Biology subjects

Freeman, M. J.

Publications and source records attributed to Freeman, M. J..

5 recordsLinked to original sources

bioRxiv2025

Understanding the Role of Pyruvate Dehydrogenase in Listeria monocytogenes Virulence

To survive within restrictive host niches, bacterial pathogens must possess finely tuned physiological adaptations. One such niche inhabited by Listeria monocytogenes (L. monocytogenes) is the host cell cytosol--a compartment characterized by significant barriers to entry, metabolic limitation, and immune surveillance. Previously, we identified L. monocytogenes transposon mutants defective for intracellular survival due to disruptions in key metabolic pathways, including cell wall biosynthesis, menaquinone production, and pyruvate metabolism. One of these mutants mapped to a central component of the pyruvate dehydrogenase (PDH) complex, pdhC::Tn. Notably, this mutant exhibits pronounced survival defects during infection, despite retaining robust growth and survival in nutrient-rich media. We go on to show that disruption of pdhA::Tn and pdhD::Tn similarly led to virulence attenuation during intra-macrophage growth, plaquing assays, and murine infections. Respiro-fermentative metabolic profiling revealed that pdhC::Tn mutants have an altered respiro-fermentative metabolism with more prominent secretion of lactate. Further, unbiased metabolomic profiling revealed a global starvation phenotype with lower levels of upper glycolytic intermediates and TCA cycle intermediates coupled with elevated intra-bacterial levels of pyruvate and lactate. We then demonstrate that PDH mutants are unable to efficiently utilize phosphotransferase (PTS)-dependent carbon sources and that their growth can be rescued using non-PTS-mediated carbon sources such as hexose phosphates. To identify genetic suppressors of PDH deficiency, we performed an EMS mutagenesis screen using fructose--a PTS-transported carbon source--as the sole carbon source. Five suppressors each contained a single independent mutation in the redox sensing regulator rex. Subsequently, we show that loss of rex restores pdhC::Tns ability to consume PTS-mediated carbon sources through the alleviation of fermentative repression. Further, pdhC::Tn suppressor mutants show restored intracellular growth, but not virulence in vivo. Together, these findings indicate that a key defect in PDH mutants is the inability to import and metabolize PTS-dependent carbon sources in the host cytosol. We posit this impairment leads to disruptions in redox balance and a shift in respiro-fermentative metabolism, ultimately contributing to the loss of intracellular fitness and virulence.

microbiology↗

bioRxiv2024

Listeria monocytogenes Requires Phosphotransferase Systems to Facilitate Intracellular Growth and Virulence

The metabolism of bacterial pathogens is exquisitely evolved to support growth and survival in the nutrient-limiting host. Many bacterial pathogens utilize bipartite metabolism to support intracellular growth by splitting carbon utilization between two carbon sources and dividing flux to distinct metabolic needs. For example, previous studies suggest that the professional cytosolic pathogen Listeria monocytogenes (L. monocytogenes) utilizes glycerol and hexose phosphates (e.g. Glucose-6-Phosphate) as catabolic and anabolic carbon sources in the host cytosol, respectively. However, the role of this putative bipartite glycerol and hexose phosphate metabolism in L. monocytogenes virulence has not been fully assessed. Here, we demonstrate that when L. monocytogenes is unable to consume either glycerol ({Delta}glpD/{Delta}golD), hexose phosphates ({Delta}uhpT), or both ({Delta}glpD/{Delta}golD/{Delta}uhpT), it is still able to grow in the host cytosol and is minimally attenuated in vivo suggesting that L. monocytogenes consumes alternative carbon source(s) in the host. An in vitro metabolic screen using BioLogs phenotypic microarrays demonstrated that both WT and PrfA* L. monocytogenes, a strain with constitutive virulence gene expression mimicking cytosolic replication, use phosphotransferase system (PTS) mediated carbon sources. These findings contrast with the existing metabolic model that cytosolic L. monocytogenes expressing PrfA does not use PTS mediated carbon sources. We next demonstrate that two independent and universal phosphocarrier proteins (PtsI [EI] and PtsH [HPr]), essential for the function of all PTS, are critical for intracellular growth and virulence in vivo. Finally, virulence phenotypes of these mutants were additive to mutants unable to consume glycerol and hexose phosphates ({Delta}glpD/{Delta}golD/{Delta}uhpT) in vivo, suggesting that hexose phosphates and glycerol are relevant metabolites in vivo in addition to those derived from PTS. Taken together, these studies indicate that PTS are critical virulence factors for the cytosolic growth and virulence of L. monocytogenes.

microbiology↗

bioRxiv2024

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression in Listeria monocytogenes

