Biology subjects

Cho, B.-K.

Publications and source records attributed to Cho, B.-K..

11 recordsLinked to original sources

bioRxiv2026

Compartmentalized glycolysis powers ATP production in primary cilia and engages mitochondria via the phosphoenolpyruvate cycle

Primary cilia are antenna-like sensory and signaling organelles present on most mammalian cells, including glucose-sensing pancreatic {beta}-cells. Here, we show that the local energetic demands of primary cilia require the ATP-producing enzyme pyruvate kinase, with loss of PKm1, but not PKm2, impairing ciliary glycolytic flux. While the entire glycolytic machinery localizes to cilia, our data indicate that mitochondria are a critical source of phosphoenolpyruvate (PEP), the high-energy glycolytic intermediate that drives the pyruvate kinase reaction. Abolishing PCK2, the mitochondrial enzyme that generates PEP, prevents cilia from sensing not only glucose but also the amino acids glutamine and leucine. Finally, by mislocalizing glycolysis, we demonstrate that primary cilia can utilize ATP generated within the cell body when glucose is limiting. These findings indicate that primary cilia, while possessing the capacity for local ATP generation, leverage a ciliary-mitochondrial signaling axis to meet their bioenergetic needs.

cell biology↗

bioRxiv2026

Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

microbiology↗

bioRxiv2025

Toward Single-Cell Control: Noise-Robust Perfect Adaptation in Biomolecular Systems

Robust perfect adaptation (RPA), whereby a consistent output level is maintained even after a disturbance, is a highly desired feature in biological systems. This property can be achieved at the population average level by combining the well-known antithetic integral feedback (AIF) loop into the target network. However, the AIF controller amplifies the noise of the output level, disrupting the single-cell level regulation of the system output and compromising the conceptual goal of stable output level control. To address this, we introduce a new regulation motif, the noise controller, which is inspired by the AIF loop but differs by sensing the output levels through the dimerization of output species. Combining this noise controller with the AIF controller successfully maintained system output noise as well as mean at their original level, even after the perturbation, thereby achieving noise RPA. Furthermore, our noise controller could reduce the output noise to a desired target value, achieving a Fano factor as small as 1, the commonly recognized lower bound of intrinsic noise in biological systems. Notably, our controller remains effective as long as the combined system is ergodic, making it applicable to a broad range of networks. We demonstrate its utility by combining the noise controller with the DNA repair system of Escherichia coli, which reduced the proportion of cells failing to initiate the DNA damage response. These findings enhance the precision of existing biological controllers, marking a key step toward achieving single-cell level regulation.

systems biology↗

bioRxiv2025

Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and mice

Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function. An appealing platform among these technologies is the auxin-inducible degron (AID) system. Although this system has been applied extensively to cell and animal models, degradation resistance to long-term auxin treatment has not been studied. With the advent of the new AID2 system, cellular toxicity due to the high concentrations of auxin required in the original AID1 system is no longer a concern, making it possible to study protein degradation over extended periods. In this study, we derived multiple miniAID-tagged knock-in human cell lines and a Ctcf-miniAID knock-in mouse strain to investigate mechanisms of degradation resistance. We revealed four independent resistance mechanisms, including a nonsense mutation in the CTCF coding sequence that removed the miniAID peptide, a missense point mutation in the miniAID coding region that disrupted ubiquitin complex targeting, and silencing of the OsTIR1 adaptor protein. Resistance to auxin degradation was also acquired in mouse primary CtcfminiAID/miniAID knock-in B-ALL cells through missense mutations of the OsTIR1(F74G) protein in vivo and ex vivo. In summary, our innovative study expands our understanding of the AID system and cautions careful consideration of design for future applications in mammalian system.

cell biology↗

bioRxiv2025

Outer Membrane Vesicles Secreted by Bacteroides fragilis Inhibit CFTR Chloride Secretion by Human Colon Organoids

The goals of this study were to develop a model to study host pathogen interactions in primary human colon organoids and to test the hypothesis that Bacteroides fragilis toxin (BFT-2) secreted in outer membrane vesicles (OMVs) modulates mucosal immunity and CFTR Cl- secretion. Since Bacteroides species reside in mucus, OMVs are likely to represent a mechanism of communication between Bacteroides and the host. Two strains of Bacteroides were studied, Enterotoxigenic Bacteroides fragilis (ETBF), which produces BFT-2, and the non-toxigenic Bacteroides fragilis strain NCTC 9343 (NTBF) that does not produce BFT-2. We also utilized two additional strains of Bacteroides fragilis: one in which bft-2 was knocked out (ETBF {Delta}bft), and one that was engineered to contain bft-2 (NTBF+bft). We report that Bacteroides fragilis OMVs reduced CFTR Cl- secretion but had no effect on tight junction or cell adhesion proteins, transepithelial resistance (TER) or cytokine secretion by primary human colon organoids. NTBF OMVs containing BFT-2 were more effective in reducing CFTR Cl- secretion than NTBF lacking BFT-2. We conclude that OMVs secreted by Bacteroides can be an important mechanism of host pathogen interactions in the colon by reducing CFTR Cl- secretion.

