Biology subjects

McFadden, M.

Publications and source records attributed to McFadden, M..

6 recordsLinked to original sources

bioRxiv2026

Salmonella Typhi asparaginase-dependent activation of GCN2 promotes bacterial killing in murine macrophages

Many intracellular pathogens stimulate host cell stress by directly or indirectly causing an imbalance in host nutrients; depletion of amino acid pools in particular can act as a danger signal to infected cells. Using a restrictive host model of Salmonella enterica serovar Typhi (S. Typhi) infection, we identify early induction the integrated stress response (ISR) by viable bacteria, but not heat-killed bacteria. Genetic deletion of the amino acid sensing ISR kinase GCN2 (also known as EIF2AK4) prevented early ISR activation during S. Typhi infection, and murine macrophages lacking GCN2 show impaired bacterial clearance and decreased cytokine output. Supplementation of wildtype C57BL/6 murine macrophages with only the non-essential amino acid asparagine was sufficient to suppress S. Typhi-induced ISR activation and deletion of S. Typhi ansB, encoding an asparaginase, prevented ISR activation during infection. Pharmacological inhibition of mammalian target of rapamycin (mTOR), the other major amino acid sensing pathway in eukaryotic cells, prevented GCN2 activation and ISR induction in murine macrophages, indicating an upstream role for mTOR in signaling to GCN2. These findings suggest a role for the ISR in macrophage innate immune responses to S. Typhi infection and highlight a potential difference in nutrient-dependent signaling between the S. Typhi-susceptible human host and the restrictive murine host centered around asparagine, mTOR, and GCN2.

immunology↗

bioRxiv2025

Genetic landscape of DNMT3A R882H reveals mechanism of aberrant oligomerization

DNA methyltransferase 3A (DNMT3A) is a de novo DNA methyltransferase that is recurrently mutated in hematological malignancies and developmental disorders. The most prevalent mutation, R882H, compromises DNMT3A activity in a dominant-negative manner, but its precise biochemical mechanism has been debated. Here, we use paired deep mutational scanning of the wild-type and R882H-mutant proteins to systematically identify mutations on a massively parallel scale that modify DNMT3A activity by suppressing, phenocopying, or selectively rescuing the dominant-negative effect of R882H. By leveraging the mutational depth and unbiased nature of the paired genetic landscapes, we uncover two distinct mechanisms that can rescue DNMT3AR882H activity, providing novel insights into the function of the R882 hotspot. First, by analyzing the effects of combinatorial mutations in the target recognition domain (TRD), we reveal that its crosstalk with the ADD regulatory domain modulates DNMT3A DNA binding and enzymatic activity, partially compensating for R882H-induced loss-of-function. Second, pairwise analysis of variant effects across the two genetic backgrounds supports the notion that R882H promotes aberrant macro-oligomerization of DNMT3A via its central dimerization interface, which accounts for its dominant-negative effect. Critically, we show that the R882 position exhibits a distinct dominant-negative signature in the genetic landscape, where positively charged residues at this position safeguard against aberrant macro-oligomerization. By performing hydrogen-deuterium exchange mass spectrometry (HDX-MS) on DNMT3A mutants, we show that R882H dramatically alters the protein dynamics of DNMT3A, rigidifying its central dimerization interface to promote oligomerization. Our data support a new model in which R882H removes the critical function of R882, where the arginine attenuates the pre-organization of the interface and subsequent oligomerization at a supramolecular assembly hotspot1. Altogether, we map the genetic landscape underpinning the DNMT3AR882H hotspot mutation, illuminating the unexpected molecular mechanism of macro-oligomerization that drives its dominant-negative effect.

biochemistry↗

bioRxiv2025

Cardioprotective effects of AMPK activation in H1N1 influenza virus infection

Cardiac complications are among the most common and severe extrapulmonary manifestations of influenza virus infection, yet they are rarely recapitulated in mouse models without immunodeficiency. We found that influenza virus A/California/04/2009 (H1N1) carrying a mouse-adaptive amino acid substitution in the PB2 protein (E158A) disseminates to the heart in WT C57BL/6 mice, where it induces inflammation, electrical dysfunction, and fibrotic remodeling. Influenza virus-infected heart tissue was significantly altered in mitochondrial metabolism, extracellular matrix, circadian rhythm, and immunity pathways. Particularly striking was activation of gene expression downstream of the mitochondrial biogenesis-promoting AMPK/PGC-1 axis, which occurred late in infection but failed to reverse the repression of mitochondria-associated genes, suggesting an insufficient or delayed compensatory response. Accordingly, we administered AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) early in infection and observed restoration of mitochondria-associated gene levels, amelioration of cardiac electrical dysfunction and fibrosis, and improvement in survival without overt effects on lung function. Overall, the advent of an immunocompetent model for severe influenza-associated cardiac dysfunction revealed activation of AMPK signaling as a host-targeted metabolic intervention for mitigating virus-induced heart pathologies

