Biology subjects

Langsley, G.

Publications and source records attributed to Langsley, G..

9 recordsLinked to original sources

bioRxiv2025

Epigenetic landscape of the H3K27me3 mark in macrophages transformed by Theileria annulata

Theileria annulata and T. parva are obligate intracellular parasites that induce bovine leukocyte transformation leading to uncontrolled proliferation and heightened dissemination of infected leukocytes. Early passage T. annulata-transformed macrophages are virulent, but with long-term culture, they become attenuated for dissemination. Loss of dissemination is restored when TGF-{beta}2-mediated signaling is re-established in attenuated macrophages, suggesting that epigenetic changes might be occurring that contribute to the attenuated phenotype. We focused on one of the important repressive histone marks, tri-methylated lysine 27 of histone H3 (H3K27me3) catalysed by the Polycomb Repressive Complex 2 and found that the global level of H3K27me3 is increased in attenuated macrophages. ChIP-seq revealed the genomic distribution of H3K27me3 is heavily remodeled from virulent to attenuated macrophages, where many genes transition from a focal peak of H3K27me3 around the transcription start site to larger chromatin domains. This transition is not paralleled by large-scale transcriptional downregulation. RNA-seq analysis following PRC2 inhibitor treatment reveals that fewer genes are derepressed in attenuated macrophages than in virulent macrophages, suggesting that broader H3K27me3 profiles do not systematically translate into increased gene silencing activity. Our findings shed light on the mechanisms underlying the dysregulation of epigenetic modifications in Theileria-induced leukocyte transformation. Significance statementTropical theileriosis is a major constraint to livestock production worldwide. Infection with Theileria annulata induces the transformation of bovine leukocytes, which subsequently exhibit similarities to human leukemia. Long-term in vitro culture of transformed leukocytes attenuates their tumorigenic properties. The molecular changes underlying this process are currently unclear. We focused on H3K27me3, a key histone tail post-translational modification that regulates chromatin structure and gene expression, and compared its genomic landscapes in virulent, highly disseminating macrophages versus attenuated macrophages with reduced dissemination potential that are used as a live vaccine against tropical theileriosis. Western blot and ChIP-seq revealed higher global levels of H3K27me3 and a striking broadening of its genomic distribution in attenuated macrophages. To our surprise, this reconfiguration is not associated with major changes in gene expression and attenuated cells appear to be less responsive to PRC2 inhibition, suggesting that large-scale broadening of H3K27me3 does not systematically translate into increased PRC2-mediated silencing activity. Despite these changes, H3K27me3 maintains the silencing of key tumor suppressor genes in both virulent and attenuated macrophages. These findings shed light on the interplay between infection, the evolution of oncogenic potential and reconfiguration of the chromatin landscape following attenuation of virulence.

microbiology↗

bioRxiv2022

Theileria annulata histone deacetylase 1 (TaHDAC1) initiates schizont to merozoite stage conversion

A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.

cell biology↗

bioRxiv2020

miR-34c-3p regulates PKA activity independent of cAMP via ablation of PRKAR2B in Theileria annulata-infected leukocytes and Plasmodium falciparum-infected erythrocytes

MicroRNAs (miRNAs) are small non-coding RNAs that can play critical roles in regulating various cellular processes including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of PKA activity in Theileria annulata infection of bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit), as a novel miR-34c-3p target gene and demonstrated how infection-induced up-regulation of miR-34c-3p in leukocytes repressed PRKAR2B expression to increase PKA activity and promote the virulent disseminating tumour phenotype of T. annulata-transformed macrophages. Finally, we demonstrate that miR-34c-3p regulation of prkar2b expression is generalizable, by confirming that Plasmodium falciparum infection of red blood cells also raises intracellular levels of miR-34c-3p and show that this negatively regulates host prkar2b expression so increasing PKA activity. Infection-induced increase in miR-34c-3p levels, therefore, represents a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. Abstract ImportanceTheileria and Plasmodium infections of leukocytes and erythrocytes; respectively, lead to an increase in host cell miR-34c-3p levels and we identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit), as a specific miR-34c-3p target gene. We demonstrate how infection-induced up-regulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity independent of fluxes in cAMP. Thus, in two different host-parasite combinations infection-induced increase in miR-34c-3p represents a novel epigenetic way of increasing in host PKA activity that contributes to the pathology of disease.

cell biology↗

bioRxiv2020

Phosphorylation Dependent Assembly of a 14-3-3 Mediated Signaling Complex During Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/ low K+) out of which 143 were Ca2+-dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using co-immunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multi-protein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+ dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multi-protein complex. Disruption of the multi-protein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood stage growth of malaria parasites. ImportanceInvasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we have used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation dependent multi-protein complex including signaling kinases assembles during the process of invasion. Disruption of this multi-protein complex impairs merozoite invasion of RBCs providing a novel approach for the development of inhibitors to block the growth of blood stage malaria parasites.