C-di-AMP is an essential second messenger in many bacteria but its levels must be regulated. Unregulated c-di-AMP accumulation attenuates the virulence of many bacterial pathogens, including those that do not require c-di-AMP for growth. However, the mechanisms by which c-di-AMP regulates bacterial pathogenesis remain poorly understood. In Listeria monocytogenes, a mutant lacking both c-di-AMP phosphodiesterases, denoted as the {Delta}PDE mutant, accumulates a high c-di-AMP level and is significantly attenuated in the mouse model of systemic infection. All key L. monocytogenes virulence genes are transcriptionally upregulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Our transcriptomic analysis revealed that the {Delta}PDE mutant is significantly impaired for the expression of virulence genes within the PrfA core regulon. Subsequent quantitative gene expression analyses validated this phenotype both at the basal level and upon PrfA activation by GSH. A constitutively active PrfA* variant, PrfA G145S, which mimics the GSH-bound conformation, restores virulence gene expression in {Delta}PDE but only partially rescues virulence defect. Through GSH quantification and uptake assays, we found that the {Delta}PDE strain is significantly depleted for GSH, and that c-di-AMP inhibits GSH uptake. Constitutive expression of gshF (encoding a GSH synthetase) does not restore GSH levels in the {Delta}PDE strain, suggesting that c-di-AMP inhibits GSH synthesis activity or promotes GSH catabolism. Taken together, our data reveals GSH metabolism as another pathway that is regulated by c-di-AMP. C-di-AMP accumulation depletes cytoplasmic GSH levels within L. monocytogenes that leads to impaired virulence program expression. IMPORTANCEC-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy, and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the mechanisms by which c-di-AMP accumulation diminishes bacterial pathogenesis are poorly understood. Such understanding will reveal the molecular function of c-di-AMP, and inform therapeutic development strategies. Here, we identify GSH metabolism as a pathway regulated by c-di-AMP that is pertinent to L. monocytogenes replication in the host. Given the role of GSH as a virulence signal, nutrient, and antioxidant, GSH depletion impairs virulence program expression and likely diminishes host adaptation.

microbiology↗

bioRxiv2020

Dissolved oxygen dynamics reveal biogeochemical tipping points driven by river corridor hydrology

Riverbeds can have an important impact on large-scale fluxes of biogeochemically active solutes in river corridor systems. The hyporheic zone is an important area of both hydrology and biogeochemistry, particularly in a hydropeaked river system where rapid variations in river stage height, hydraulic gradients, and residence times occur. We measured several biogeochemical and hydrological parameters at three different subsurface depths in the riverbed of the Columbia River in Washington state, a complex hydropeaked river system with significant subsurface heterogeneity. During the study, episodes of significant dissolved oxygen (DO) change were observed. The DO signal changes were the most apparent, compared to more modest changes in other biogeochemical markers. While DO is often associated with biological activity, we ultimately found that the notable DO excursions were associated with hydrologic gradients. Here we describe hydrologic perturbations and biogeochemical responses in terms of hydrobiogeochemical regimes. Two different forms of DO response to hydraulic gradient perturbation were observed, defining different hydrobiogeochemical regimes. The system tips abruptly from one regime to the other, exhibiting threshold behavior that is uncaptured in current estimations of cumulative influences of subsurface processes from reach to earth system scales. Plain language summaryThe water in a river is constantly flowing in and out of the rivers bed. While it is in the bed, microbes process environmentally and ecologically important nutrients and other compounds. The exchange of water between the river and the riverbed is largely controlled by the stage of the river. Many processes, both natural and human, can cause rapid changes in the river stage. These rapid stage changes can cause abrupt changes in the riverbeds ability to process nutrients and other compounds. In this study, we use dissolved oxygen as a window into riverbed microbial and chemical processing. We see that relatively rapid changes in river stage can cause abrupt changes in the concentrations of chemicals in the riverbed, indicating fundamental changes in how the riverbed processes are occurring. Understanding these abrupt and fundamental changes is important to our overall understanding of the ecological function of river corridor systems. 3 key pointsO_LIA hydrobiogeochemical regime is defined by the functional form of the relationship of a hydrologic perturbation and biogeochemical marker. C_LIO_LIShort-term hydrologic perturbations drive tipping points between distinct hydrobiogeochemical regimes. C_LIO_LILinear and hysteretic dissolved oxygen dynamics define distinct regimes as observed throughout the hyporheic zone. C_LI

biochemistry↗

bioRxiv2019

Club cell TRPV4 as a damage sensor driving lung allergic inflammation

Airway epithelium is the first body surface to contact inhaled irritants and report danger. We studied how epithelial cells recognize and respond to protease, which is a critical component of many allergens that provoke asthma. In a murine model, the aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp. elicited helper T (Th) cell-dependent lung eosinophilia. Bronchiolar club cells responded rapidly to Alp1 by coordinating the accumulation of allergic immune cells in the lung. Alp1 degraded bronchiolar cell junctions, and club cells within the bronchioles propagated this signal via calcium and calcineurin to incite inflammation. In two human cohorts, we linked fungal sensitization and asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 was also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium and calcineurin to initiate Th cell sensitization.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC=\"FIGDIR/small/773382v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (24K):\norg.highwire.dtl.DTLVardef@c03f95org.highwire.dtl.DTLVardef@18e54b6org.highwire.dtl.DTLVardef@15ba395org.highwire.dtl.DTLVardef@1509e5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