Biology subjects

Yildiz, M. S.

Publications and source records attributed to Yildiz, M. S..

6 recordsLinked to original sources

bioRxiv2025

Saturation mutagenesis map of generalist versus specialist adaptations of β-lactamase to novel antibiotics

The evolution of {beta}-lactamase proteins is shaped by the need to maintain enzymatic activity against previously prevalent {beta}-lactam antibiotics while expanding substrate range against new classes of antibiotics. Using saturation mutagenesis and sequence-barcoding-based quantification, we comprehensively mapped the response of the fitness landscape of TEM-1 {beta}-lactamase, which evolved against penicillin-class antibiotics, to mutational perturbations against six diverse {beta}-lactam antibiotics. This systematic panel of antibiotic substrates, including representatives from penicillin, cephalosporin, and monobactam classes, allowed us to classify resistance mutations into two categories. Generalist mutations conferred resistance to multiple antibiotics and were consistently restricted to three positions critical for substrate recognition and catalytic function R164, G238, and E240. These substitutions produced broad spectrum resistance through mechanisms such as expansion of the active site and improved substrate accommodation. In contrast, specialist mutations conferred resistance to only a single antibiotic and exhibited much wider positional diversity. Ceftazidime selection yielded the greatest number of distinct specialist mutations, which were frequently found in flexible or peripheral regions including the omega loop. One especially unexpected finding was the identification of the E166P variant. E166 is a catalytic residue required for deacylation during hydrolysis, and substitutions at this site are generally assumed to abolish function. However, E166P conferred a significant increase in ceftazidime resistance despite eliminating activity against penicillins. Molecular dynamics simulations and mutational analysis revealed that the E166P mutant employs an alternative catalytic mechanism, involving residue N132, rather than the canonical pathway. Together, our findings reveal, at the molecular level, how specialist mutations open up a wide range of diverse and idiosyncratic solutions at the expense of generalizability. These insights may inform strategic design of antibiotic administration protocols to systematically lower pathogenic evolvability.

evolutionary biology↗

bioRxiv2025

Higher-order epistasis drives evolutionary unpredictability toward novel antibiotic resistance

The rapid evolution of extended-spectrum {beta}-lactamases (ESBLs) represents a global health threat, undermining the efficacy of {beta}-lactams, the most extensively used antibiotic class. To elucidate the evolutionary dynamics underlying {beta}-lactam resistance, we constructed a comprehensive combinatorial mutant library comprising all 55,296 possible TEM-1 {beta}-lactamase variants integrating 18 clinically observed mutations across 13 key residues. Over eight million empirical fitness measurements were obtained under selection pressure with both a native antibiotic substrate (ampicillin) and a novel antibiotic (aztreonam), establishing the largest experimentally determined fitness landscape for antibiotic resistance to date. Through graph-theoretic and epistatic analyses, we discovered that selection with ampicillin resulted in weak epistasis, with mutants rarely surpassing the fitness of the wild-type enzyme. Conversely, aztreonam selection elicited extensive higher-order epistasis, generating a rugged fitness landscape characterized by increased phenotypic unpredictability. Interpretable machine-learning analyses identified context-dependent epistatic interactions necessary for achieving high-level aztreonam resistance. Further evolutionary statistical analyses, including direct coupling analysis and latent generative landscapes, showed that top-performing TEM-1 variants consistently adhered to conserved epistatic patterns found in naturally occurring {beta}-lactamases. Our findings demonstrate that higher-order epistasis critically shapes fitness landscape ruggedness when enzymes adapt to novel substrates, whereas adaptations to native substrates exhibit predictably smoother landscapes. This integrated experimental and computational framework provides a foundation for predictive evolutionary pharmacology, enabling assessments of newly developed {beta}-lactams or emerging {beta}-lactamase variants for their potential contribution to ESBL evolution. Importantly, incorporating graph-theoretically informed evolutionary constraints can strategically disrupt evolutionary pathways, presenting a viable approach to mitigate the rise of antibiotic resistance.

evolutionary biology↗

bioRxiv2025

Context-dependent evolutionary dynamics toward bacterial survival during sequential antibiotic treatment

To overcome the antibiotic resistance problem, exploiting reproducible evolutionary tradeoffs is considered key for designing evolution-proof antibiotic therapies. Yet the predictability of resistance evolution has been viewed as limited, given the often idiosyncratic genetic trajectories observed in laboratory evolution experiments. To address this, we partially mimicked clinical antibiotic pharmacodynamics by imposing strong selection and evolved Escherichia coli under single or sequential antibiotic treatment. Under single-antibiotic selection, endpoint resistance, persistence, and tolerance phenotypes were reproducible, but populations evolving in parallel frequently followed divergent genetic trajectories. Remarkably, sequential antibiotic use redirected these divergent paths toward genotypic and phenotypic convergence, driven by extinction of resistance-conferring mutations when switching to the next effective antibiotic. Single-cell RNA sequencing demonstrated that evolved cultures contain cells occupying distinct metabolic niches and include more cells in states with lower translational activity and higher expression of toxin-antitoxin genes. These findings provide evolutionary insights to inform clinically effective antibiotic treatment strategies that employ sequential treatment.