microbiology↗

bioRxiv2025

Gasdermin E is Dispensable for H1N1 Influenza Virus Pathogenesis in Mice

Targeting cell death pathways, including pyroptosis and necroptosis, has been shown to mitigate influenza virus infection severity. Here, we examined whether pyroptosis specifically driven by the pore-forming protein gasdermin E (GSDME) is involved in regulating influenza virus infection outcomes. We found that Gsdme-/- mice showed similar weight loss and survival in severe A/PR/8/34 (H1N1) virus infections compared to WT counterparts. Likewise, lung dysfunction, histopathological damage, viral titers, and inflammatory cytokine levels were similar in the two groups. Global transcriptomic analysis also revealed similar inflammatory and antiviral gene expression programs in WT versus Gsdme-/- mouse lungs at baseline and in response to infection. To confirm the generality of these findings, we infected mice with 2009 pandemic H1N1 virus and again observed similar weight loss, mortality, and lung dysfunction in WT and Gsdme-/- mice. Our results overall demonstrate that GSDME contributes negligibly to the host response against H1N1 influenza virus, refining our understanding of cell death pathways in influenza pathogenesis.

immunology↗

bioRxiv2024

Exposure to low doses of Batrachochytrium dendrobatidis reveals variation in resistance in the Critically Endangered southern corroboree frog

Chytridiomycosis poses a significant extinction threat to many amphibians, including the critically endangered southern corroboree frog (Pseudophryne corroboree). Captive breeding programs have become essential to maintain populations while effective long-term conservation strategies are developed. Understanding the variation in susceptibility to chytridiomycosis within this species is essential in exploring the potential for selective breeding to enhance disease resistance. In this study, we conducted a large-scale Batrachochytrium dendrobatidis (Bd) exposure experiment involving 972 juvenile P. corroboree selected to ensure a broad genetic representation of the species. Three replicate experiments were conducted under uniform conditions, to assess individual susceptibility and compare results across replicate experiments. Significant variation was observed within and between experiments, with individual survival rates ranging from 44-74% across experiments, influenced notably by the zoo in which frogs were bred. Remarkably, 21-47% of exposed frogs remained Bd-negative, suggesting potential innate resistance. Infection intensity correlated positively with body condition, in one experiment, while age and size showed inconsistent effects on survival and infection rates across experiments, but younger and smaller frogs were more susceptible to infection and had lower survival. Among frogs that became infected, none cleared infection, with most progressing to terminal stages within an average of 69 days (ranging from 33 to 97 days). However, a few individuals maintained stable infection loads without displaying clinical signs of chytridiomycosis. This observed phenotypic variation in P. corroboree responses to Bd highlights the potential for selective breeding to improve survival outcomes in this species. The dataset generated from this study will be instrumental in guiding breeding strategies that strengthen conservation efforts for this critically endangered species.

zoology↗

bioRxiv2019

A metabolic pathway for bile acid dehydroxylation by the gut microbiome

The gut microbiota synthesize hundreds of molecules, many of which are known to impact host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at ~500 M and are known to block C. difficile growth1, promote hepatocellular carcinoma2, and modulate host metabolism via the GPCR TGR53. More broadly, DCA, LCA and their derivatives are a major component of the recirculating bile acid pool4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Despite the clear impact of DCA and LCA on host physiology, incomplete knowledge of their biosynthetic genes and a lack of genetic tools in their native producer limit our ability to modulate secondary bile acid levels in the host. Here, we complete the pathway to DCA/LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the 8-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a non-producing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool, and provide a road map for deorphaning and engineering pathways from the microbiome as a critical step toward controlling the metabolic output of the gut microbiota.

microbiology↗