Biology subjects

Fisher, T.

Publications and source records attributed to Fisher, T..

10 recordsLinked to original sources

bioRxiv2026

Kingdom-wide Discovery of Type VI Secretion Effectors via AI Uncovers Three New Bacterial Toxins

The type VI secretion system (T6SS) is a molecular harpoon used by 19% of bacteria to inject toxic proteins into microbial or host cells. Despite its widespread distribution, many T6SS effector (T6Es) genes remain unidentified. Here, we developed an XGBoost classifier integrating 42 genetic and biochemical features to identify novel T6Es. Applying our model to 2,559 T6SS-encoding bacterial genomes revealed 19,958 T6E candidates. We validated our computational predictions and confirmed that three effector candidates, having new 3D structures, demonstrated cytotoxicity to bacteria and yeast, mostly in the bacterial periplasm. For two of these, cognate immunity proteins neutralizing toxicity have been validated. T6EC11, a new peptidoglycan degrading effector, quickly leads to spherical E. coli, with inner membrane separation, and gradual peptidoglycan degradation in a unipolar manner, demonstrating an intriguing moon-like structural phenotype. T6EC12, an effector toxic to bacteria and yeast, carries a conserved zinc-binding motif. Mutations the motif markedly reduced toxicity, suggesting that T6EC12 represents a novel metalloprotease. Our study expands the known T6E repertoire, and provides thousands of T6E predictions in all T6SS-encoding bacteria.

microbiology↗

bioRxiv2026

A targeted bispecific TGFBR2 antagonist antibody demonstrates cell selectivity and enhanced potency on human fibroblasts

Beyond its critical roles in development and tissue homeostasis, TGF{beta} signaling promotes key aspects of cancer progression and is a primary driver of fibrosis. Although blocking TGF{beta} signaling has great therapeutic potential for cancer and fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), preclinical and clinical studies revealed that non-specific alteration of the pathway can have severe adverse consequences; therefore, inhibiting TGF{beta} signaling in a cell-type specific manner may avoid systemic toxic effects while preserving potential therapeutic effects. The parasitic helminth Heligmosomoides polygyrus has evolved cell-type-targeted modulators of TGF{beta} signaling. With insights from the development of other targeted signaling modulators and using the worm proteins as a guide, we sought to develop a human-fibroblast-targeted TGFBR2 antagonist. Here, we report mechanistic insights into the targeted worm TGFBR2 antagonist TGM6 and fusion proteins containing the TGM6 targeting domains. We created a bispecific antibody TGFBR2 antagonist that binds PDGFRA as a targeting receptor and demonstrates cell selectivity and enhanced potency in fibroblasts. Our findings suggest a viable path for developing targeted TGF{beta} signaling antagonists as therapeutics for cancer and tissue fibrosis.

cell biology↗

bioRxiv2026

A modular platform for scalable recombinant production of highly toxic bacterial proteins

Bacterial protein toxins constitute a vast and largely untapped reservoir of antimicrobial activities with substantial therapeutic and biotechnological potential. However, their intrinsic toxicity frequently prevents stable recombinant expression in bacterial hosts, creating a major bottleneck for biochemical characterization, structural analysis, and development as antimicrobial agents. Here, we present a modular platform for the scalable recombinant production of highly toxic bacterial proteins based on transient intramolecular toxin neutralization. The strategy covalently links each toxin to its cognate immunity protein, promoting neutralization during biosynthesis while permitting recovery of the native toxin through site-specific proteolytic cleavage. Using this approach, we produced multiple previously intractable polymorphic toxin domains that could not be obtained using conventional inducible expression, toxin-immunity co-expression, or bacterial cell-free systems. We further streamlined the production workflow through intracellular protease-mediated cleavage, reducing the purification process from four steps to two and increasing protein recovery. To address cases in which native immunity proteins were insufficient, we incorporated computational protein design to engineer improved toxin-binding partners, enabling production of an additional toxin that remained refractory to the original platform. Purified toxins retained enzymatic activity following denaturation and refolding, confirming recovery of functional proteins and enabling identification of a previously uncharacterized nuclease activity. Together, these findings establish a scalable and adaptable microbial biotechnology platform for the production of intrinsically toxic proteins. The integration of transient intramolecular neutralization with computational engineering provides a route toward systematic production and characterization of toxic proteins for antimicrobial discovery, structural biology, protein engineering, and future biotechnological applications.

biochemistry↗

bioRxiv2024

R-Spondin Mimetic, SZN-043, Induced Proliferation and Wnt Activity, Two Features Deficient in Human Alcohol-Associated Liver Disease

Liver regeneration is impaired in patients suffering from alcohol-associated liver (ALD) diseases. Wnt ligands and their FZD receptors are dysregulated in diseased livers. R-spondin and their receptors are known to regulate Wnt activity via the stabilization of FZD receptors. Here, we investigated the components of the Wnt and R-Spondin-signaling pathways and their activity in patients with ALD. We found that while hepatocytes retained high levels of differentiation markers such as ASGR1 and ASGR2, the expression of two R-spondin co-receptors, LGR4 and LGR5, and of Wnt target genes, CYP1A2 and others, were strongly reduced. SZN-043, a hepatocyte-targeted R-Spondin mimetic, is a new investigational drug that stimulates the physiological Wnt repair pathway and proliferation of hepatocytes. Here, we show that SZN-043 induced hepatocyte proliferation in all models tested, including humanized mouse livers, a chronic-binge alcohol-induced liver injury, and a CCl4-induced fibrosis mouse model. Altogether, SZN-043 could be beneficial for the treatment of ALD.

