Biology subjects

Sibley, L. D.

Publications and source records attributed to Sibley, L. D..

26 records · Page 2Linked to original sources

bioRxiv2021

Reduced calcium storage blunts calcium signaling in Toxoplasma bradyzoites and impedes motility and egress

Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e. tachyzoites) and for establishing chronic infection (i.e. bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened calcium responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with down-regulation of calcium ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus calcium rapidly restored their intracellular Ca2+ and ATP stores leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.

microbiology↗

bioRxiv2020

Insulinase like protease 1 contributes to macrogamont formation in Cryptosporidium parvum

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is a M16A clan member and contains a signal peptide, an N-terminal domain with the HxxEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1 positive vesicles often subtend large vacuoles resembling wall forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo. Our findings reveal that INS1 functions in formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice. ImportanceCryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. Absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, hence readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, may be useful for producing attenuated vaccines to induce immunity without causing disease.

microbiology↗

bioRxiv2020

Gut metabolites influence susceptibility of neonatal mice to cryptosporidiosis.

The protozoan parasite Cryptosporidium is a leading cause of diarrheal disease in those with compromised or under-developed immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection, and then tested the isolated effects of these metabolites on C. parvum invasion and growth. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, while long-chain unsaturated fatty acids enhance C. parvum invasion. The influence of these two classes of metabolites on C. parvum infection likely reflects the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Hence, gut metabolites, either from diet or produced by the microbiota, play an important role in the early susceptibility to cryptosporidiosis seen in young animals. ImportanceCryptosporidium occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development, and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced growth of C. parvum in vitro and may increase susceptibility to infection in young mice. Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium is unable to synthesize de novo. The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids provide further insight into reliance on fatty acid salvage and metabolism of this enteric parasite.

microbiology↗

bioRxiv2020

Toxoplasma gondii secreted effectors co-opt host repressor complexes to inhibit necroptosis

During infection, Toxoplasma gondii translocates effector proteins directly into its host cell to subvert various signaling pathways. Here we characterize a novel secreted effector that localizes to the host cell nucleus where it modulates NCoR/SMRT repressor complex levels to repress interferon regulated genes involved in cell death. Type I and type II interferons upregulate many genes including protein kinase R (PKR), inducing formation of the necrosome complex that activates Mixed Lineage Kinase Domain Like Pseudokinase (MLKL) to execute necrotic cell death. Toxoplasma NCoR/SMRT modulator (TgNSM) acts together with another secreted effector TgIST, previously shown to down-modulate IFN-{gamma} signaling to block immune functions. Together TgNSM and TgIST block IFN driven expression of PKR and MLKL, thus preventing host cell necroptotic death. The mechanism of action of TgNSM highlights a previously unappreciated role of NCoR/SMRT in regulation of necroptosis, assuring survival of intracellular cysts, and maintenance of chronic infection.

microbiology↗

bioRxiv2019

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii.

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of T. gondii, a related apicomplexan parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing drug concentrations and whole genome sequencing identified several non-conservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acids changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild type parasites in the presence of drug, despite not changing EC50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondrial genome, including genes encoding cytochrome b and cytochrome oxidase I. Consistent with prior studies in yeast and mammalian tumor cells that implicate the mitochondrion as a target of artemisinins, treatment of wild type parasites with artemisinin decreased mitochondrial membrane potential, and resistant parasites showed altered morphology and decreased membrane potential. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition in T. gondii.\n\nSignificanceArtemisinins provide important therapeutic agents for treatment of malaria and have potential for use in other infections and in cancer. Their use is threatened by the potential for resistance development, so understanding their mechanism of action and identifying genetic changes that alter sensitivity are important for improving clinical outcomes. Our findings suggest that mutations in novel targets can contribute to the emergence of parasites with increased tolerance to artemisinin treatment and that such mutations can confer a fitness advantage even in the absence of a notable shift in EC50. Our findings also support the idea that inhibition of mitochondrial function may be an important target in T. gondii, as previously suggested by studies in yeast and human cancer cells.

microbiology↗

bioRxiv2019

Toxoplasma gondii infection drives conversion of NK Cells into ILC1s

Innate lymphoid cells (ILCs) were originally classified based on their cytokine profiles, placing natural killer (NK) cells and ILC1s together, but recent studies support their separation into different lineages at steady-state. However, tumors may induce NK cell conversion into ILC1-like cells that are limited to the tumor microenvironment and whether this conversion occurs beyond this environment remains unknown. Here we describe Toxoplasma gondii infection converts NK cells into cells resembling steady-state ILC1s that are heterogeneous and distinct from both steady-state NK cells and ILC1s in uninfected mice. Most toxoplasma-induced ILC1s were Eomes-dependent, indicating that NK cells can give rise to Eomes- Tbet-dependent ILC1-like cells that circulate widely and persist independent of ongoing infection. Moreover, these changes appear permanent, as supported by epigenetic analyses. Thus, these studies markedly expand current concepts of NK cells, ILCs, and their potential conversion.

immunology↗

bioRxiv2019

Assays for monitoring Toxoplasma gondii infectivity in the laboratory mouse

Toxoplasma is a widespread parasite of animals including many rodents that are a natural part of the transmission cycle between cats, which serve as the definitive host. Although wild rodents, including house mice, are relatively resistant, laboratory mice are highly susceptible to infection. As such, laboratory mice and have been used to compare pathogenesis of natural variants, and to evaluate the contributions of both host and parasite genes to infection. Protocols are provided here for evaluating acute and chronic infection with different parasite strains in laboratory mice. These protocols should provide uniform standards for evaluating natural variants and attenuated mutants and for comparing outcomes across different studies and between different laboratories.

microbiology↗

bioRxiv2018

Evaluation of current and emerging anti-malarial medicines for inhibition of Toxoplasma gondii growth in vitro

Toxoplasma gondii is a common zoonotic infection of humans and estimates indicate that 1-2 billion people are chronically infected. Although largely asymptomatic, chronic infection poses risk of serious disease due to reactivation should immunity decline. Current therapies for toxoplasmosis only control acute infection caused by actively proliferating tachyzoites but do not eradicate the chronic tissue cyst stages. As well, there are considerable adverse side effects of the most commonly used therapy of combined sulfadiazine and pyrimethamine. Targeting the folate pathway is also an effective treatment for malaria, caused by the related parasites Plasmodium spp., suggesting common agents might be used to treat both infections. Here we evaluated currently approved and newly emerging medicines for malaria to determine if such compounds might also prove useful for treating toxoplasmosis. Surprisingly, the majority of anti-malarial compounds being used currently or in development for treatment of malaria were only modestly effective at inhibiting in vitro growth of T. gondii tachyzoites. These findings suggest that many essential processes in P. falciparum that are targeted by anti-malarial compounds are either divergent, or non-essential in T. gondii, thus limiting options for repurposing of current antimalarial medicines for toxoplasmosis.

microbiology↗