Biology subjects

Cegelski, L.

Publications and source records attributed to Cegelski, L..

7 recordsLinked to original sources

bioRxiv2026

Capsular polysaccharide can sensitize bacteria to non-antibiotic drugs

Medications administered over long durations, such as phenothiazine antipsychotics, accumulate in the gut at concentrations that affect microbial growth. However, the bacterial features influencing sensitivity to these non-antibiotics remain poorly understood. Bacterial capsular polysaccharides (CPSs) typically confer protection against environmental stressors, including chemical, viral, and immunological pressures within the gut. But their roles under non-antibiotic drug pressure are unknown. Here, we show that the K5 CPS of Escherichia coli Nissle 1917 (EcN) sensitizes it to thioridazine (TDZ) and related antipsychotics. Among a panel of E. coli strains grown in minimal medium, EcN exhibited the highest sensitivity to TDZ. Experimentally evolving EcN under gut-relevant TDZ concentrations selected for resistant populations with convergent variants affecting the CPS locus, and TDZ-resistant clones correspondingly had lower CPS expression than their drug-susceptible counterparts. Genetic, transcriptomic, and phenotypic analyses confirmed that the K5 CPS enhances, rather than mitigates, TDZ sensitivity. These findings demonstrate that canonically protective surface structures can become vulnerabilities under non-antibiotic pharmaceutical pressure. Human medications may therefore inadvertently shape the expression and evolution of bacterial surface structures in the gastrointestinal tract, challenging presumptions of CPS-mediated environmental protection.

microbiology↗

bioRxiv2025

Cross-Molecular Active Learning for the Discovery of Antimicrobial Polyacrylamides

Antimicrobial resistance poses an urgent and increasing threat to global health. The development of new antimicrobials is crucial. Synthetic copolymers are attractive as a potential solution, because they can be produced at scale and designed to mimic antimicrobial peptides and act as broad-spectrum antimicrobials capable of evading resistance mechanisms. This work leverages a cross-molecular machine learning pipeline, trained on antimicrobial peptides, to develop potent antimicrobial polymers to combat Escherichia coli, which were then synthesized and validated experimentally. One candidate copolymer was further characterized and shown to permeabilize the bacterial membrane, which is associated with decreased resistance. Furthermore, this copolymer demonstrated remarkable synergy in eradicating biofilm-associated E. coli when combined with a first-line clinical drug regimen, reducing the amount needed to eradicate bacteria in biofilms by three orders of magnitude. These results demonstrate promise for potentiating antibacterial activity of currently available antibiotics, treating serious and complicated infections, and combatting antimicrobial resistance.

microbiology↗

bioRxiv2024

Polyacrylamide-based Antimicrobial Copolymers Immune to Typical Resistance Mechanisms to Replace or Rescue Antibiotics

Antibiotics save countless lives each year and have dramatically improved human health outcomes since their introduction in the 20th century. Unfortunately, bacteria are now developing resistance to antibiotics at an alarming rate, with many new strains of "superbugs" showing simultaneous resistance to multiple classes of antibiotics. To mitigate the global burden of antimicrobial resistance, we must develop new antibiotics that are broadly effective, safe, and highly stable for the purpose of global access. In this manuscript, we report the development of polyacrylamide-based copolymers as a novel class of broad-spectrum antibiotics with efficacy against several critical pathogens. We demonstrate that these copolymer drugs are selective for bacteria over mammalian cells, indicating a favorable safety profile. We show that they kill bacteria through a membrane disruption mechanism, which allows them to overcome traditional mechanisms of antimicrobial resistance. Finally, we demonstrate their ability to rehabilitate an existing small-molecule antibiotic that is highly subject to resistance development by improving its potency and eliminating the development of resistance in a combination treatment. This work represents a significant step towards combatting antimicrobial resistance.

bioengineering↗

bioRxiv2024

Vibrio cholerae RbmB is an α-1,4-polysaccharide lyase with biofilm-dispersal activity against Vibrio Polysaccharide (VPS)

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, usually including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogen Vibrio cholerae assembles and secretes an exopolysaccharide called VPS (Vibrio polysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved in Vibrio cholerae biofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPS in vitro under physiological conditions. Analysis of the cleavage products using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the -1,4 linkage between D-galactose and a modified L-guluronic acid) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified-guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB from Vibrio cholerae and the related aquatic species Vibrio coralliilyticus are both able to disperse living Vibrio cholerae biofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes. Author summaryBiofilms are protective sheaths produced by many bacteria that play important roles in survival in the environment and in hosts during infection. Understanding how biofilms form and disperse is essential in the fight against harmful bacterial pathogens. Vibrio cholerae is a historically significant human pathogen that forms biofilms made of a polysaccharide called VPS and secreted accessory proteins. Within the V. cholerae biofilm gene cluster is a gene encoding a protein called RbmB, which is known to play a role in biofilm dispersal. Using a combination of biochemical and analytical tools, we show that RbmB indeed cleaves VPS into 4- and 8-monosaccharide fragments at a unique site and demonstrate that it falls into the family of polysaccharide lyase enzymes (as opposed to the more common hydrolases). Furthermore, recombinant RbmB disperses living V. cholerae biofilms further indicating that it may play a role in VPS processing or degradation.

biochemistry↗

bioRxiv2024

Glycogen phase separation drives macromolecular rearrangement and asymmetric division in E. coli

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during transition phase. These asymmetries were coupled with asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and 13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.

microbiology↗

bioRxiv2024

Molecular insights into phosphoethanolamine cellulose formation and secretion

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsCs TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.

biophysics↗

bioRxiv2018

Respiratory heterogeneity shapes biofilm formation and host colonization in uropathogenic Escherichia coli

Biofilms are multicellular bacterial communities encased in a self-secreted extracellular matrix comprised of polysaccharides, proteinaceous fibers, and DNA. Organization of these components lends spatial organization to the biofilm community such that biofilm residents can benefit from the production of common goods, while being protected from exogenous insults. Spatial organization is driven by the presence of chemical gradients, such as oxygen. Here we quantified and localized the expression of two Escherichia coli cytochrome oxidases in cells found in the biofilm state and defined their contribution to biofilm architecture. These studies elucidated a role for the high-affinity quinol oxidase cytochrome bd in matrix production and biofilm resident protection. Cytochrome bd was the most abundantly expressed respiratory complex in the biofilm community and was localized in the biofilm interior. Depletion of the cytochrome bd-expressing subpopulation led to decreased extracellular matrix and increased sensitivity of the community to exogenous stresses. Interrogation of the distribution of cytochrome oxidases in the planktonic state revealed that [~]15% of the population expresses cytochrome bd at atmospheric oxygen concentration, and this population dominates during acute urinary tract infection. These data point towards a bet-hedging mechanism in which heterogeneous expression of respiratory complexes ensures respiratory plasticity of E. coli across diverse host niches.

microbiology↗