pathology↗

bioRxiv2024

Generation of morphologically distinct astrocyte subtypes from human iPSCs reveals a link between glial shape, function and reactivity

Astrocyte morphology in vivo is heterogeneous across different subtypes and dynamically changes in response to various stimuli. However, several questions on the mechanistic links between shape and function remain unanswered. Here, we developed an efficient protocol to generate pure populations of morphologically distinct human astrocytes in vitro, which we used for a systematic analysis of shape-function relationships. We performed a structural, molecular, and functional characterization of these populations and highlighted how their distinct morphologies mirror distinct functional and transcriptional patterns at the population level. We were also able to both correlate gene expression profiles of these morphologically distinct astrocyte subtypes with in vivo astrocytes in the human brain, and to validate our findings with primary isolated murine astrocytes in vitro. Moreover, we show that the observed morphological differences are correlated with changes in key cytoskeletal proteins, which offers a potential link to the observed functional differences. Finally, we demonstrated that different morphological subtypes of astrocytes have distinct reactivity responses to a common stimulus. This study offers a glimpse into the shape-function dynamics of human astrocytes, highlighting potential mechanistic links between cytoskeletal usage and astrocyte function, while also providing tools and datasets that will be useful for further studies into human glial biology in health and disease.

neuroscience↗

bioRxiv2024

Virus Entry is a Major Determinant of HCMV Latency in Monocytes

Human cytomegalovirus (HCMV) infection can result in either productive or latent infection, the latter enabling life-long viral persistence. Monocytes support latent infection but become permissive to productive infection upon differentiation into macrophages. The molecular basis for these differentiation-driven differences has been largely attributed to chromatin-mediated repression of the viral genome. Using metabolic labeling of newly synthesized RNA during the early stages of infection, we observed markedly lower viral transcription in monocytes compared to macrophages. Unbiased comparison of the two cell types revealed that this difference is partly due to reduced viral entry in monocytes: fewer viruses enter, and correspondingly fewer genomes reach the nucleus. Indeed, ectopic expression of known HCMV entry receptors in monocytes enhanced viral entry and consequently facilitated productive infection, demonstrating that these cells can support full replication if entry is efficient. We further identified integrin {beta}3 as a surface protein upregulated upon differentiation that plays an important role in HCMV entry into macrophages, partially accounting for the observed differences in entry efficiency. Finally, we show that cells receiving fewer viral genomes are the ones that establish latent infection and have the capacity to reactivate. Overall, our findings reveal that entry is a previously unrecognized factor contributing to latent infection in monocytes, adding a critical layer to the paradigm of HCMV latency.

microbiology↗

bioRxiv2023

A virally encoded high resolution screen of cytomegalovirus host dependencies

Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins role along the infection cycle, providing a roadmap for in-depth dissection of host-herpesvirus interactions.

microbiology↗

bioRxiv2022

Parsing the role of NSP1 in SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite its urgency, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. SARS-CoV-2 leads to shutoff of cellular protein synthesis and over-expression of nsp1, a central shutoff factor in coronaviruses, inhibits cellular gene translation. However, the diverse molecular mechanisms nsp1 employs as well as its functional importance in infection are still unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant in which nsp1 does not bind ribosomes, we untangle the effects of nsp1. We uncover that nsp1, through inhibition of translation and induction of mRNA degradation, is the main driver of host shutoff during SARS-CoV-2 infection. Furthermore, we find the propagation of nsp1 mutant virus is inhibited specifically in cells with intact interferon (IFN) response as well as in-vivo, in infected hamsters, and this attenuation is associated with stronger induction of type I IFN response. This illustrates that nsp1 shutoff activity has an essential role mainly in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover the central role it plays in SARS-CoV-2 pathogenesis, explicitly through blockage of the IFN response.

microbiology↗

bioRxiv2021

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is unknown. Here we demonstrate that S. pombe Lem2, an NE protein, regulates nuclear exosome-mediated RNA degradation. Lem2 deletion causes accumulation of non-coding RNAs and meiotic transcripts. Indeed, an engineered exosome substrate RNA shows Lem2-dependent localization to the nuclear periphery. Lem2 does not directly bind RNA, but instead physically interacts with the exosome-targeting MTREC complex and promotes RNA recruitment. The Lem2-assisted pathway acts independently of nuclear bodies where exosome factors assemble, revealing that multiple spatially distinct degradation pathways exist. The Lem2 pathway is environmentally responsive: nutrient availability modulates Lem2 regulation of meiotic transcripts. Our data indicate that Lem2 recruits exosome co-factors to the nuclear periphery to coordinate RNA surveillance and regulates transcripts during the mitosis-to-meiosis switch.

molecular biology↗

bioRxiv2019

Discovering precise temporal patterns in large-scale neural recordings through robust and interpretable time warping

Though the temporal precision of neural computation has been studied intensively, a data-driven determination of this precision remains a fundamental challenge. Reproducible spike time patterns may be obscured on single trials by uncontrolled temporal variability in behavior and cognition, or may not even be time locked to measurable signatures in either behavior or local field potentials (LFP). To overcome these challenges, we describe a general-purpose time warping framework that reveals precise spike-time patterns in an unsupervised manner, even when spiking is decoupled from behavior or is temporally stretched across single trials. We demonstrate this method across diverse systems: cued reaching in nonhuman primates, motor sequence production in rats, and olfaction in mice. This approach flexibly uncovers diverse dynamical firing patterns, including pulsatile responses to behavioral events, LFP-aligned oscillatory spiking, and even unanticipated patterns, like 7 Hz oscillations in rat motor cortex that are not time-locked to measured behaviors or LFP.

neuroscience↗