microbiology↗

bioRxiv2019

Rab11A regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of DG and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile parasites suggesting that a Rab11A-dependent apically polarized transport of cargo regulates parasite motility.

microbiology↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗

bioRxiv2018

TGF-b2, catalase activity, H2O2 output and metastatic potential of diverse types of tumour

Theileria annulata is a protozoan parasite that infects and transforms bovine macrophages causing a myeloid-leukaemia-like disease called tropical theileriosis. TGF-{beta}2 is highly expressed in many cancer cells and is significantly increased in Theileria-transformed macrophages, as are levels of Reactive Oxygen Species (ROS), notably H2O2. Here, we describe the interplay between TGF-{beta}2 and ROS in cellular transformation. We show that TGF-{beta}2 drives expression of catalase to reduce the amount of H2O2 produced by T. annulata-transformed bovine macrophages, as well as by human lung (A549) and colon cancer (HT-29) cell lines. Theileria-transformed macrophages attenuated for dissemination express less catalase and produce more H2O2, but regain both virulent migratory and matrigel traversal phenotypes when stimulated with TGF-{beta}2, or catalase that reduce H2O2 output. Increased H2O2 output therefore, underpins the aggressive dissemination phenotype of diverse tumour cell types, but in contrast, too much H2O2 can dampen dissemination.

cancer biology↗

bioRxiv2018

Theileria highjacks JNK2 into a complex with the macroschizont GPI-anchored surface protein p104

Theileria is a unique apicomplexan parasite capable of transforming its host cell into a disseminating tumour. Constitutive JNK activity characterizes bovine T and B cells infected with T. parva, and B cells and macrophages infected with T. annulata. Here, we show that T. annulata manipulates JNK activation by recruiting JNK2, and not JNK1, to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. In silico analysis identified 3 potential JNK-binding motifs in the previously characterized GPI-anchored macroschizont surface protein (p104), and we demonstrate here that JNK2 is recruited to the parasite via physical interaction with p104. A cell penetrating peptide harbouring a p104 JNK-binding motif also conserved in T. parva p104 competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Sequestration of JNK2 depended on PKA-mediated phosphorylation of the conserved JNK-binding motif and upon disruption of the p104/JNK2 complex loss of JNK2 resulted in diminished matrigel traversal of T. annulata-transformed macrophages. Loss of JNK2 also resulted in upregulation of small mitochondrial ARF that promoted autophagy consistent with cytosolic sequestration of JNK2 sustaininf not only JNK2, but also nuclear JNK1 levels that combined contribute to both survival and dissemination of Theileria-transformed macrophages.\n\nAuthor SummaryTheileria annulata parasites infect and transform their host bovine leukocytes into tumourlike cells that disseminate throughout infected animals causing a widespread disease called tropical theileriosis. Virulence has been ascribed to the parasites ability to constitutively activate leukocyte c-Jun N-terminal Kinase (JNK) leading to permanent induction of Matrix Metallo-Proteinase 9 (MMP9) that promotes transformed macrophage dissemination. In attenuated live vaccines JNK-mediated AP-1-driven transcriptional activity is reduced so dampening dissemination. However, in leukocytes JNK exists as two isoforms JNK1 and JNK2 and here, we show for the first time that in T. annulata-transformed macrophages they have different subcellular localisations and perform separate functions. Surprisingly, JNK2 associates with the parasite and is not in the nucleus like JNK1. JNK2 is hijacked by the parasite and sequestered in a complex with a macroschizont surface protein called p104. Upon forced complex dissociation JNK2 gets degraded and its loss negatively affects infected macrophage survival and ability to disseminate.

microbiology↗

bioRxiv2018

GZMA and RASGRP1 are novel tumor suppressors that counter dissemination of Theileria annulata-transformed macrophages

Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumors that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria-induced transformation. Dataset comparisons highlighted a small set of genes associated with Theileria-transformed leukocyte dissemination. The roles of Granzyme A (GZMA) and RAS guanyl-releasing protein 1 (RASGRP1) were verified by CRISPR/Cas9-mediated knock-down. Knocking down of GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in Rag2/{gamma}C mice confirming their role as dissemination suppressors in vivo. We further evaluated the roles of GZMA and RASGRP1 in human B-lymphoma cells by comparing the transcriptome of 934 human cancer cell lines to that of Theileria-transformed bovine host cells. We confirmed dampened dissemination potential of human B-lymphoma cells that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and RASGRP1 have a novel tumor suppressor function in both T. annulata-infected bovine host cells and in human B-lymphomas.\n\nSummaryWe compared the transcriptomes of Theileria annulata transformed B-lymphocytes to 934 human cancer cell lines and provide functional evidence for shared tumor suppressor roles for GZMA and RASGRP1 in controlling the dissemination phenotype of both human B lymphomas and Theileria-transformed leukocytes.

molecular biology↗