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

González, Lisandro J.; Bahr, Guillermo; González, Mariano M.; Vila, Alejandro J.

doi:10.1038/s41589-023-01319-0

Cited by 8 publications

(4 citation statements)

References 55 publications

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“…3 – 5 ). Fast degradation of CAZ is crucial to bacterial fitness, although in vivo resistance could also be affected by other factors, such as in vivo stability, protein production and translocation into the periplasm 32 , 33 . The excellent correlation of the k cat / K M and IC 50 values in the burst phase, however, suggests that the resistance increases are dominated by enhanced burst-phase activity.…”

Section: Resultsmentioning

confidence: 99%

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase

Fröhlich,

Bunzel,

Buda

et al. 2024

Nat Catal

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Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a β-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme’s conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

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Section: Resultsmentioning

confidence: 99%

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase

Fröhlich,

Bunzel,

Buda

et al. 2024

Nat Catal

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“…However, non-metalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. Zn(II)-binding renders NlpI is refractory to degradation by quenching the flexibility of this region (41). Prc, a bacterial periplasmic protease, and its homologues have also been directly demonstrated to be involved in the pathogenesis of Gram-negative bacterial infections.…”

Section: Discussionmentioning

confidence: 99%

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

Mobley,

Anderson,

Moricz

et al. 2024

Preprint

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There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from an unregulated immune response to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Each mutant in each species was used to cochallenge C57BL/6 mice via tail vein injection along with the respective wild-type strain to determine competitive indices for each fitness gene or operon. Among the five species, we found three fitness factor genes, that when mutated, attenuated the mutant for all species in the spleen and liver (tatC, ruvA, gmhB). Nine additional fitness factor genes or operons were validated as outcompeted by wild-type in three or four bacterial species in the spleen (xerC, wzxE, arcA, prc, apaGH, atpG, lpdA, ubiH, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

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“…Interactions with other bacterial proteins can also influence MBL evolu-tion. One study has shown that the availability of Zn(II) exerts evolutionary pressure on MBLs because less ordered conformations of nonmetalated NDM-1 can be recognized by periplasmic proteases, causing many NDM variants to contain hydrophobic mutations that induce rigidity, thereby preventing protease detection [56].…”

Section: Understanding the β-Lactamase Targetsmentioning

confidence: 99%

Drug Discovery in the Field of β-Lactams: An Academic Perspective

Jacobs,

Consol,

Chen

2024

Antibiotics

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β-Lactams are the most widely prescribed class of antibiotics that inhibit penicillin-binding proteins (PBPs), particularly transpeptidases that function in peptidoglycan synthesis. A major mechanism of antibiotic resistance is the production of β-lactamase enzymes, which are capable of hydrolyzing β-lactam antibiotics. There have been many efforts to counter increasing bacterial resistance against β-lactams. These studies have mainly focused on three areas: discovering novel inhibitors against β-lactamases, developing new β-lactams less susceptible to existing resistance mechanisms, and identifying non-β-lactam inhibitors against cell wall transpeptidases. Drug discovery in the β-lactam field has afforded a range of research opportunities for academia. In this review, we summarize the recent new findings on both β-lactamases and cell wall transpeptidases because these two groups of enzymes are evolutionarily and functionally connected. Many efforts to develop new β-lactams have aimed to inhibit both transpeptidases and β-lactamases, while several promising novel β-lactamase inhibitors have shown the potential to be further developed into transpeptidase inhibitors. In addition, the drug discovery progress against each group of enzymes is presented in three aspects: understanding the targets, screening methodology, and new inhibitor chemotypes. This is to offer insights into not only the advancement in this field but also the challenges, opportunities, and resources for future research. In particular, cyclic boronate compounds are now capable of inhibiting all classes of β-lactamases, while the diazabicyclooctane (DBO) series of small molecules has led to not only new β-lactamase inhibitors but potentially a new class of antibiotics by directly targeting PBPs. With the cautiously optimistic successes of a number of new β-lactamase inhibitor chemotypes and many questions remaining to be answered about the structure and function of cell wall transpeptidases, non-β-lactam transpeptidase inhibitors may usher in the next exciting phase of drug discovery in this field.

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

Cited by 8 publications

References 55 publications

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a β-lactamase

Fitness Factor Genes Conserved within the Multi-species Core Genome of Gram-negative Enterobacterales Species Contribute to Bacteremia Pathogenesis

Drug Discovery in the Field of β-Lactams: An Academic Perspective

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