Biology subjects

Hagen, S. J.

Publications and source records attributed to Hagen, S. J..

11 recordsLinked to original sources

bioRxiv2024

Enteric glia regulate Paneth cell secretion and intestinal microbial ecology

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions [1]. To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express PLP1 in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

neuroscience↗

bioRxiv2023

Jump-start and push-start: Mutual activation through crosstalk in coupled quorum sensing pathways

Many bacterial species are able to coordinate population-wide phenotypic responses through the exchange of diffusible chemical signals, a behavior known as quorum sensing. A quorum sensing bacterium may employ multiple types of chemical signals and detect them using inter-connected pathways that crosstalk with each other. While there are many hypotheses for the advantages of sensing multiple signals, the prevalence and functional significance of crosstalk between the sensing pathways are much less understood. Here we explore the effect of intra-cellular signal crosstalk on a simple model of a quorum sensing circuit. The model captures key aspects of typical quorum sensing pathways, including detection of multiple signals that crosstalk at the receptor and promoter levels, positive feedback, and hierarchical positioning of sensing pathways. We find that a variety of behaviors can be tuned by modifying crosstalk and feedback strengths. These include activation or inhibition of one output by the non-cognate signal, broadening of dynamic range of the outputs, and the ability of either the upstream or downstream branch to modulate the feedback circuit of the other branch. Our findings show how crosstalk between quorum sensing pathways can be viewed not solely as a detriment to the flow of information but also as a mechanism that enhances the functional range of the full regulatory system: When positive feedback systems are coupled through crosstalk, several new modes of activation or deactivation become possible.

systems biology↗

bioRxiv2023

Therapy Development for Microvillus Inclusion Disease using Patient-derived Enteroids

Microvillus Inclusion Disease (MVID), caused by loss-of-function mutations in the motor protein Myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid-base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex Immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking anti-diarrheal drug, Crofelemer, dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. Inhibition of Notch signaling with the {gamma}-secretase inhibitor, DAPT, recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum- and glucocorticoid-induced protein kinase 2 (SGK2), and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID. Conflict-of-interest statementThe authors have declared that no conflict of interest exists.

cell biology↗

bioRxiv2020

Spatial correlations and distribution of competence gene expression in biofilms of Streptococcus mutans

Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment causes spatial clustering of S. mutans virulence behaviors, by creating microniches where competence and related phenotypes are concentrated. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized, competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. CSP triggers a Poisson-like, spatially random, comX response from a subpopulation of cells that is homogeneously dispersed. XIP elicits a population-wide response. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.

biophysics↗

bioRxiv2020

Thioesterase Superfamily Member 1 Undergoes Stimulus-coupled Reorganization to Regulate Metabolism

In brown adipose tissue, cold exposure promotes thermogenesis, in large part by increasing mitochondrial {beta}-oxidation of lipid droplet-derived fatty acids. This process is suppressed by thioesterase superfamily member 1 (Them1), a long chain fatty acyl-CoA thioesterase that is highly upregulated by cold ambient temperatures. Them1 reduces fatty acid availability for {beta}-oxidation in mitochondria and limits thermogenesis by cellular mechanisms that are not well defined. We show that Them1 regulates metabolism by undergoing marked intracellular conformational changes that occur in response to {beta}-adrenergic stimulation. Mechanistically, Them1 formed puncta that were localized near LD and mitochondria in an immortalized brown adipose cell line. In response to stimulation by norepinephrine, Them1 was phosphorylated by PKC{beta} at S15, which specifically inhibited puncta formation and resulted in a diffuse intracellular localization. This change in Them1 localization also occurred after stimulation in vivo. Puncta formation activated Them1 metabolic activity in vitro, as evidenced by suppression of oxygen consumption following {beta}-adrenergic stimulation. We show by correlative light and electron microscopy that puncta are biomolecular condensates (also known as membraneless organelles) that typically form by phase separation. Them1 contains one intrinsically disordered region at the N-terminus with multiple interacting motifs that is frequently observed in phase-separating proteins. Phosphorylation, which is known to disrupt phase separation and aggregation, results in a diffuse Them1 localization. Our data thus establish that Them1 forms intracellular biomolecular condensates that limit fatty acid oxidation and suppress thermogenesis. During a period of energy demand, the condensates are disrupted by phosphorylation to allow for maximal thermogenesis. The stimulus-coupled reorganization of Them1 thus provides fine-tuning of thermogenesis and energy expenditure.

