Biology subjects

Swale, C.

Publications and source records attributed to Swale, C..

7 recordsLinked to original sources

bioRxiv2026

Genome-wide chromatin reset at the onset of leg regeneration in pancrustaceans

Regeneration requires injured tissues to selectively reactivate certain developmental programs while triggering regeneration-specific mechanisms and responding to the immediate consequences of tissue damage. How chromatin states are reorganized during this transition, and whether the underlying sequence of regulatory events is conserved across regenerating animals, remain poorly understood. We address these questions in the two-spotted cricket Gryllus bimaculatus, in which limb regeneration progressively declines during nymphal development and is absent in adults, and extend our analysis to the crustacean Parhyale hawaiensis. We first establish a chromosome-scale genomic and epigenomic framework for the cricket and then follow chromatin and transcriptional changes after limb amputation. Amputation triggers a striking genome-wide shutdown of enhancer-associated chromatin, marked by loss of accessible regulatory regions and depletion of H3K27ac and H3K4me1. This is followed, in regeneration-competent tissue, by selective reopening of a restricted population of regulatory elements and strong induction of the pioneer transcription factor Zelda (GbZelda). The GbZelda locus is unusually marked by both H3K27me3 and H3K9me3 in intact leg tissue; after injury, H3K9me3 is reduced broadly, whereas H3K27me3 is selectively relieved in regenerating nymphs. Finally, analysis of Parhyale reveals the same sequence of chromatin events during appendage regeneration: an initial enhancer shutdown followed by selective regulatory reopening concomitant with Zelda upregulation. Thus, rather than restoring the pre-injury regulatory landscape wholesale, appendage regeneration in these two pancrustacean models proceeds through a conserved sequence of genome-wide chromatin resetting, selective regulatory reopening and activation of a pioneer transcription factor.

developmental biology↗

bioRxiv2025

Mechanistic insights into the activity of Benzoxaboroles against Cryptosporidium parvum: CPSF3 targeting and resistance implications

Benzoxaboroles are emerging as promising treatments against a broad range of protozoan parasites, including Trypanosoma, Leishmania, Plasmodium, Toxoplasma, and Cryptosporidium. We previously demonstrated that the benzoxaborole compound AN3661 inhibits the endonuclease activity of CPSF3 in Cryptosporidium hominis, likely by disrupting pre-mRNA processing, which in turn limits parasite growth. In this study, we further explored its mode of action and found that a specific mutation (Y385N) in CPSF3 of C. parvum confers strong resistance to AN3661. Interestingly, this mutation does not affect the parasites sensitivity to two other benzoxaboroles, AN13762 and AN7973, which are also thought to target CpCPSF3. All three compounds interfered with mRNA processing in C. parvum, consistent with inhibition of CPSF3 complex activity, and showed parasiticidal activity, especially during the late stages of merogony, blocking parasite egress. Two of them also impaired gamogony. In T. gondii, AN7973 remained effective against several strains carrying mutations within TgCPSF3 that confer resistance to AN3661 and AN13762, suggesting that it might represent an alternative chemotype targeting CPSF3 with the potential to overcome resistance. Notably, AN7973 successfully controlled severe infection in susceptible mice challenged with the AN3661-resistant C. parvum strain carrying the CpCPSF3Y385N mutation. Finally, we extended the known antiparasitic spectrum of AN3661 and AN7973 to include Eimeria tenella and Giardia duodenalis, two important pathogens in veterinary and human health. Altogether, our findings refine the understanding of CPSF3-targeting benzoxaboroles, identify alternative chemotypes with the potential to bypass resistance, and support their potential use in combination therapies to delay or prevent the emergence of drug resistance. Author summaryProtozoa are single-celled parasites that cause significant morbidity and mortality worldwide, affecting both humans and animals. The development of new targeted therapies with highly selective compounds requires a detailed understanding of their mode of action and precise interactions with parasite targets. Benzoxaboroles are a potent class of molecules active against Cryptosporidium, with compound AN3661 known to inhibit the endonuclease activity of the Cleavage and Polyadenylation Specificity Factor 3 (CPSF3) in Cryptosporidium hominis. Here, we show that this inhibitory effect is critical during both asexual and sexual developmental stages and that tyrosine at position 385 of C. parvum CPSF3 plays a key role in its inhibition. A mutation at this site confers resistance to AN3661 both in vitro and in vivo, but not to two other benzoxaboroles, AN13762 and AN7973. Notably, all three compounds disrupted pre-mRNA processing in Cryptosporidium, consistent with inhibition of the CPSF3 complex. Using Toxoplasma, a related protozoan that allows more efficient genetic manipulation, we found that none of the six mutations conferring resistance to compounds AN3661 and AN13762 conferred resistance to compound AN7973. AN7973, which strongly inhibits both C. parvum and T. gondii, may therefore represent an alternative chemotype targeting CPSF3. Furthermore, we demonstrated that the antiparasitic spectrum of AN3661and AN7973 extends to include E. tenella and G. duodenalis, two important pathogens in veterinary and human health. Altogether, our results refine the understanding of three CPSF3-targeting benzoxaboroles in Cryptosporidium and identify compound AN7973 as capable of overcoming resistance to AN3661, thereby supporting the rationale for combination therapies to prevent the emergence of drug resistance.

