Biology subjects

Butterworth, S.

Publications and source records attributed to Butterworth, S..

9 recordsLinked to original sources

bioRxiv2026

Proteome-wide crosslinking mass spectrometry reveals novel components of essential complexes in Toxoplasma

Protein-protein interactions underpin nearly all cellular processes, yet systematic definition of these networks remains limited outside a few model organisms. As a result, the architectures of essential complexes in many divergent lineages remain poorly characterized. Here we developed a high-coverage crosslinking mass spectrometry framework to map the proteome-wide interactome of the model apicomplexan parasite Toxoplasma gondii. From 29,624 crosslinked peptide pairs, we resolved a network of 2,859 protein-protein interactions that we integrated with structural modeling to resolve interaction interfaces. We identified and validated previously unrecognized components of essential protein complexes, including a structurally distinct ATP synthase subcomplex containing a highly divergent, apicomplexan-specific subunit essential for parasite fitness. Beyond revealing unexpected diversification of core mitochondrial machinery, these findings provide a general strategy to define the molecular architecture of divergent organisms and represent a foundational resource for hypothesis generation, structural inference, and discovery of lineage-specific vulnerabilities in pathogen biology.

microbiology↗

bioRxiv2025

NAMPT activity plays a key role in driving autoimmune processes that characterize type 1 diabetes development in mice

Type 1 diabetes (T1D) is characterised by destruction of pancreatic beta cells by islet-infiltrating cytotoxic lymphocytes, and elevated intra-islet secretion of pro-inflammatory cytokines. However, the underlying pathophysiological mechanisms remain incompletely understood. We hypothesised that abnormal elevation of islet NAD, via activation of NAMPT, plays a key role in driving islet autoimmune processes in T1D. Here, we report that NAMPT inhibition protects against pro-inflammatory cytokine (IL-1{beta}, TNF and IFN{gamma}) mediated beta-cell dysfunction and apoptosis in isolated mouse and human islets. RNAseq revealed that NAMPT inhibition blocked cytokine-mediated gene expression linked to pro-inflammatory responses and leukocyte migration. In vivo, diabetes was induced in CD1 mice via multiple low dose streptozotocin (MLDS) injection. MLDS mice were administered the NAMPT inhibitor FK866 (10 mg/kg; IP) or saline equivalent for 16 days. These experiments demonstrated that NAMPT inhibition improved glycaemic control and beta-cell function and insulin content in MLDS mice. FK866 also reduced proportions of islet-residing TNF-producing CD4+T-cells and F4/80+macrophages, proliferation of spleen-derived CD4+ and CD8+T-cells, and proliferation of islet-derived CD4+T-cells and F4/80+macrophages. Finally, we report that NAMPT inhibition was able to block pro-inflammatory cytokine-mediated migration of cytotoxic CD8+T-cells into isolated islets, using an in vitro transwell platform. This data supports a key immunomodulatory role for NAMPT in islet autoimmunity. NAMPT inhibition may represent a novel therapeutic approach for T1D. The effects of increased NAD levels on islet inflammation require in-depth characterisation, and caution should be exercised with regard to use of NAD boosting supplements, particularly in individuals at risk of developing T1D.

cell biology↗

bioRxiv2024

Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-{gamma}-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While {Delta}MYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues {Delta}MYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell. SIGNIFICANCE STATEMENTPooled CRISPR screens are powerful tools to interrogate gene function in a high-throughput manner. Genes conferring fitness advantages or disadvantages upon disruption can be identified by sequencing. However, in Toxoplasma gondii pooled CRISPR screens in mice, fitness defects for some selected mutants drastically diverge from those observed in single-strain infections. Here, we show that a growth defect of a single Toxoplasma gene deletion mutant is rescued if co-infected with wildtype parasites. These results shine light on Toxoplasmas ability to subvert the host response beyond the infected cell, and highlight an important limitation of pooled CRISPR screens in mice. This limitation is probably encountered in CRISPR screens in general where paracrine effects occur.

microbiology↗

bioRxiv2023

Mapping host-microbe transcriptional interactions by dual perturb-seq

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonisation. Here, we combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA-sequencing to identify the molecular mediators of these transcriptional interactions, a method we term dual perturb-seq. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterised effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify a novel effector, TgSOS1, that is necessary for sustained host STAT6 signalling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a novel tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.

molecular biology↗

bioRxiv2022

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma stimulated human cells

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFN{gamma}-activated human cells. Independent screens were carried out using two Toxoplasma strains which differ in virulence in mice, leading to the identification of effectors required for survival in IFN{gamma}-activated human cells. We identify the secreted protein GRA57 and two other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFN{gamma}-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFN{gamma} in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70 and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that deletion of this trimeric complex renders parasites hypersensitive to remaining ubiquitination, resulting in increased parasite clearance.

microbiology↗

bioRxiv2022

Stable and ancient endocytic structures navigate the complex pellicle of apicomplexan parasites

Apicomplexan parasites have an immense impact on humanity, but their basic cellular processes are often poorly understood. The sites of endocytosis, the conservation of this process with other eukaryotes, and its functions across Apicomplexa are major unanswered questions. Yet endocytosis in Plasmodium is implicated in antimalarial drug failure. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. Moreover, conserved molecular adaptation of this structure is seen in Apicomplexa, including the kelch-domain protein K13 central to malarial drug-resistance. We determine that an essential function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata, likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

cell biology↗

bioRxiv2022

Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.

microbiology↗

bioRxiv2020

A subcellular atlas of Toxoplasma reveals the functional context of the proteome

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to novel, highly specialized cell compartments and structures. These adaptations drive their recognition and non-destructive penetration of hosts cells and the elaborate reengineering of these cells to promote growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty that defines these new cell organizations and their functions. Consequently, half of apicomplexan proteins are unique and uncharacterized, and these cells are, therefore, very poorly understood. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition to these cells and their novel compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

cell biology↗

bioRxiv2018

DeepCDpred: Inter-residue Distance and Contact Predictionfor Improved Prediction of Protein Structure.

Rapid, accurate prediction of protein structure from amino acid sequence would accelerate fields as diverse as drug discovery, synthetic biology and disease diagnosis. Massively improved prediction of protein structures has been driven by improving the prediction of the amino acid residues that contact in their 3D structure. For an average globular protein, around 92% of all residue pairs are non-contacting, therefore accurate prediction of only a small percentage of inter-amino acid distances could increase the number of constraints to guide structure determination. We have trained deep neural networks to predict inter-residue contacts and distances. Distances are predicted with an accuracy better than most contact prediction techniques. Addition of distance constraints improved de novo structure predictions for test sets of 158 protein structures, as compared to using the best contact prediction methods alone. Importantly, usage of distance predictions allows the selection of better models from the structure pool without a need for an external model assessment tool. The results also indicate how the accuracy of distance prediction methods might be improved further.

bioinformatics↗