microbiology↗

bioRxiv2024

Synergistic and antagonistic drug-drug interactions are prevalent but not conserved across acute myeloid leukemia cell lines

Acute myeloid leukemia (AML) is the most prevalent type of leukemia in adults. Despite advancements in medicine, the standard treatment that utilizes a combination of cytarabine and daunorubicin for AML has remained the same for decades. Combination drug therapies are proven to be an effective way to achieve targeted efficacy while minimizing drug dosage along with the unintended side effects. However, a systematic survey of synergistic potential of drug-drug interactions in the context of AML pathology is currently lacking. Here we examine the interactions between 15 frequently used cancer drugs across distinct AML cell lines and demonstrate that synergistic and antagonistic drug-drug interactions are widespread but not conserved across these cell lines. Notably, enasidenib (AG-221) and venetoclax (ABT-199), recently approved anticancer agents, exhibited the highest counts of synergistic interactions and the fewest antagonistic ones. In contrast, 6-Thioguanine (6-TG), a purine analog, was involved in the highest number of antagonistic interactions. The interactions we report here cannot be attributed solely to the inherent synergistic or antagonistic natures of these three drugs, as each drug we examined was involved in several synergistic or antagonistic interactions in the cell lines we tested. Moreover, we observed that these drug-drug interactions are not conserved across cell lines, suggesting that the success of combination therapies might vary depending on AML genotypes. For instance, we found that a single mutation in the TF1 cell line could dramatically alter drug-drug interactions, even turning synergistic interactions into antagonistic ones, as seen with AG-221 and cladribine A (2CdA). Our findings provide a preclinical survey of the potential synergistic effects revealing the complexity of the problem in vitro. However, the exploitable synergistic regimes in clinical scenarios remain to be explored. We anticipate these results to be an insightful guideline for future clinical studies, aiming to refine chemotherapy regimens and ultimately enhance patient outcomes.

cancer biology↗

bioRxiv2024

Elucidating TolC Protein Dynamics: Structural Shifts Facilitate Efflux Mediated β-lactam Resistance

Efflux-mediated {beta}-lactam resistance represents a significant public health challenge, limiting the efficacy of various {beta}-lactam antibiotics against numerous clinically relevant pathogenic bacteria. Structural and functional analyses have revealed that the efflux protein TolC in several Gram-negative bacteria serves as a conduit for antibiotics, bacteriocins, and phages, affecting bacterial susceptibility and virulence. In this study, we conducted a comprehensive examination of the efflux of {beta}-lactam drugs mediated by TolC, employing extensive experimental and computational analyses. Our computational investigations into the molecular dynamics of drug-free TolC revealed critical unidirectional movements of the trimeric TolC and identified residues significantly involved in TolC opening. To corroborate these findings, we performed a whole-gene-saturation mutagenesis assay, systematically mutating each residue of TolC to 19 other amino acids and measuring the fitness effects of these mutations under {beta}-lactam-induced selection. The {beta}-lactams oxacillin, piperacillin, and carbenicillin were selected for this study because they are effluxed by the AcrAB-TolC complex with varying efficiencies. This approach clarified the similarities and differences in the efflux processes of the three {beta}-lactam antibiotics through the trimeric TolC. Further analysis of TolCs efflux mechanism for these {beta}-lactam antibiotics via steered molecular dynamics simulations revealed the existence of general and drug-specific mechanisms employed by TolC. We identified key positions at the periplasmic entry of TolC whose altered dynamics influence long-range efflux motions as allosteric modulators. Our findings provide valuable insights into the structural dynamics of TolC, establishing a foundation for understanding the key mechanisms behind multidrug resistance and principles for designing new antibiotics and antibiotic derivatives capable of circumventing the bacterial efflux mechanism.

biophysics↗

bioRxiv2023

Susceptible bacteria survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance

In vitro systems have provided great insight into the mechanisms of antibiotic resistance. Yet, in vitro approaches cannot reflect the full complexity of what transpires within a host. As the mammalian gut is host to trillions of resident bacteria and thus a potential breeding ground for antibiotic resistance, we sought to better understand how gut bacteria respond to antibiotic treatment in vivo. Here, we colonized germ-free mice with a genetically barcoded antibiotic pan-susceptible Escherichia coli clinical isolate and then administered the antibiotic cefepime via programmable subcutaneous pumps which allowed for closer emulation of human parenteral antibiotic pharmacokinetics/dynamics. After seven days of antibiotics, we were unable to culture E. coli from feces. We were, however, able to recover barcoded E. coli from harvested gastrointestinal (GI) tissue, despite high GI tract and plasma cefepime concentrations. Strikingly, these E. coli isolates were not resistant to cefepime but had acquired mutations - most notably in the wbaP gene, which encodes an enzyme required for the initiation of the synthesis of the polysaccharide capsule and lipopolysaccharide O antigen - that increased their ability to invade and survive within intestinal cells, including cultured human colonocytes. Further, these E. coli mutants exhibited a persister phenotype when exposed to cefepime, allowing for greater survival to pulses of cefepime treatment when compared to the wildtype strain. Our findings highlight a mechanism by which bacteria in the gastrointestinal tract can adapt to antibiotic treatment by increasing their ability to persist during antibiotic treatment and invade intestinal epithelial cells where antibiotic concentrations are substantially reduced.

microbiology↗