Biology subjects

Ng, W.-L.

Publications and source records attributed to Ng, W.-L..

10 recordsLinked to original sources

bioRxiv2026

Signaling input from the polar flagellum uncouples quorum sensing from cell-density regulation in Vibrio species

Quorum sensing (QS) is traditionally recognized as a signaling mechanism that monitors cell density to coordinate bacterial group behaviors. Although the architecture of many QS pathways and their dependence on cell density are well established, the impact from other physiological cues on QS response is largely unclear. We demonstrate here that flagellar integrity profoundly influences QS responses across multiple Vibrio species. Systematic disruption of flagellar components in Vibrio cholerae revealed that loss of the hook cap protein FlgD induces QS sRNAs Qrr1-4 production independent of cell density and the canonical QS regulator LuxO. This bypass mechanism also overrides normal cell density-dependent regulation of several QS-controlled phenotypes. Notably, {Delta}flgD mutation restores intestinal colonization of {Delta}luxO mutants in an infant mouse model, indicating that flagellar defects reshape V. cholerae QS during infection. This bypass mechanism is evolutionarily conserved as {Delta}flgD mutation induced LuxO-independent qrr expression in multiple Vibrio species. The FlrBC two-component system, essential for flagellar gene expression, is required for the activation of qrr expression in {Delta}flgD mutants. FlgD specifically interacts with the FlrB histidine kinase but not the FlrC response regulator, and a hyperactive FlrC variant is sufficient to drive qrr expression even in cells with FlgD. These findings suggest that not only FlrBC senses cytoplasmic FlgD levels to monitor flagellar completeness to direct flagellar gene expression, this signaling system also functions as a link to bypass the canonical cell-density control of QS by integrating cellular structural information to coordinate group behaviors.

microbiology↗

bioRxiv2025

A Phage Ejectosome Protein Moonlights to Inhibit CBASS Anti-Viral Defense System

A family of prokaryotic antiviral systems collectively known as the cyclic oligonucleotide-based signaling system (CBASS) is found in many bacterial species. Phage defense in CBASS is mediated by the action of a specific effector activated by the cognate cyclic oligonucleotide, often resulting in abortive infection. In the prototypical Type II-A CBASS, phage defense is initiated by the production of cyclic GMP-AMP (cGAMP) by enzymes known as cGAS/DncV-like nucleotidyltransferases (CD-NTases) within the bacterial host, and subsequent activation of the CapV phospholipase by cGAMP leads to cell lysis, thereby limiting phage replication within the host population. Phage proteins that degrade cGAMP, sequester cGAMP, or prevent cGAMP synthesis have been identified in phages to evade CBASS protection. However, some phages are resistant to CBASS protection despite not encoding these proteins, suggesting that additional counter-defense mechanisms exist. To identify new anti-CBASS mechanisms in phages, we exploited bacterial host sensitivity to the antimicrobial compound sulfamethoxazole (SMX) as an indirect read-out for CBASS activity. Bacteria with active CBASS are more sensitive to SMX; therefore, we postulated that any viral proteins produced inside the bacterial host that target CBASS could increase bacterial SMX resistance. Using this screen, we identified that the Gp15 protein from the vibriophage ICP3 increases SMX resistance in Vibrio cholerae El Tor biotype that encodes an active CBASS. Gp15 is an essential virion protein involved in DNA ejection and conserved in many phages. Expression of Gp15 of another coliphage increases SMX resistance in Escherichia coli expressing an active CBASS, and it allows CBASS-sensitive phage to infect the bacterial host with CBASS, demonstrating an important role of Gp15 in phage defense evasion. Unlike other known anti-CBASS proteins, Gp15 does not degrade or sequester cGAMP, and it does not reduce the cellular level of cGAMP. Gp15 also does not interfere with CapV binding to cGAMP. Instead, Gp15 directly inhibits cGAMP-activated CapV serine hydrolase activity in a stoichiometric manner. Together, our study reveals a new mechanism for CBASS antagonism, in which a phage DNA ejection protein moonlights to inhibit a CBASS effector, thereby evading anti-phage defense.

microbiology↗

bioRxiv2025

A small periplasmic protein governs broad physiological adaptations in Vibrio cholerae via regulation of the DbfRS two-component system

