Biology subjects

Boyle, J. P.

Publications and source records attributed to Boyle, J. P..

12 recordsLinked to original sources

bioRxiv2025

A single-cell atlas of Toxoplasma sexual development identifies regulators of gametogenesis

Toxoplasma gondii undergoes sexual development exclusively in the feline intestine, a process critical for genetic diversity and population expansion. Recent studies have identified genes critical in suppressing presexual development and metabolic differences in felines that may promote sexual development, but to date the gene regulatory networks driving development in the cat are unknown. To investigate this, we performed single-cell transcriptomics on parasites isolated from cat intestines, using fluorescent reporter strains and flow cytometry. From 15,068 cells across two experiments, we identified rare populations, including cells that bear all of the hallmarks of gametes. Candidate genes emerging from this study were tested via CRISPR-Cas9 Perturb-seq, identifying AP2X6 as a regulator of macrogametocyte development. Our single-cell data extends what is known about gene expression changes throughout sexual development and should be useful to those in the field working towards inducing gametogenesis, mating, and oocyst production in vitro.

microbiology↗

bioRxiv2024

Toxoplasma gondii infection misdirects placental trophoblast lineage specification

Pregnancy is a critical point of vulnerability to infection and other insults that could compromise proper fetal development. The placenta acts as a protective and nutrient-permeable barrier to most infectious agents, but a few are capable of bypassing its defenses. Remarkably little is known about how exposure to these select pathogens might impact ongoing placental development. Here we demonstrate that Toxoplasma gondii entirely misdirects the developmental program of trophoblast stem cells. Infection of progenitor cytotrophoblasts prevents fusion and differentiation to infection-resistant syncytiotrophoblast. Rather, T. gondii elicits a unique transcriptional identity that polarizes cytotrophoblasts to the infection-permissive extravillous trophoblast fate. Strong evidence of developmental disruption is found in multiple orthogonal models, including trophoblast stem cells, trophoblast organoids, and chorionic villi. Manipulation of cell fate by the parasite is most dramatic in trophoblast organoids, where we see robust outgrowth of HLA-G(+) extravillous trophoblasts. Collectively, these data show that Toxoplasma antagonizes differentiation of an infection-resistant cell type by inducing formation of an infection-permissive cell type, therefore potentiating its own transmission to the fetus.

developmental biology↗

bioRxiv2023

Human trophoblast stem cells can be used to model placental susceptibility to Toxoplasma gondii and highlight the critical importance of the trophoblast cell surface in pathogen resistance

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance to understand these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by TEM and SEM, a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYT were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.

microbiology↗

bioRxiv2021

ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii reveals novel virulence factors implicated in host cell invasion

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen with significant impact in children and during pregnancy. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we utilize a biochemical fractionation strategy coupled with mass spectrometry to predict interactions by correlation profiling. Key to this approach is the integration of additional datasets based on gene co-expression as well as phylogenetic profiles that eliminate poorly supported interactions and reduce the number of false positive interactions. In addition to a supervised machine learning strategy, we employed an unsupervised approach in data integration, based on similarity network fusion, to overcome the deficit of high-quality training data in non-model organisms. The resulting high confidence network, we term ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering of this network identifies 93 protein complexes, predicting both novel complexes as well as new components for previously known complexes. In particular, we identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.

microbiology↗

bioRxiv2020

Evolutionary loss of inflammasomes in carnivores to facilitate carriage of zoonotic infections

Zoonotic infections, such as COVID-19, reside in animal hosts before jumping species to humans. The Carnivora, like mink, carry many zoonoses yet how diversity in host immune genes across species impact upon pathogen carriage are poorly understood. Here we describe a progressive evolutionary downregulation of pathogen sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerisation domain leucine rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing lytic cell death and the formation of an NLRP3-caspase-8 containing inflammasome that inefficiently processes interleukin-1{beta} (IL-1{beta}). Inflammasomes regulate gut immunity, but the carnivorous diet is antimicrobial suggesting a tolerance to the loss of these immune pathways. The consequences of systemic inflammasome downregulation, however, can reduce the host sensing of specific pathogens such that they can reside undetected in the Carnivora.

immunology↗

bioRxiv2020

Toxoplasma gondii GRA28 is required for specific induction of the regulatory chemokine CCL22 in human and mouse cells.

