Biology subjects

Kester, J. C.

Publications and source records attributed to Kester, J. C..

4 recordsLinked to original sources

bioRxiv2026

A mixer tap mechanism in Mycobacterium tuberculosis regulates a critical metabolic node in response to nutrient availability

Mycobacterium tuberculosis persists within the host by exploiting metabolic flexibility underpinned by the tricarboxylic acid (TCA) cycle-glyoxylate shunt junction, where isocitrate dehydrogenase (ICD) and isocitrate lyase (ICL) compete to direct carbon flux between energy production and carbon conservation. Yet, how nutrient availability regulates flux partitioning at this junction is unknown. We show that among the four gatekeeping isoforms of isocitrate lyase (ICL1/2) and isocitrate dehydrogenase (ICD1/2), only icl1 transcription substantially responds to carbon substrate switching. Systematic metabolite screening revealed that pyruvate, oxaloacetate, and glyoxylate are novel allosteric activators of ICD2, while concurrently inhibiting ICL1/2 at their physiological concentrations. To resolve how these regulatory layers integrate, we developed an experimental 1H NMR-based flux assay that combines recombinant enzymes with metabolomics-informed metabolite conditions to quantify flux partitioning under defined states. This systems-level framework reveals that flux partitioning emerges from coordinated balancing of enzyme abundance and protein-metabolite effector combinations that shift in a time-dependent, nutrient-responsive manner. This dynamic, multi-layered regulatory framework, analogous to a mixer-tap mechanism, offers new strategies for therapeutic disruption of M. tuberculosis metabolism.

biochemistry↗

bioRxiv2021

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized envelope biosynthesis. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by other experimental methods. Furthermore, we speculate that some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains each expressing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed fluorescence patterns similar to those of IMD proteins and, consistent with this co-localization, we had previously identified 20 of these using a proteomics approach. Of the nine remaining IMD candidate proteins, three were confirmed to be associated with the IMD while some others appear to be lipid droplet-associated. Taken together, our newly devised strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. ImportanceThe intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis, that likely contribute to the function of the IMD. How can we find more IMD-associated proteins that escaped proteomic detection? Here, as an alternative approach, fluorescence microscope images of 523 proteins were screened to identify IMD-associated proteins. We confirmed the IMD association of previously identified proteins and discovered three additional proteins associated with the IMD. Together, subcellular fractionation, proteomics, and fluorescence microscopy form a robust combination to more rigorously define IMD proteins, which will aid future investigations to decipher the synthesis, maintenance and functions of this membrane domain.

microbiology↗

bioRxiv2019

C. difficile-associated antibiotics prime the host for infection by a microbiome-independent mechanism.

The most clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Though most broad-spectrum antibiotics significantly disrupt the structure of the gut microbiota, only particular ones increase CDAD risk, suggesting additional factors might increase the risk from certain antibiotics. Here we show that commensal-independent effects of antibiotics collectively prime an in vitro germ-free human gut for CDAD. We found a marked loss of mucosal barrier and immune function with CDAD-associated antibiotic pretreatment distinct from pretreatment with an antibiotic unassociated with CDAD, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent host changes in the increased risk of CDAD with specific antibiotics. Our findings are contrary to the previously held belief that antibiotics allow for CDAD solely through disruption of the microbiome. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD, and to impact human tissue culturing protocols.

molecular biology↗

bioRxiv2019

Application of a gut-immune co-culture system for the study of N-glycan-dependent host-pathogen interactions of Campylobacter jejuni

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the Nlinked protein glycosylation (Pgl) pathway in Campylobacter jejuni pathogenicity. The gutimmune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin producing goblet cells, human enterocytes, and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host-pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylationdeficient strain 11168 {Delta}pglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168{Delta}pglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168{Delta}pglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome, is enabled by application of the gut-immune model and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

microbiology↗