microbiology↗

bioRxiv2024

Spatial proteomics of Onchocerca volvulus with pleomorphic neoplasms shows local and systemic dysregulation of protein expression

Onchocerca volvulus is the agent of onchocerciasis (river blindness) and targeted by WHO for elimination though mass drug administration with ivermectin. A small percentage of adult worms develop pleomorphic neoplasms (PN) that are positively associated with the frequency of ivermectin treatment. Worms with PN have a lower life expectancy and a better understanding about the proteins expressed in PN, and how PN affect protein expression in different tissues could help to elucidate the mechanisms of macrofilaricidal activity of ivermectin. Within a clinical trial of drug combinations that included ivermectin, we detected 24 (5.6%) O. volvulus females with PN by histology of paraffin embedded nodules. To assess the protein inventory of the neoplasms and to identify proteins that may be associated with tumor development, we used laser capture microdissection and highly sensitive mass spectrometry analysis. Neoplasm tissue from three female worms was analyzed, and compared to normal tissues from the body wall, uterus and intestine from the same worms, and to tissues from three females without PN. The healthy females showed all intact embryogenesis. In PN worms, 151 proteins were detected in the body wall, 215 proteins in the intestine, 47 proteins in the uterus and 1,577 proteins in the neoplasms. Only the uterus of one PN female with some stretched intrauterine microfilariae had an elevated number of proteins (601) detectable, while in the uteri of the healthy females 1,710 proteins were detected. Even in tissues that were not directly affected by PN (intestine, body wall), fewer proteins were detected compared to the corresponding tissue of the healthy controls. Immunolocalization of the calcium binding protein OvDig-1 (OVOC8391) confirmed the detection in PN by mass spectrometry. In conclusion we identified proteins that are potentially linked to the development of PN, and systemic dysregulation of protein expression may contribute to worm mortality. Author summaryOnchocerca volvulus, the causative agent of onchocerciasis (river blindness), is targeted for elimination by WHO. The primary strategy involves mass administration of ivermectin. A small proportion of adult female worms develop pleomorphic neoplasms (PN). Here, we used laser capture microdissection and highly sensitive mass spectrometry analysis to determine the protein inventory of PN to identify proteins that may be associated with tumor development. Neoplasm tissue from female worms was analyzed, and compared to normal tissue from the body wall, uterus and intestine from the same worms, and to tissues from females without PN. When compared, PN and healthy control (HC) worms display a different set of proteins, the PN tissue being the one with the highest number of proteins (1,390). From these, 594 were not present in any HC worm tissue. Despite the large number of proteins identified in PN tissue, their low abundance suggests also in PN dysregulation of protein expression. Immunolocalization of a calcium binding protein detected in PN confirmed the mass spectrometry results. In conclusion, we have developed a system to analyze the proteome of O. volvulus from nodule sections and identified proteins that are potentially linked to the development of PN and may contribute to worm mortality.

molecular biology↗

bioRxiv2022

Zur and Zinc Increase Expression of E. coli Ribosomal Protein L31 Through RNA-Mediated Repression of the Repressor L31p

Bacteria can adapt in response to numerous stress conditions. One such stress condition is zinc depletion. The zinc-sensing transcription factor Zur regulates the way enteric bacteria respond to severe changes in zinc availability. Under zinc sufficient conditions, Zn-loaded Zur (Zn2-Zur) is well-known to repress transcription of genes encoding zinc uptake transporters and paralogues of a few ribosomal subunits. Here, we report the discovery and mechanistic basis for the ability of Zur to up-regulate expression of the ribosomal protein L31 in response to zinc in E. coli. Through genetic mutations and reporter gene assays, we find that Zur achieves the up-regulation of L31 through a double repression cascade by which Zur first represses the transcription of L31p, a zinc-lacking paralogue of L31, which in turn represses the translation of L31. Mutational analyses show that translational repression by L31p requires an RNA hairpin structure within the l31 mRNA and involves the N-terminus of the L31p protein. This work uncovers a new genetic network that allows bacteria to respond to host-induced nutrient limiting conditions through a sophisticated ribosomal protein switching mechanism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/493739v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@6a6281org.highwire.dtl.DTLVardef@6942f7org.highwire.dtl.DTLVardef@ab7ce8org.highwire.dtl.DTLVardef@957d55_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

An rRNA fragment in extracellular vesicles secreted by human airway epithelial cells increases the fluoroquinolone sensitivity of P. aeruginosa

