Biology subjects

Koh, A.

Publications and source records attributed to Koh, A..

25 records · Page 2Linked to original sources

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↗

bioRxiv2022

An actin-like filament from Clostridium botulinum exhibits a novel mechanism of filament dynamics

Here, we report the discovery of a ParM protein from Clostridium botulinum (CBgs-ParM), which forms a double-stranded polar filament. CBgs-ParM shares many similarities in its basic filament architecture with actin, however, Pi release after nucleotide hydrolysis induces a large lateral strand shift of ~2.5 nm. We identified the ParR (CBgs-ParR) that acts as a nucleation factor in the initial stage of polymerization, similar to ParR from Escherichia coli plasmid R1. CBgs-ParR also functions as a depolymerization factor, probably by recognizing the structural change in the CBgs-ParM filament after Pi release. Comparison with CBH-ParM, another ParM from Clostridum botulinum, showed that subunit-subunit interacting regions largely differ, preventing co-polymerization, implying a selection pressure in evolution to prevent interference between different ParMRC systems.

biophysics↗

bioRxiv2022

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal anti-tumor immunity

Gut microbiota are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anti-cancer immune responses, however, are largely unknown. Here, we find that ICT induces translocation of specific endogenous gut microbiota into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, gut microbiota activated dendritic cells (DCs) traffic a selective subset of gut bacteria to mesenteric lymph nodes (MLN) and promote optimal anti-tumor T-cell responses in both the tumor draining lymph nodes (TDLN) and the primary tumor. Antibiotic treatment resulted in decreased gut microbiota translocation into MLN and TDLN, diminished polyfunctional effector CD8+ T cell responses, and attenuated response to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anti-cancer immunity. One sentence summaryFollowing immune checkpoint blockade therapy, dendritic cells traffic gut microbiota into secondary lymphoid organs, promoting optimal extraintestinal anti-cancer immunity.

immunology↗

bioRxiv2021

The composition and function of Enterococcus faecalismembrane vesicles

Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, host immune evasion, and cross-species communication. MVs are frequently being discharged from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host-pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe, and functionally characterize MVs from E. faecalis. Here we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane, and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.

microbiology↗

bioRxiv2020

How to make a dial not a switch: Control of histidine kinase conformation by an ABC-transporter facilitates need-based activation of antibiotic resistance

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

microbiology↗

bioRxiv2019

VAMPr: VAriant Mapping and Prediction of antibiotic resistance via explainable features and machine learning

Antimicrobial resistance (AMR) is an increasing threat to public health. Current methods of determining AMR rely on inefficient phenotypic approaches, and there remains incomplete understanding of AMR mechanisms for many pathogen-antimicrobial combinations. Given the rapid, ongoing increase in availability of high density genomic data for a diverse array of bacteria, development of algorithms that could utilize genomic information to predict phenotype could both be useful clinically and assist with discovery of heretofore unrecognized AMR pathways. To facilitate understanding of the connections between DNA variation and phenotypic AMR, we developed a new bioinformatics tool, variant mapping and prediction of antibiotic resistance (VAMPr), to (1) derive gene ortholog-based sequence features for variants; (2) interrogate these explainable gene-level variants for their known or novel associations with AMR; and (3) build accurate models to predict AMR based on whole genome sequencing data. Following the Clinical & Laboratory Standards Institute (CLSI) guidelines, we curated the publicly available sequencing data for 3,393 bacterial isolates from 9 species along with AMR phenotypes for 29 antibiotics. We detected 14,615 variant genotypes and built 93 association and prediction models. The association models confirmed known genetic antibiotic resistance mechanisms, such as blaKPC and carbapenem resistance consistent with the accurate nature of our approach. The prediction models achieved high accuracies (mean accuracy of 91.1% for all antibiotic-pathogen combinations) internally through nested cross validation and were also validated using external clinical datasets. The VAMPr variant detection method, association and prediction models will be valuable tools for AMR research for basic scientists with potential for clinical applicability.

bioinformatics↗

bioRxiv2017

The C-Terminal Domain Of ParB Is Critical For Dynamic DNA Binding And Bridging Interactions Which Condense The Bacterial Centromere

The ParB protein forms DNA bridging interactions around parS to form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring at parS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

biochemistry↗