemporary Antimicrobial Agents and ß-lactamase Inhibitors.

Background: Elizabethkingia anophelis is an emerging Gram-negative nonlactose fermenter in the health care setting, where it causes life-threatening infections in immunocompromised patients. We aimed to characterize the molecular mechanisms of antimicrobial resistance and evaluate the utility of contemporary antibiotics with the intent to offer targeted therapy against an uncommonly encountered pathogen. Methods: Whole-genome sequencing (WGS) was conducted to accurately identify isolate species and elucidate the determinants of ß-lactam resistance. Antimicrobial susceptibility testing was performed using broth microdilution and disk diffusion assays. To assess the functional contribution of the major metallo-ß-lactamase (MBL) encoding genes to the resistance profile, bla BlaB was cloned into pBCSK(-) phagemid vector and transformed into Escherichia coli DH10B. Results: WGS identified the organism as E. anophelis. MBL genes bla BlaB-1 and bla GOB-26 were identified, in addition to bla CME-2, which encodes for an extended-spectrum ß-lactamase (ESBL). Plasmids were not detected. The isolate was nonsusceptible to all commonly available ß-lactams, carbapenems, newer ß-lactam ß-lactamase inhibitor combinations, and to the combination of aztreonam (ATM) with ceftazidime-avibactam (CAZ-AVI). Susceptibility to the novel siderophore cephalosporin cefiderocol was determined. A BlaB-1 transformant E. coli DH10B isolate was obtained and demonstrated increased minimum inhibitory concentrations to cephalosporins, carbapenems, and CAZ-AVI, but not ATM. Conclusions: Using WGS, we accurately identified and characterized an extensively drug-resistant E. anophelis in an immunocompromised patient. Rapid evaluation of the genetic background can guide accurate susceptibility testing to better inform antimicrobial therapy selection.

Increased prevalence of canine echinococcosis a decade after the discontinuation of a governmental deworming program in Tierra del Fuego, Southern Chile.

Hydatid disease is a neglected zoonotic parasitic disease caused by cysts of the tapeworm Echinococcus granulosus. Canids, especially domestic dogs, are definitive hosts of the parasite and are the most pragmatic targets for control programs. A governmental dog deworming campaign was established in 1979 to control hydatidosis in southern Chile, which succeeded in reducing the prevalence of canine echinococcosis in Tierra del Fuego province from 68.4% (in 1978) to 1.2% (in 2002). In 2004, however, the program was dismantled to reduce costs, and since then, no follow-up echinococcosis monitoring has been conducted. We surveyed 356 domestic dogs and interviewed owners or workers at 45 ranches in Chilean Tierra del Fuego during the summer of 2015-2016. Faecal flotation was employed to detect Taeniidae eggs, and PCR was used to test faecal samples for Echinococcus granulosus. Taeniidae eggs and Echinococcus sp. DNA were detected in the faeces of 45.4% (147/324) and 6.9% (23/331) of dogs, respectively. Infrequent dog deworming and the presence of culpeo foxes (Lycalopex culpaeus) were significant predictors of the prevalence of Echinococcus sp. DNA and Taeniidae eggs. Furthermore, the presence of introduced chilla foxes (Lycalopex griseus), the municipality, and several operational characteristics of ranches (number of sheep, frequency of sheep slaughter, number of dogs, frequency of removal of dog faeces, feeding of dogs with sheep viscera) were also predictive of the prevalence of Taeniidae eggs. Our findings reveal an ongoing risk of echinococcosis with pathogen maintenance in ranch dogs in Chilean Tierra del Fuego, and in the absence of adequate control programmes, there is a tangible risk of re-emergence of hydatid disease as a public health concern.

The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro.

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu+ and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu+-transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.

"Vermellogens" and the Development of CB[8]-Based Supramolecular Switches Using pH-Responsive and Non-Toxic Viologen Analogues.

We present herein the "vermellogens", a new class of pH-responsive viologen analogues, which replace the direct linking between para-substituted pyridinium moieties within those by a hydrazone functional group. A series of such compounds have been efficiently synthesized in aqueous media by hydrazone exchange reactions, displaying a marked pH-responsivity. Furthermore, the parent N,N'-dimethylated "vermellogen": the "red thread", an analogue of the herbicide paraquat and used herein as a representative model of the series, showed anion-recognition abilities, non-reversible electrochemical behavior, and non-toxicity of the modified bis-pyridinium core. The host-guest chemistry for the "red thread" with the CB[7,8] macrocyclic receptors has been extensively studied experimentally and by dispersion corrected density functional theory methods, showing a parallel behavior to that previously described for the herbicide but, crucially, swapping the well-known redox reactive capabilities of the viologen-based inclusion complexes by acid-base supramolecular responsiveness.

Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a Cystic Fibrosis Patient Treated with ß-Lactams.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C ß-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC ß-lactamase is the main mechanism driving resistance in this notorious pathogen to ß-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the ß-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions.

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M ß-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried ß-sheet. The two mutations located in this ß-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M ß-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.

Genomic heterogeneity underlies multidrug resistance in Pseudomonas aeruginosa: A population-level analysis beyond susceptibility testing.

BACKGROUND: Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. METHODS: Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. RESULTS: The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. CONCLUSIONS: The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

Human Serum Proteins and Susceptibility of Acinetobacter baumannii to Cefiderocol: Role of Iron Transport.

Cefiderocol, a recently introduced antibiotic, has a chemical structure that includes a cephalosporin that targets cell wall synthesis and a chlorocatechol siderophore moiety that facilitates cell penetration by active iron transporters. Analysis of the effect that human serum, human serum albumin, and human pleural fluid had on growing Acinetobacter baumannii showed that genes related to iron uptake were down-regulated. At the same time, ß-lactamase genes were expressed at higher levels. The minimum inhibitory concentrations of this antimicrobial in A. baumannii cells growing in the presence of human serum, human serum albumin, or human pleural fluid were higher than those measured when these fluids were absent from the culture medium. These results correlate with increased expression levels of ß-lactamase genes and the down-regulation of iron uptake-related genes in cultures containing human serum, human serum albumin, or human pleural fluid. These modifications in gene expression could explain the less-than-ideal clinical response observed in patients with pulmonary or bloodstream A. baumannii infections. The exposure of the infecting cells to the host's fluids could cause reduced cefiderocol transport capabilities and increased resistance to ß-lactams. The regulation of genes that could impact the A. baumannii susceptibility to cefiderocol, or other antibacterials, is an understudied phenomenon that merits further investigation.

Electrochemical characterization of an engineered red copper protein featuring an unprecedented entropic control of the reduction potential.

Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear CuA sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.

Deciphering the evolution of metallo-ß-lactamases: A journey from the test tube to the bacterial periplasm.

Understanding the evolution of metallo-ß-lactamases (MBLs) is fundamental to deciphering the mechanistic basis of resistance to carbapenems in pathogenic and opportunistic bacteria. Presently, these MBL-producing pathogens are linked to high rates of morbidity and mortality worldwide. However, the study of the biochemical and biophysical features of MBLs in vitro provides an incomplete picture of their evolutionary potential, since this limited and artificial environment disregards the physiological context where evolution and selection take place. Herein, we describe recent efforts aimed to address the evolutionary traits acquired by different clinical variants of MBLs in conditions mimicking their native environment (the bacterial periplasm) and considering whether they are soluble or membrane-bound proteins. This includes addressing the metal content of MBLs within the cell under zinc starvation conditions and the context provided by different bacterial hosts that result in particular resistance phenotypes. Our analysis highlights recent progress bridging the gap between in vitro and in-cell studies.

Metallo-ß-lactamases and a tug-of-war for the available zinc at the host-pathogen interface.

Metallo-ß-lactamases (MBLs) are zinc-dependent hydrolases that inactivate virtually all ß-lactam antibiotics. The expression of MBLs by Gram-negative bacteria severely limits the therapeutic options to treat infections. MBLs bind the essential metal ions in the bacterial periplasm, and their activity is challenged upon the zinc starvation conditions elicited by the native immune response. Metal depletion compromises both the enzyme activity and stability in the periplasm, impacting on the resistance profile in vivo. Thus, novel inhibitory approaches involve the use of chelating agents or metal-based drugs that displace the native metal ion. However, newer MBL variants incorporate mutations that improve their metal binding abilities or stabilize the metal-depleted form, revealing that metal starvation is a driving force acting on MBL evolution. Future challenges require addressing the gap between in cell and in vitro studies, dissecting the mechanism for MBL metalation and determining the metal content in situ.