Biology subjects

Demey, L. M.

Publications and source records attributed to Demey, L. M..

5 recordsLinked to original sources

bioRxiv2023

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion

A major challenge faced by bacteria is infection by bacteriophage (phage). Abortive infection is one strategy for combating phage in which an infected cell kills itself to limit phage replication, thus protecting neighboring kin. One class of abortive infection systems is the cyclic oligonucleotide based anti-phage signaling system (CBASS) which relies on two core enzymatic activities; an oligo-nucleotide cyclase that is activated following phage infection and a cyclic-oligo-nucleotide sensitive effector whose activity kills the infected cell. However, the mechanisms behind the deployment and activation of these lethal CBASS systems prior-to and following infection have largely remained a mystery. While exploring unique genomic features of the current pandemic Vibrio cholerae biotype El Tor for clues underlying its pandemic success we found its CBASS was spuriously activated by the folate biosynthesis inhibitor sulfamethoxazole, but only after the population had reached a high-cell density. This population density dependent activity revealed that transcription of both the oligo-nucleotide cyclase, dncV, and the CBASS phospholipase effector, capV, is enhanced at high-cell density by quorum sensing. Together, these results demonstrate that the V. cholerae CBASS is deployed when the environment is densely populated and activated in response to a perturbation in folate biosynthesis. SignificanceTo counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the 7th V. cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

microbiology↗

bioRxiv2022

An essential host dietary fatty acid promotes TcpH inhibition of TcpP proteolysis enabling virulence gene expression in Vibrio cholerae

Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated indirectly by two single-pass membrane-localized transcription regulators (MLTR), ToxR and TcpP, that promote expression of the transcription activator toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. Absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that a dietary fatty acid, -linolenic acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs; also known as lipid rafts) in a mechanism requiring the TcpH transmembrane domain. Taken together our data support a model where V. cholerae cells use exogenous -linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity. Significance StatementV. cholerae continues to pose a significant global burden on health infection millions of people every year resulting in [~]100,000 deaths annually. The importance of toxT gene expression in V. cholerae pathogenesis has been well established. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction, in the presence of -linolenic acid, an essential dietary fatty acid. Here we identify a physiological relevant host factor that stimulates V. cholerae pathogenicity via TcpH-dependent antagonism of TcpP proteolysis.

microbiology↗

bioRxiv2021

Oxidative respiration through the bd-I and cbb3 oxidases is required for Vibrio cholerae pathogenicity and proliferation in vivo.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host. Author SummaryThe bacterium that causes cholera, Vibrio cholerae, can grow with or without oxygen. When growing without oxygen it may use other molecules that serve the same purpose as oxygen, acting as a terminal electron acceptor in an energy-generating process known as respiration. Given the largely anaerobic nature of the gastrointestinal tract, and the lack of significant inflammation during cholera infection, a process that can stimulate elevated levels of oxygen and other terminal electron acceptors, we sought to understand the respiratory mechanisms of V. cholerae during infection. We used a powerful genome-editing method to construct mutant strains of V. cholerae lacking some or all of the complement of proteins required for aerobic or anaerobic respiration. By analyzing these mutants in the laboratory and in intestinal colonization of infant mice, we determined that the ability to respire without oxygen is completely dispensable for V. cholerae to thrive during infection. We determined that two of the four oxygen-dependent respiration mechanisms are essential for V. cholerae to grow during infection, with the other two dispensable for wild type levels of colonization.

microbiology↗

bioRxiv2021

Independent Promoter Recognition by TcpP Precedes Cooperative Promoter Activation by TcpP and ToxR

Cholera is a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. To reach the surface of intestinal epithelial cells, proliferate, and cause disease, V. cholerae tightly regulates the production of virulence factors such as cholera toxin (ctxAB) and the toxin co-regulated pilus (tcpA-F). ToxT is directly responsible for regulating these major virulence factors while TcpP and ToxR indirectly regulate virulence factor production by stimulating toxT expression. TcpP and ToxR are membrane-localized transcription activators (MLTAs) required to activate toxT expression. To gain a deeper understanding of how MLTAs identify promoter DNA while in the membrane, we tracked the dynamics of single TcpP-PAmCherry molecules in live cells using photoactivated localization microscopy and identified heterogeneous diffusion patterns. Our results provide evidence that: 1) TcpP exists in three biophysical states (fast diffusion, intermediate diffusion, and slow diffusion); 2) TcpP transitions between these different diffusion states; 3) TcpP molecules in the slow diffusion state are interacting with the toxT promoter; and 4) ToxR is not essential for TcpP to localize the toxT promoter. These data refine the current model of cooperativity between TcpP and ToxR in stimulating toxT expression and demonstrate that TcpP locates the toxT promoter independent of ToxR.

microbiology↗

bioRxiv2019

SMAUG: Analyzing single-molecule tracks with nonparametric Bayesian statistics

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory (SPT) dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of SPT datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.\n\nStatement of SignificanceSuper-resolution microscopy allows researchers access to the motions of individual molecules inside living cells. However, due to experimental constraints and unknown interactions between molecules, rigorous conclusions cannot always be made from the resulting datasets when model fitting is used. SMAUG (Single-Molecule Analysis by Unsupervised Gibbs sampling) is an algorithm that uses Bayesian statistical methods to uncover the underlying behavior masked by noisy datasets. This paper outlines the theory behind the SMAUG approach, discusses its implementation, and then uses simulated data and simple experimental systems to show the efficacy of the SMAUG algorithm. Finally, this paper applies the SMAUG method to two model living cellular systems--one bacterial and one mammalian--and reports the dynamics of important membrane proteins to demonstrate the usefulness of SMAUG to a variety of systems.

biophysics↗