Lung infection by antibiotic resistant strains of Pseudomonas aeruginosa is a well-known concern for immunocompromised hosts including people with lung diseases such as cystic fibrosis. We have previously demonstrated that extracellular vesicles (EVs) secreted by primary human airway epithelial cells (AEC) deliver miRNA let-7b-5p to P. aeruginosa where it suppresses biofilm formation and increases sensitivity to beta-lactam antibiotics. In this study we used RNA-seq to characterize the small RNA (sRNA) content of EVs secreted by AEC and demonstrate transfer of multiple distinct RNA fragments from EVs to P. aeruginosa. Bioinformatic predictions reveal that several sRNAs may target all three subunits of the fluoroquinolone efflux pump MexHI-OpmD, an effect predicted to increase antibiotic sensitivity to fluoroquinolone antibiotics. Exposure of P. aeruginosa to EVs resulted in a significant reduction in the protein levels of MexH (-48%), MexI (-50%) and OpmD (-35%). Moreover, EVs reduced planktonic growth of P. aeruginosa in the presence of the fluoroquinolone antibiotic ciprofloxacin by 20%. A mexGHI-opmD deletion mutant of P. aeruginosa phenocopied this increased sensitivity to ciprofloxacin. Finally, we found that a fragment of an 18S rRNA external transcribed spacer that was transferred to P. aeruginosa by EVs was sufficient to reduce planktonic growth of P. aeruginosa in the presence of ciprofloxacin, to reduce the minimum inhibitory concentration (MIC) of P. aeruginosa for ciprofloxacin by over 50%, and to significantly reduce protein levels of MexH and OpmD. In conclusion, an rRNA fragment secreted by AEC in EVs increases the ciprofloxacin sensitivity of P. aeruginosa by targeting and down-regulating the fluoroquinolone efflux pump MexHI-OpmD. A combination of rRNA fragments and ciprofloxacin packaged in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections. Author SummaryAccording to the World Health Organization and the U.S. Centers for Disease Control and Prevention the development of antibiotic resistant strains of bacteria, including Pseudomonas aeruginosa, are a significant global threat to human health. Thus, development of new approaches to eliminate antibiotic resistant infections is required. In this study, we report that lung epithelial cells secrete extracellular vesicles (EVs) that fuse with and deliver small rRNAs to P. aeruginosa, and that the rRNAs increase the sensitivity of P. aeruginosa to the antibiotic ciprofloxacin by reducing protein levels of the drug efflux pump MexHI-OpmD. We identified one rRNA fragment that by itself significantly reduced the protein levels of MexH and OpmD and increased the ability of ciprofloxacin to kill P. aeruginosa. We propose that developing synthetic vesicles containing a combination of the rRNA that inhibits antibiotic efflux pumps and ciprofloxacin would benefit patients with antibiotic resistant P. aeruginosa infections.

microbiology↗

bioRxiv2021

Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs; 34 of them were found to be DNA-binding protein. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. These TFs were found to have various roles in regulating primary cellular processes in E. coli. Taken together, this study: (1) significantly expands the number of confirmed TFs, close to the estimated total of about 280 TFs; (2) predicts the putative functions of the newly discovered TFs, and (3) confirms the functions of representative TFs through mutant phenotypes.

systems biology↗

bioRxiv2019

A multifaceted cellular damage repair and prevention pathway promotes high level tolerance to β-lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two-component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high-level {beta}-lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that {beta}-lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the {Delta}vxrAB mutant. Mutating major iron uptake systems drastically increased {Delta}vxrAB tolerance to {beta}-lactams. We propose that VxrAB reduces antibiotic-induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against {beta}-lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganisms ability to counteract diverse antibiotic-induced stresses promotes high-level antibiotic tolerance, and highlights the complex secondary responses elicited by antibiotics.

microbiology↗

bioRxiv2018

Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655

Transcriptional regulation enables cells to respond to environmental changes. Yet, among the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, 185 have been experimentally identified and only a few tens of them have been fully characterized by ChIP methods. Understanding the remaining TFs is key to improving our knowledge of the E. coli transcriptional regulatory network (TRN). Here, we developed an integrated workflow for the computational prediction and comprehensive experimental validation of TFs using a suite of genome-wide experiments. We applied this workflow to: 1) identify 16 candidate TFs from over a hundred candidate uncharacterized genes; 2) capture a total of 255 DNA binding peaks for 10 candidate TFs resulting in six high-confidence binding motifs; 3) reconstruct the regulons of these 10 TFs by determining gene expression changes upon deletion of each TF; and 4) determine the regulatory roles of three TFs (YiaJ, YdcI, and YeiE) as regulators of L-ascorbate utilization, proton transfer and acetate metabolism, and iron homeostasis under iron limited condition, respectively. Together, these results demonstrate how this workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs in parallel.

microbiology↗