cell biology↗

bioRxiv2020

Allosteric regulation of Thioesterase Superfamily Member 1 by free fatty acids and lysophosphatidylcholine

Non-shivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally upregulated in brown adipose tissue upon cold exposure and suppresses thermogenesis to conserve energy reserves. Them1 hydrolyzes long-chain fatty acyl-CoAs, preventing their use as fuel for thermogenesis. Them1 contains a C-terminal StAR-related lipid transfer domain (StarD) with unknown ligand or function. By complementary biophysical approaches, we show that StarD binds to long-chain fatty acids, products of Them1s enzymatic reaction, as well lysophosphatidylcholine (LPC), which activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the StarD and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the StarD functions to localize Them1 near lipid droplets. These findings define the role of the StarD as a lipid sensor that allosterically regulates Them1 activity and localization.

biochemistry↗

bioRxiv2019

Carbohydrate and PepO control bimodality in competence development by Streptococcus mutans

In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence signaling peptide (CSP) induces bimodal expression of comX, with only a fraction of cells in the population becoming transformable. Curiously, bimodal comX activation in response to CSP is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but in defined media lacking small peptides CSP induces no response in comX. In addition, growth on certain sugars other than glucose increases the proportion of the population that activates comX in response to CSP, relative to growth on glucose. By investigating the connection between media and bimodal comX expression, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, which is the origin of comX bimodality. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the intracellular XIP/ComS signal. Deletion of pepO eliminates bimodality in comX, leading to a unimodal comX response to CSP in defined and complex media. We also find that CSP upregulates comR in a carbohydrate source-dependent fashion, providing an additional stimulus to the ComRS feedback system. Our data provide mechanistic insight into how CSP regulates the bistable competence circuit and explain the puzzle of growth medium-dependence in S. mutans competence regulation.

biophysics↗

bioRxiv2019

Environmental Triggers of lrgA Expression in Streptococcus mutans

The lrgAB and cidAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, including oxidative stress tolerance, biofilm formation, and autolysis. The regulation of cidAB and lrgAB is still not understood, as these operons show opposite patterns of expression as well as a complex dependence on growth conditions. We have used a microfluidic approach, together with single-cell imaging of a fluorescent gene reporter, to identify with greater precision the cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. lrgA activates very abruptly at stationary phase, with a high degree of synchrony across the population. We find this activation is controlled by a small number of inputs that are sensitive to growth phase: Extracellular pyruvate, glucose, and molecular oxygen. Further, activation of lrgA appears to be self-limiting, so that lrgA is strongly expressed only for a very short interval of time. Consequently, lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies showing that homologs of lrgAB are linked, together with lytST, to the reimport of pyruvate for anaerobic fermentative growth.\n\nImportanceThe function and regulation of cidAB and lrgAB in Streptococcus mutans is not understood, although these operons have been clearly linked to stress responses and they show a complex dependence on environmental inputs and growth phase. Identifying specific environmental cues that trigger activation of lrgAB has been difficult owing to the cells own modification of key inputs such as glucose and oxygen: In S. mutans the lrgAB operon is strongly upregulated at the end of exponential phase, where growth conditions in a bulk culture become poorly defined. Here we have used microfluidics to apply precise control of environmental inputs to S. mutans and identify specific chemical cues that activate lrgAB. We find that rigorously anaerobic conditions and the presence of extracellular pyruvate are sufficient to induce lrgAB expression, suggesting that lrgAB is timed to activate just as pyruvate fermentation becomes favorable.

biophysics↗

bioRxiv2018

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

AbstractEntry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX.\n\nAuthor SummaryHeterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

microbiology↗

bioRxiv2018

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

microbiology↗

bioRxiv2017

Intercellular communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses and ecological competitiveness. One example is activation of natural genetic competence by ComR and the comX-inducing peptide (XIP) in Streptococcus mutans. Although the competence pathway can be activated by addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signal molecule has not been rigorously tested. Co-culture model systems were developed that included a \"sender\" strain that overexpressed the XIP precursor (ComS) and a \"responder\" strain harboring a GFP reporter fusion to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using i) planktonic culture systems, ii) cells suspended in an agarose matrix or iii) cells growing in biofilms. XIP was shown to be freely diffusible and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with endogenously-produced peptide(s) in populations in various environments and physiologic states.\n\nIMPORTANCEThe comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids and addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence and evolution of a ubiquitous human pathogen and related organisms.

microbiology↗