Biology subjects

Balogh, D.

Publications and source records attributed to Balogh, D..

7 recordsLinked to original sources

bioRxiv2023

Antibiotics of the future are prone to resistance in Gram-negative pathogens

Despite the ongoing development of new antibiotics, the future evolution of bacterial resistance may render them ineffective. We demonstrate that antibiotic candidates currently under development are as prone to resistance evolution in Gram-negative pathogens as clinically employed antibiotics. Resistance generally stems from both genomic mutations and the transfer of antibiotic resistance genes from microbiomes associated with humans, both factors carrying equal significance. The molecular mechanisms of resistance overlap with those found in commonly used antibiotics. Therefore, these mechanisms are already present in natural populations of pathogens, indicating that resistance can rapidly emerge through selection of pre-existing bacterial variants. However, certain combinations of antibiotics and bacterial strains are less prone to developing resistance, emphasizing the potential of narrow-spectrum antibacterial therapies that could remain effective. Our comprehensive framework allows for predicting future health risks associated with bacterial resistance to new antibiotics.

microbiology↗

bioRxiv2022

Phase separated ribosome nascent chain complexes paused in translation are capable to continue expression of proteins playing role in genotoxic stress response upon DNA damage

EDTA- and RNase-resistant ribonucleoprotein complexes of arrested ribosomes with protruding nascent polypeptide chains have recently been described in yeast and human cells. These complexes have been termed assemblysomes, a type of soluble condensates distinct from other known granules. Here, we use bioinformatics to identify additional proteins that likely form assemblysomes during translation. We characterize soluble condensates of the DNA helicase Sgs1, one such identified protein and a key player in the repair of DNA double-strand breaks in yeast. We show that paused ribosome-associated nascent chains of Sgs1 in condensates are able to resume translation upon UV irradiation, consistent with the return of mRNA to the ribosome pool. By extending our studies to human cell lines, we found that EDTA-resistant pellets of ribosomes from the human prostate cancer cell line DU145 are sensitive to treatment with 1,6-hexanediol, which is known to dissolve liquid-liquid phase-separated condensates. In addition, transmission electron microscopy shows that 1,6-hexanediol dissolves ring ribosomal structures from the cytoplasm of radioresistant A549 cells while making the cells more sensitive to X-rays. These results suggest that the stress response is based on a conserved mechanism involving the regulated return of phase-separated paused ribosome-nascent chain complexes to translating ribosomes.

molecular biology↗

bioRxiv2021

Proteome-wide landscape of solubility limits in a bacterial cell

Proteins are prone to aggregate when they are expressed above their solubility limits, a phenomenon termed supersaturation. Aggregation may occur as proteins emerge from the ribosome or after they fold and accumulate in the cell, but the relative importance of these two routes remain poorly known. Here, we systematically probed the solubility limits of each Escherichia coli protein upon overexpression using an image-based screen coupled with machine learning. The analysis suggests that competition between folding and aggregation from the unfolded state governs the two aggregation routes. Remarkably, the majority (70%) of insoluble proteins have low supersaturation risks in their unfolded states and rather aggregate after folding. Furthermore, a substantial fraction ([~]35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our proteome-wide study provides empirical insights into the molecular determinants of protein aggregation routes in a bacterial cell.

molecular biology↗

bioRxiv2021

Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation under heat stress

Listeria monocytogenes exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1:1 at heat shock conditions, while almost no complex formation occurred below 4{degrees}C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed L. monocytogenes ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain via whole proteome mass-spectrometry (MS) at 37 {degrees}C and 42 {degrees}C. While {Delta}clpP1 strain only altered the expression of very few proteins, {Delta}clpP2 and {Delta}clpP1/2 strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and thus functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.

biochemistry↗

bioRxiv2020

Improved bacterial recombineering by parallelized protein discovery

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, Multiplex Automated Genome Engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call Serial Enrichment for Efficient Recombineering (SEER). By performing SEER in E. coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

microbiology↗

bioRxiv2019

Cryo-EM structure of the ClpXP protein degradation machinery

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXPs complex mode of action and provides a structural framework for future pharmacological applications.

molecular biology↗

bioRxiv2018

Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

Multi-targeting antibiotics, i.e. single compounds capable to inhibit two or more bacterial targets offer a promising therapeutic strategy, but information on resistance evolution against such drugs is scarce. Gepotidacin is an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an over 2000-fold increment in resistance, while individually none of these mutations affect resistance significantly. Alarmingly, gepotidacin-resistant strains are found to be as virulent as the wild-type K. pneumoniae strain in a murine model, and extensive cross-resistance was demonstrated between gepotidacin and ciprofloxacin, a fluoroquinolone antibiotic widely employed in clinical practice. This suggests that numerous fluoroquinolone-resistant pathogenic isolates carry mutations which would promote the evolution of clinically significant resistance against gepotidacin in the future. We conclude that prolonged antibiotic usage could select for mutations that serve as stepping-stones towards resistance against antimicrobial compounds still under development. More generally, our research indicates that even balanced multi-targeting antibiotics are prone to resistance evolution.

microbiology↗