RESUMO
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.
Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Antibacterianos/farmacologia , Antibacterianos/química , beta-Lactamases/química , Carbapenêmicos , Bactérias Gram-NegativasRESUMO
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.
Assuntos
Antibacterianos , Ácidos Borínicos , Ácidos Carboxílicos , Antibacterianos/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , Ácidos Borínicos/farmacologia , Resistência beta-Lactâmica , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Peptídeo Hidrolases , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Estabilidade Proteica , Proteólise , Peptídeo Hidrolases/metabolismo , Antibacterianos , Testes de Sensibilidade MicrobianaRESUMO
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.
Assuntos
Doenças do Cão , Equinococose , Echinococcus granulosus , Doenças dos Ovinos , Animais , Cães , Ovinos , Chile/epidemiologia , Raposas , Prevalência , Óvulo , Equinococose/epidemiologia , Equinococose/veterinária , Zoonoses , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Doenças dos Ovinos/epidemiologiaRESUMO
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.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Humanos , Cefalosporinase/genética , Fibrose Cística/microbiologia , Ceftazidima/farmacologia , Infecções por Pseudomonas/microbiologia , Pseudomonas/metabolismo , Testes de Sensibilidade Microbiana , beta-Lactamases/metabolismo , Cefalosporinas/farmacologia , Cefalosporinas/uso terapêutico , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêuticoRESUMO
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.
Assuntos
Ceftazidima , Escherichia coli , Antibacterianos/farmacologia , Ceftazidima/química , Ceftazidima/farmacologia , Cefalosporinas/farmacologia , Escherichia coli/genética , Solventes/farmacologia , beta-Lactamases/metabolismoRESUMO
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.
Assuntos
Cobre , Thermus thermophilus , Cobre/química , Transporte de Elétrons , Ligantes , Oxirredução , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMO
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.
Assuntos
Bactérias , Periplasma , beta-Lactamases , Antibacterianos/química , Bactérias/enzimologia , Bactérias/metabolismo , Carbapenêmicos , Periplasma/enzimologia , Periplasma/metabolismo , beta-Lactamases/químicaRESUMO
Due to their superior tolerability and efficacy, ß-lactams are the most potent and prescribed class of antibiotics in the clinic. The emergence of resistance to those antibiotics, mainly due to the production of bacterial enzymes called ß-lactamases, has been partially solved by the introduction of ß-lactamase inhibitors, which restore the activity of otherwise obsolete molecules. This solution is limited because currently available ß-lactamase inhibitors only work against serine ß-lactamases, whereas metallo-ß-lactamases continue to spread, evolve, and confer resistance to all ß-lactams, including carbapenems. Furthermore, the increased use of antibiotics to treat secondary bacterial pneumonia in severely sick patients with COVID-19 might exacerbate the problem of antimicrobial resistance. In this Personal View, we summarise the main advances accomplished in this area of research, emphasise the main challenges that need to be solved, and the importance of research on inhibitors for metallo-B-lactamases amidst the current pandemic.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana , Saúde Global , Inibidores de beta-Lactamases/uso terapêutico , beta-Lactamas/farmacologia , Bactérias/enzimologia , Bactérias/patogenicidade , COVID-19/complicações , COVID-19/microbiologia , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , HumanosRESUMO
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.