microbiology↗

bioRxiv2025

Hydra domain drives SNF2L multimerization and marks ISWI diversification in parasites

ISWI chromatin remodelers are conserved regulators of nucleosome positioning and chromatin accessibility across eukaryotes, yet their evolutionary diversification is poorly understood. In the apicomplexan parasite Toxoplasma gondii, we identify Hydra, a previously unrecognized globular domain embedded within TgSNF2L, one of two ISWI paralogues. Hydra is structurally unique, lacking homology to any known protein fold, and represents a lineage-specific insertion in an otherwise structurally conserved protein family. Biochemical analyses reveal that the isolated Hydra domain self-assembles into stable oligomers, undergoing reversible equilibrium with its monomeric form. Cryo-electron microscopy analysis reveals discrete globular assemblies, though little consistency could be obtained, suggesting a highly dynamic complex. Deletion of Hydra from full-length TgSNF2L disrupts its intrinsic ability to form higher order oligomers in solution, yielding predominantly monomeric and dimeric species. Functionally, the Hydra-driven multimerization of TgSNF2L modulates its availability for chromatin engagement in response to cell-cycle cues. Hydra thus represents the first reported structural innovation within the ISWI family.

molecular biology↗

bioRxiv2023

GRA47 and GRA72 are Toxoplasma gondii pore-forming proteins that influence small molecule permeability of the parasitophorous vacuole

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identified GRA72 as synthetically lethal with GRA17. Deleting GRA72 produced similar phenotypes to{Delta} gra17 parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion of GRA47 resulted in an aberrant bubble vacuole morphology with reduced small molecule permeability, mirroring the phenotype observed in GRA17 and GRA72 knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, {Delta}gra47 parasites and parasites expressing GRA47 with a histidine mutation had reduced in vitro proliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets against Toxoplasma infections. IMPORTANCEToxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the parasitophorous vacuole membrane (PVM), which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVMs permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47, or those with a histidine mutation, have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against Toxoplasmosis.

microbiology↗

bioRxiv2023

Oxidation-sensitive cysteines drive IL-38 amyloid formation

Cytokines of the interleukin (IL)-1 family are widely expressed in epithelial surfaces, including the epidermis, where they play a key role in the maintenance of barrier integrity and host defense. A recent report associated the IL-1 family member IL-33 with stress granules (SGs) in epithelial cells. Formation of SGs is promoted by the aggregation of proteins harboring low complexity regions (LCRs). In this study, using computational analyses, we predicted the presence of LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR and localized to Ras GTPase-activating protein binding protein 1 (G3BP1) positive SGs, as well as to G3BP1 negative intracellular protein condensates in keratinocytes exposed to oxidative stress (OS). In addition, we identified two highly aggregation-prone amyloid core (AC) motifs in the IL-38 LCR and detected the formation of amyloid IL-38 aggregates in response to OS in cells and in vitro. Disulfide bond mapping, in silico modelling and the analysis of specific cysteine mutants supported a model in which specific oxidation-sensitive cysteines act as redox switches to modify the conformation of IL-38 and thus the surface exposure of its ACs, shuttling it from a soluble state into biomolecular condensates. Finally, the presence of IL-38 granules in human epidermal layers highly exposed to environmental OS suggests that oxidation-induced formation of amyloid aggregates, as a previously unrecognized intrinsic biological property of IL-38, may be physiologically relevant at this epithelial barrier.

cell biology↗

bioRxiv2023

In vitro production of cat-restricted Toxoplasma pre-sexual stages by epigenetic reprogramming

Summary paragraphSexual reproduction of Toxoplasma gondii, which is restricted to the small intestine of felids, is sparsely documented, due to ethical concerns surrounding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1. In this study, we found that transcription factors AP2XII-1 and AP2XI-2, expressed in tachyzoite stage that causes acute toxoplasmosis, can silence genes necessary for merozoites, a developmental stage critical for sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a drastic change in the transcriptional program, promoting a complete transition from tachyzoites to merozoites. Pre-gametes produced in vitro under these conditions are characterized by specific protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit the epigenitors MORC and HDAC31, which in turn restrict the accessibility of chromatin to the transcriptional machinery. Thus, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. This effective in vitro culture of merozoites paves the way to explore Toxoplasma sexual reproduction without the need to infect kittens and has potential for the development of therapeutics to block parasite transmission.

microbiology↗

bioRxiv2021

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning in Toxoplasma

Correct 3end processing of mRNAs is regarded as one of the regulatory cornerstones of gene expression. In a parasite that must answer to the high regulatory requirements of its multi-host life style, there is a great need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report on our findings in T. gondii of an m6A-dependent 3end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. We bring Nanopore direct RNA sequencing-based evidence of the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs. HighlightsO_LIm6A is recognized in apicomplexan and plants by CPSF4, a member of the cleavage and polyadenylation complex machinery. C_LIO_LIThe structural insight behind the specificity of the binding of m6A by the CPSF4 YTH subunit are solved by high resolution crystal structures. C_LIO_LIThe m6A-driven 3end polyadenylation pathway protects transcriptome integrity by restricting transcriptional read-throughs and RNA chimera formation in apicomplexan parasites and plants. C_LI

microbiology↗