Toxoplasma gondii is an intracellular protozoan pathogen of humans that can cross the placenta and result in adverse pregnancy outcomes and long-term birth defects. The mechanism used by T. gondii to cross the placenta are unknown but complex interactions with the host immune response are likely to play a role in dictating infection outcomes during pregnancy. Prior work showed that T. gondii infection dramatically and specifically increases the secretion of the immunomodulatory chemokine CCL22 in human placental cells during infection.. Given the important role of this chemokine during pregnancy, we hypothesized that CCL22 induction was driven by a specific T. gondii-secreted effector. Using a combination of bioinformatics and molecular genetics, we have now identified T. gondii GRA28 as the gene product required for CCL22 induction. GRA28 is secreted into the host cell where it localizes to the nucleus, and deletion of this gene results in reduced CCL22 placental cells as well as a human monocyte cell line. The impact of GRA28 on CCL22 production is also conserved in mouse immune and placental cells both in vitro and in vivo. Moreover, parasites lacking GRA28 are impaired in their ability to disseminate throughout the animal, suggesting a link between CCL22 induction and the ability of the parasite to cause disease. Overall these data demonstrate a clear function for GRA28 in altering the immunomodulatory landscape during infection of both placental and peripheral immune cells, and show a clear impact of this immunomodulation on infection outcome. AUTHOR SUMMARYToxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in HIV/AIDS patients and can also cross the placenta and infect the developing fetus. We have found that placental and immune cells infected with T. gondii secrete significant amounts of a chemokine (called "CCL22") that is critical for immune tolerance during pregnancy. In order to better understand whether this is a response by the host or a process that is driven by the parasite, we have identified a T. gondii gene that is absolutely required to induce CCL22 production in human cells, indicating that CCL22 production is a process driven almost entirely by the parasite rather than the host. Consistent with its role in immune tolerance, we also found that T. gondii parasites lacking this gene are less able to proliferate and disseminate throughout the host. Taken together these data illustrate a direct relationship between CCL22 levels in the infected host and a key parasite effector, and provide an interesting example of how T. gondii can directly modulate host signaling pathways in order to facilitate its growth and dissemination.

microbiology↗

bioRxiv2020

Transcriptional profiling reveals T cells cluster around neurons injected with Toxoplasma gondii proteins

Toxoplasma gondiis tropism for and persistence in the CNS underlies the symptomatic disease Toxoplasma causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and infects in vivo. This predilection for neurons suggests that Toxoplasmas persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on Toxoplasma-host cell interactions has been done in vitro and in non-neuronal cells. We address this gap by utilizing our Toxoplasma-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA-seq, we isolated and transcriptionally profiled Toxoplasma-injected neurons (TINs), Bystander neurons (nearby non-Toxoplasma injected neurons), and neurons from uninfected mice (controls). These profiles show that TINs transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that Toxoplasma transcripts were primarily found in the TINs transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells.

bioinformatics↗

bioRxiv2020

Third generation sequencing revises the molecular karyotype for Toxoplasma gondii and identifies emerging copy number variants in sexual recombinants.

Toxoplasma gondii is an obligate intracellular parasite that has a significant impact on human health, especially in the immunocompromised. This parasite is also a useful genetic model for intracellular parasitism given its ease of culture in the laboratory and relevant animal models. However, as for many other eukaryotes, the T. gondii genome is incomplete, containing hundreds of sequence gaps due to the presence of repetitive and/or uncloneable sequences that prevent complete telomere-to-telomere de novo chromosome assembly. Here, we report the first use of single molecule DNA sequencing to generate near complete de novo genome assemblies for T. gondii and its near relative, N. caninum. Using the Oxford Nanopore Minion platform, we dramatically improved the contiguity of the T. gondii genome (N50 of [~]6.6Mb) and increased overall assembled sequence compared to current reference sequences by [~]2 Mb. Multiple complete chromosomes were fully assembled as evidenced by clear telomeric repeats on the end of each contig. Interestingly, for all of the Toxoplasma gondii strains that we sequenced (RH, CTG, IIxIII F1 progeny clones CL13, S27, S21, and S26), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome. This was due to a repeatable and consistent fusion of chromosomes VIIb and VIII. These data were further validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. In addition revising the molecular karyotype we were also able to resolve hundreds of repeats derived from both coding and non-coding tandem sequence expansions. For well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38, we were also able to accurately determine the precise gene count, order and orientation using established assembly approaches and the most likely primary sequence of each using our own assembly correction scripts tailored to correcting homopolymeric run errors in tandem sequence arrays. Finally, when we compared the T. gondii and N. caninum assemblies we found that while the 13 chromosome karyotype was conserved, we determined that previously unidentified large scale translocation events occurred in T. gondii and N. caninum since their most recent common ancestry.

