Biology subjects

Frohlich, C.

Publications and source records attributed to Frohlich, C..

7 recordsLinked to original sources

bioRxiv2026

Mechanistic origins and evolutionary erosion of collateral sensitivity in a β-lactamase

As antibiotic discovery stalls, exploiting collateral sensitivity, where resistance to one drug increases sensitivity to another, offers a promising route to extend the lifespan of existing drugs. However, the molecular origins and robustness of such trade-offs at the level of single resistance determinants remain poorly understood. Here, we examined a previously evolved trajectory of the {beta}-lactamase OXA-48 to Q4 (A33V/F72L/T212A/S213A), which confers a 40-fold increase in ceftazidime resistance when expressed in Escherichia coli. This evolution coincided with the emergence of collateral sensitivity to piperacillin (27-fold reduction), likely caused by the introduction of F72L. We challenged the stability of this collateral sensitivity network by subjecting Q4 to directed evolution under co-selective pressure from both ceftazidime and piperacillin. This selected for the substitution V120G, which alleviated the piperacillin trade-off while maintaining elevated resistance to ceftazidime in genetic backgrounds harboring F72L. Structural and computational analyses revealed that, during evolution from OXA-48 to Q4, F72L introduced substantial conformational changes in the {Omega}-loop, likely leading to less productive piperacillin binding poses. V120G counteracted the effect of F72L by decreasing the {Omega}-loops conformational freedom, thereby partially restoring piperacillin resistance. Finally, we show that other substitutions at position 120 can exert similar mitigating effects. Taken together, our results provide a mechanistic understanding of how adaptive solutions both generate and erode collateral sensitivity, knowledge crucial for predicting the long-term stability of these networks. IMPORTANCEThe evolution of antimicrobial resistance to one drug can also increase bacterial susceptibility to other drugs, a phenomenon known as collateral sensitivity. Understanding the mechanisms underlying these effects may help clinicians design antimicrobial treatment strategies that limit the emergence of resistance. However, for clinical applications, collateral effects must be stable and robust over a certain evolutionary time. While many studies have focused on the stability of collateral effects at the bacterial population level, their stability during the evolution of a single resistance determinant has remained largely unknown. Here, we studied the molecular origins of collateral sensitivity arising during the evolution of the {beta}-lactamase OXA-48 and robustness of this network under co-selection. By linking resistance evolution to changes in protein structure, we illuminate the role of active site loop dynamics in shaping both the collateral effect and its mitigation. Such results are important for understanding collateral sensitivity-based strategies and how they may shape the evolutionary trajectories of resistance enzymes.

molecular biology↗

bioRxiv2025

Evolution tunes functional sub-state interconversion to boost enzyme function

Enzymes do not operate as static structures, but continuously fluctuate between different conformations. Enzymes therefore dynamically sample conformations with varying catalytic activity. However, it remains largely unexplored whether evolution can exploit the conformational dynamics between sub-states to improve activity. Here, we dissect the evolutionary trajectory of the {beta}-lactamase OXA-48 toward improved ceftazidime hydrolysis. Evolution relieved conformational bottlenecks by promoting alternate functional sub-states, gradually shifting the rate-limiting step from substrate binding to sub-state interconversion, and finally to the chemical step. Reorganization of the conformational landscape enhanced OXA-48s ability to hydrolyze ceftazidime and introduced a trade-off in its native activity against meropenem. This trade-off stemmed from catalytic incompatibility between the native and the evolved sub-state populations. Our findings highlight the transitions between functional sub-states as a mechanism of natural selection, shaping functional divergence and offering new strategies for enzyme and antibiotic engineering.

molecular biology↗

bioRxiv2023

The Biofilm Lifestyle Shapes the Evolution of β-Lactamases

The evolutionary relationship between the biofilm lifestyle and antibiotic resistance enzymes remains a subject of limited understanding. Here, we investigate how {beta}-lactamases affect biofilm formation in Vibrio cholerae and how selection for a biofilm lifestyle impacts the evolution of these enzymes. Seven genetically diverse {beta}-lactamases expressed in V. cholerae displayed a strong inhibitory effect on biofilm production, ranging from 17% to 61%. To understand how natural evolution affects this antagonistic pleiotropy under biofilm selecting conditions, we randomly mutagenized one {beta}-lactamase and selected for elevated biofilm formation. Our results revealed that biofilm evolution selects for mutations predominantly clustered around the {beta}-lactamases active site, yielding functional variants still proficient in {beta}-lactam hydrolysis without biofilm inhibition. Mutational analysis of evolved variants demonstrated that restoration of biofilm development could be achieved either independent of enzymatic function or by actively leveraging enzymatic activity to increase biofilm formation. Taken together, the biofilm lifestyle can impose a profound selective pressure on antimicrobial resistance enzymes. Shedding light on such evolutionary interplays is of great importance to understand the various factors driving antimicrobial resistance. Impact statement{beta}-lactamases inhibit biofilm formation and the selection for increased biofilm production can mitigate this antagonistic pleiotropic effect. The emergence of {beta}-lactamase variants avoiding biofilm inhibition strongly suggests that the biofilm lifestyle affects the evolutionary fate of these enzymes.