Two-component signaling pathways allow bacteria to sense and respond to environmental changes, yet the sensory mechanisms of many remain poorly understood. In the pathogen Vibrio cholerae, the DbfRS two-component system controls the biofilm lifecycle, a critical process for environmental persistence and host colonization. Here, we identified DbfQ, a small periplasmic protein encoded adjacent to dbfRS, as a direct modulator of pathway activity. DbfQ directly binds the sensory domain of the histidine kinase DbfS, shifting it toward phosphatase activity and promoting biofilm dispersal. In contrast, outer membrane perturbations, caused by mutations in lipopolysaccharide biosynthesis genes or membrane-damaging antimicrobials, activate phosphorylation of the response regulator DbfR. Transcriptomic analyses reveal that DbfR phosphorylation leads to broad transcriptional changes spanning genes involved in biofilm formation, central metabolism, peptidoglycan synthesis, and cellular stress responses. Constitutive DbfR phosphorylation imposes severe fitness costs in an infection model, highlighting this pathway as a potential target for anti-infective therapeutics. We find that dbfQRS-like genetic modules are widely present across bacterial phyla, underscoring their broad relevance in bacterial physiology. Collectively, these findings establish DbfQ as a new class of periplasmic regulator that influences two-component signaling and bacterial adaptation.

microbiology↗

bioRxiv2024

Surface remodeling and inversion of cell-matrix interactions underlies community recognition and dispersal in Vibrio cholerae biofilms

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. While it is commonly assumed that biofilm-dwelling cells are glued together by the matrix, how the cell-matrix interaction depends on the specific biochemistry of the matrix components and how this interaction varies during biofilm growth remains unclear. Here, we investigated cell-matrix interactions in Vibrio cholerae (Vc), the causative agent of cholera. We combine genetics, microscopy, simulation, and biochemical tools to show that Vc cells are not attractive to Vibrio polysaccharide (VPS), the main matrix component, but they can be bridged with each other and to the VPS network through crosslinking by Bap1. Downregulation of VPS and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results suggest a new conceptual model in understanding the intricate cell-matrix interaction as the major driver for biofilm development, which is potentially generalizable to certain other biofilm-forming species and exopolysaccharides.

biophysics↗

bioRxiv2023

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules

Protein O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of O-GlcNAcylation homeostasis has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of O-GlcNAcylation challenging. To address this, we have developed first-in-class heterobifunctional small molecules, named O-GlcNAcylation targeting chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2, and EZH2 in cellulo by recruiting O-GlcNAc transferase (OGT), with temporal and magnitude control. Mass spectrometry data revealed that OGTACs induced site-selective O-GlcNAcylation of BRD4. Overall, OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

cell biology↗

bioRxiv2022

The Vibrio cholerae Seventh Pandemic islands act in tandem to defend against a circulating phage

The current circulating pandemic El Tor biotype of Vibrio cholerae has persisted for over sixty years and is characterized by its acquisition of two unique genomic islands called the Vibrio Seventh Pandemic Islands 1 and 2 (VSP-I and VSP-II). However, the functions of most of the genes on VSP-I and VSP-II are unknown and the advantages realized by El Tor through these two islands are not clear. Recent studies have broadly implicated these two mobile genetic elements with phage defense. Still, protection against phage infection through these islands has not been observed directly in any V. cholerae El Tor biotype. Here we report the isolation of a circulating phage from a cholera patient stool sample and demonstrate that propagation of this phage in its native host is inhibited by elements in both VSP-I and VSP-II, providing direct evidence for the role of these genomic islands in phage defense. Moreover, we show that these defense systems are regulated by quorum sensing and active only at certain cell density. Finally, we have isolated a naturally occurring phage variant that is resistant to the defense conferred by the VSP islands, illustrating the countermeasures used by phages to evade these defense mechanisms. Together, this work demonstrates a functional role for the VSPs in V. cholerae and highlights the key regulatory and mechanistic insights that can be gained by studying anti-phage systems in their native contexts. SIGNIFICANCE (AUTHOR SUMMARY)The current pandemic strain of Vibrio cholerae carries two unique genomic islands. How these two islands confer evolutionary advantage to the pathogen is unknown. We show here the identification of a circulating phage that is sensitive to the defense systems present on these two islands and demonstrate how phage variants can evade these defenses. Our studies provide the first direct evidence showing the importance of these genomic islands in defending against phage in their native environments; and in doing so provide novel insight into the mechanisms of these highly conserved defense elements.

microbiology↗

bioRxiv2021

Impact of a human gut microbe on Vibrio cholerae host colonization through biofilm enhancement