genomics↗

bioRxiv2019

Parasite manipulation of the host cell cycle as a means to block inflammatory signaling and promote intracellular replication

Toxoplasma gondii and Hammondia hammondi are closely-related coccidian intracellular parasites that differ in their ability to cause disease in animal and (likely) humans. The role of the host response in these phenotypic differences is not known and to address this we performed a transcriptomic analysis of a monocyte cell line (THP-1) infected with these two parasite species. The pathways altered by infection were shared between species ~95% the time, but the magnitude of the host response to H. hammondi was significantly higher compared to T. gondii. Accompanying this divergent host response was an equally divergent impact on the cell cycle of the host cell. In contrast to T. gondii, H. hammondi infection induces cell cycle arrest via pathways linked to DNA-damage responses and cellular senescence and robust secretion of multiple chemokines that are known to be a part of the senescence associated secretory phenotype (SASP). Remarkably T. gondii-conditioned media can suppress the SASP response during H. hammondi infection, and this suppression is accompanied by a corresponding increase in the replication rate of H. hammondi in recipient cells. Taken together our data suggest that T. gondii manipulation of the host cell cycle provides a novel mechanism to avoid stress and/or DNA-damage induced responses by the host cell, and that this ability has a direct impact on parasite replication rate both within the host cell as well as in bystander cells.

cell biology↗

bioRxiv2017

Human placental syncytiotrophoblasts restrict Toxoplasma gondii vertical transmission at two distinct stages and induce CCL22 in response to infection

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human mid-gestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at distinct stages of the parasite lytic cycle--at the time of attachment and also during intracellular replication. Utilizing comparative RNAseq transcriptional profiling, we also show that human placental trophoblasts at both mid- and late-stages of gestation induce the chemokine CCL22 in response to T. gondii infection, which relies on the secretion of parasite effector(s). Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy, and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.\n\nSignificance statementToxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetal-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection through the induction of the chemokine CCL22. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy and identify the placental-enriched signaling pathways induced in response to infection.

microbiology↗

bioRxiv2017

Hammondia hammondi has a developmental program in vitro that mirrors its stringent two host life cycle

Hammondia hammondi is the nearest relative of Toxoplasma gondii, but unlike T. gondii is obligately heteroxenous. We have compared H. hammondi and T. gondii development in vitro and identified multiple H. hammondi-specific growth states. Despite replicating slower than T. gondii, H. hammondi was resistant to pH-induced tissue cyst formation early after excystation. However, in the absence of stress H. hammondi spontaneously converted to a terminally differentiated tissue cyst stage while T. gondii did not. Cultured H. hammondi could infect new host cells for up to 8 days following excystation, and this period was exploited to generate stably transgenic H. hammondi. Coupled with RNAseq analyses, our data clearly show that H. hammondi zoites grow as stringently regulated life stages that are fundamentally distinct from T. gondii tachyzoites and bradyzoites.

microbiology↗

bioRxiv2016

Structure-based alignment of human caspase recruitment domains provides a framework for understanding their function

Intracellular signalling is driven by protein-protein interactions. Members of the Death Domain superfamily mediate protein-protein interactions in both cell death and innate immune signalling pathways. They drive the formation of macromolecular complexes that act as a scaffold for protein recruitment and downstream signal transduction. Death Domain family members have low sequence identity, complicating their identification and predictions of their structure and function. We have taken all known human caspase recruitment domains (CARDs), a subfamily of the Death Domain superfamily, and generated a structure-guided sequence alignment. This alignment has enabled the identification of 14 positions that define the hydrophobic core and present a template for the identification of novel CARD sequences. We identify a conserved salt bridge in over half of all human CARDs and find a subset of CARDs likely to be regulated by tyrosine phosphorylation in their type I interface. Our alignment highlights that the CARDs of NLRC3 and NLRC5 are likely to be pseudodomains that have lost some of their original functionality. Together these studies demonstrate the benefits of structure-guided sequence alignments in understanding protein functionality.

biochemistry↗