microbiology↗

bioRxiv2023

Epistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution

The molecular mechanisms by which epistasis boosts enzyme activity remain elusive, undermining our ability to predict the evolution of pathogens and engineer novel biocatalysts. Here, we reveal how directed evolution of a {beta}-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 enzymes conformational dynamics. Kinetic, structural, and dynamical analyses reveal that epistasis was driven by distinct effects of each mutation on the catalytic cycle. The first mutation acquired during evolution increases protein flexibility and accelerates substrate binding, which is rate-limiting in the wild-type enzyme. The ensuing 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 positive synergy boosting enzyme activity.

evolutionary biology↗

bioRxiv2022

Evolution of β-lactamase mediated cefiderocol resistance

Cefiderocol is a novel siderophore {beta}-lactam with improved hydrolytic stability toward {beta}-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by multi-drug resistant Gram-negatives. Using directed evolution on a wide variety of different {beta}-lactamases, such as KPC-2 and CTX-M-15 (Ambler class A), NDM-1 (class B), CMY-2 (class C) and OXA-48 (class D), we studied the role of cefiderocol during {beta}-lactamase-mediated resistance development. First, we investigated how the expression of different {beta}-lactamases causes changes in cefiderocol susceptibility. In a low-copy number vector, we found that OXA-48 and KPC-2 conferred non or marginal decreases in cefiderocol susceptibility, respectively. On the contrary, CMY-2, CTX-M-15 and NDM-1 substantially decreased cefiderocol susceptibility by 16-, 8- and 32-fold, respectively. Second, we determined the evolutionary potential of these enzymes to adapt to increasing concentrations of cefiderocol. Our data show that with the acquisition of only 1 to 2 mutations, all {beta}-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime-avibactam as well as functional trade-offs against different {beta}-lactam drugs. Taken together, contemporary {beta}-lactamases of all Ambler classes can potentially contribute to cefiderocol resistance development and can acquire mutations allowing them to adapt to increasing cefiderocol concentration. At the same time, resistance development caused clinically important cross-resistance, especially against ceftazidime combinations. SummaryDespite the reported higher stability of cefiderocol against {beta}-lactamase hydrolysis, we show that the expression of {beta}-lactamases from different Ambler classes significantly contributes to cefiderocol resistance and that these enzymes have the evolutionary potential to evolve towards increasing cefiderocol concentrations.

molecular biology↗

bioRxiv2020

Cryptic β-lactamase evolution is driven by low β-lactam concentrations

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary {beta}-lactamases is limited. Using the wide-spread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to develop clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-minimum inhibitory concentrations (sub-MIC) of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MIC of ceftazidime, they showed strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies towards ceftazidime. These changes are likely caused by enhanced flexibility of the {Omega}- and {beta}5-{beta}6 loops. In conclusion, low-level concentrations of {beta}-lactams can drive the evolution of {beta}-lactamases through cryptic phenotypes which may act as stepping-stones towards clinical resistance.

evolutionary biology↗

bioRxiv2019

Exploring the Relationship between Geomagnetic Activity and Human Heart Rate Variability

Both geomagnetic and solar activity fluctuate over time and have been proposed to affect human physiology. One physiological measurement that has been previously investigated in this context, heart rate variability (HRV), has substantial health implications regarding the ability to adapt to stressors and has been shown to be altered in many cardiovascular and neurological disorders. Intriguingly, previous work found significant, strong correlations between HRV and geomagnetic/solar activity. In an attempt to replicate these findings, we simultaneously measured HRV from 20 healthy participants during a thirty-day period. In agreement with previous work, we found several significant correlations between HRV and geophysical time-series. However, after correction for autocorrelation, which is inherent in time-series, the only significant results were an increase in very low frequency during higher local geomagnetic activity and a geomagnetic anticipatory decrease in heart rate a day before higher global geomagnetic activity. Both correlations were very low. The loss of most significant effects after this correction suggests that previous findings may be a result of autocorrelation. A further note of caution is required since our and the previous studies in the field do not correct for multiple comparisons given the exploratory analysis strategy. We thus conclude that the effects of geomagnetic and solar activity are (if present) most likely of very small effect size and question the validity of the previous studies given the methodological concerns we have uncovered in our work.

physiology↗