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by Vibrio cholerae, the etiological agent of the diarrheal disease cholera. A commensal bacterium, Paracoccus aminovorans, was previously identified in high abundance in stool collected from individuals infected with V. cholerae when compared to stool from uninfected persons. However, if and how P. aminovorans interacts with V. cholerae has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of V. cholerae to affect the disease outcome is unclear. Here we show that P. aminovorans and V. cholerae together form dual-species biofilm structures at the air-liquid interface, with previously uncharacterized novel features. Importantly, the presence of P. aminovorans within the murine small intestine enhances V. cholerae colonization in the same niche that is dependent on the Vibrio exopolysaccharide (VPS) and other major components of mature V. cholerae biofilm. These studies illustrate that dual-species biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections. Significance StatementWhile ample evidence suggests that the outcome of some enteric infections can be affected by the intestinal microbiota, how specific gut microbes change the behaviors of a pathogen is unclear. Here we characterize the interaction between Vibrio cholerae and Paracoccus aminovorans, a gut microbe known to increase in abundance in the intestines during active V. cholerae infection in humans. These two bacteria form a dual-species biofilm structure at the air-liquid interface, and the gut microbe increases the host colonization efficiency of V. cholerae. Importantly, our study identifies a previously unknown mechanism of gut microbe-pathogen interaction that has the potential to alter the disease outcome.

microbiology↗

bioRxiv2020

Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

microbiology↗

bioRxiv2019

Zonation-dependent single-endothelial cell transcriptomic changes in the aged brain

With advances in single-cell genomics, molecular signatures of cells comprising the brain vasculature are revealed in unprecedented detail1,2, yet the ageing-associated cell subtype transcriptomic changes which may contribute to neurovascular dysfunction in neurodegenerative diseases3-7 remain elusive. Here, we performed single-cell transcriptomic profiling of brain endothelial cells (EC) in young adult and aged mice to characterize their ageing-associated genome-wide expression changes. We identified zonation-dependent transcriptomic changes in aged brain EC subtypes, with capillary ECs exhibiting the most transcriptomic alterations. Pathway enrichment analysis revealed altered immune/cytokine signaling in ECs of all vascular segments, while functional changes impacting the blood-brain barrier (BBB) and glucose/energy metabolism were most prominently implicated in ECs of the capillary bed - the primary site where ECs and other neurovascular unit (NVU) cell types closely interact and coordinate to regulate BBB and cerebral blood flow in health and diseased conditions8-17. Furthermore, an overrepresentation of Alzheimers disease (AD)-associated genes identified from GWAS studies was evident among the human orthologs of differentially expressed genes of aged capillary ECs but not other EC subtypes. Importantly, for numerous EC-enriched differentially expressed genes with important functional roles at the BBB and/or association with AD, we found concordant expression changes in human aged or AD brains. Finally, we demonstrated that treatment with exenatide, a glucagon-like peptide-1 receptor (GLP-1R) agonist, strongly reverses transcriptomic changes in ECs and largely reduces BBB leakage in the aged brain. Thus, our study provides insights into detailed transcriptomic alterations underlying brain EC ageing that are complex with subtype specificity yet amenable to pharmacological interventions.

neuroscience↗

bioRxiv2019

Ethanolamine regulates CqsR quorum-sensing signaling in Vibrio cholerae

The pathogen that causes cholera, Vibrio cholerae, uses the cell-cell communication process known as quorum sensing (QS) to regulate virulence factor production and biofilm formation in response to changes in population density and complexity. QS is mediated through the detection of extracellular chemical signals called autoinducers. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified as receptors to activate the key QS regulator LuxO at low cell density. At high cell density, detection of autoinducers by these receptors leads to deactivation of LuxO, resulting in population-wide gene expression changes. While the cognate autoinducers that regulate the activity of CqsS and LuxQ are known, the signals that regulate CqsR have not been determined. Here we show that the common metabolite ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR in vitro and induces the high cell-density QS response through CqsR kinase inhibition in V. cholerae cells. We also identified residues in the CqsR CACHE domain important for ethanolamine detection and signal transduction. Moreover, mutations disrupting endogenous ethanolamine production in V. cholerae delay the onset of, but do not abolish, the high cell-density QS gene expression. Finally, we demonstrate that modulation of CqsR QS response by ethanolamine occurs inside animal hosts. Our findings suggest that V. cholerae uses CqsR as a dual-function receptor to integrate information from the self-made signals as well as exogenous ethanolamine as an environmental cue to modulate QS response.\n\nIMPORTANCEMany bacteria use quorum sensing to regulate cellular processes that are important for their survival and adaptation to different environments. Quorum sensing usually depends on the detection on chemical signals called autoinducers made endogenously by the bacteria. We show here ethanolamine, a common metabolite made by various bacteria and eukaryotes, can modulate the activity of one of the quorum-sensing receptors in Vibrio cholerae, the etiological agent of the disease cholera. Our results raise the possibility that V. cholerae or other quorum-sensing bacteria can combine environmental sensing and quorum sensing to control group behaviors.

microbiology↗