A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues.

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.

Microsporidia dressing up: the spore polaroplast transport through the polar tube and transformation into the sporoplasm membrane.

Microsporidia are obligate intracellular parasites that infect a wide variety of hosts including humans. Microsporidian spores possess a unique, highly specialized invasion apparatus involving the polar filament, polaroplast, and posterior vacuole. During spore germination, the polar filament is discharged out of the spore forming a hollow polar tube that transports the sporoplasm components including the nucleus into the host cell. Due to the complicated topological changes occurring in this process, the details of sporoplasm formation are not clear. Our data suggest that the limiting membrane of the nascent sporoplasm is formed by the polaroplast after microsporidian germination. Using electron microscopy and 1,1'-dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine perchlorate staining, we describe that a large number of vesicles, nucleus, and other cytoplasm contents were transported out via the polar tube during spore germination, while the posterior vacuole and plasma membrane finally remained in the empty spore coat. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were also found to localize in the region of the polaroplast and posterior vacuole in mature spores and in the discharged polar tube, which suggested that the polaroplast during transport through the polar tube became the limiting membrane of the sporoplasm. The analysis results of Golgi-tracker green and Golgi marker protein syntaxin 6 were also consistent with the model of the transported polaroplast derived from Golgi transformed into the nascent sporoplasm membrane.IMPORTANCEMicrosporidia, which are obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection by the microsporidia is their unique invasion apparatus which includes the polar filament, polaroplast, and posterior vacuole. When the mature spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that transports the sporoplasm components including the microsporidian nucleus into host cells. Details of the structural difference between the polar filament and polar tube, the process of cargo transport in extruded polar tube, and the formation of the sporoplasm membrane are still poorly understood. Herein, we verify that the polar filament evaginates to form the polar tube, which serves as a conduit for transporting the nucleus and other sporoplasm components. Furthermore, our results indicate that the transported polaroplast transforms into the sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of the polar tube and origin of the sporoplasm membrane, which provide important clarification of the microsporidian infection mechanism.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Memory-like NK Cells Are a Critical Component of Vaccine-Induced Immunity to Trypanosoma cruzi Infection.

Chagas disease by Trypanosoma cruzi infection is a major public health issue. The available therapeutic agents have limited efficacy and significant side effects. A reliable vaccine would reduce the threat of T. cruzi infections and prevent Chagas disease. Understanding the immune response to this infection would improve vaccine design. We previously demonstrated that adoptively transferred NK cells from mice immunized with highly attenuated T. cruzi, GFP-DDDHA strain, provided potent protection in naive recipients against secondary lethal challenge with various wild-type (WT) strains. To understand the importance of NK cells in protecting mice against T. cruzi infection, we performed an in-depth characterization of NK cell phenotype, responses, and memory-like traits during acute infections due to GFP-DDDHA and WT strains and in immunized mice during a recall response to a WT lethal challenge. NK cells robustly expanded and became more mature and cytolytic during the GFP-DDDHA strain immunization. NK cells in immunized mice responded more robustly after WT lethal challenge than during an acute primary WT infection. In addition, protection by immunization with the GFP-DDDHA strain is significantly weakened in NK cell-deficient mice and did not prevent parasitemia from WT lethal challenge, indicating that NK cells with memory-like traits were a critical component for early control of WT lethal challenge. Prior T. cruzi vaccine development studies have not included studies of this rapid NK response. These findings provide insights into overcoming existing challenges in developing a safe and effective vaccine to prevent this infection.

Susceptibility of Toxoplasma gondii to autophagy in human cells relies on multiple interacting parasite loci.

IMPORTANCE: Autophagy is a process used by cells to recycle organelles and macromolecules and to eliminate intracellular pathogens. Previous studies have shown that some stains of Toxoplasma gondii are resistant to autophagy-dependent growth restriction, while others are highly susceptible. Although it is known that autophagy-mediated control requires activation by interferon gamma, the basis for why parasite strains differ in their susceptibility is unknown. Our findings indicate that susceptibility involves at least five unlinked parasite genes on different chromosomes, including several secretory proteins targeted to the parasite-containing vacuole and exposed to the host cell cytosol. Our findings reveal that susceptibility to autophagy-mediated growth restriction relies on differential recognition of parasite proteins exposed at the host-pathogen interface, thus identifying a new mechanism for cell-autonomous control of intracellular pathogens.

Toxoplasma gondii scavenges mammalian host organelles through the usurpation of host ESCRT-III and Vps4A.

Intracellular pathogens exploit cellular resources through host cell manipulation. Within its nonfusogenic parasitophorous vacuole (PV), Toxoplasma gondii targets host nutrient-filled organelles and sequesters them into the PV through deep invaginations of the PV membrane (PVM) that ultimately detach from this membrane. Some of these invaginations are generated by an intravacuolar network (IVN) of parasite-derived tubules attached to the PVM. Here, we examined the usurpation of host ESCRT-III and Vps4A by the parasite to create PVM buds and vesicles. CHMP4B associated with the PVM/IVN, and dominant-negative (DN) CHMP4B formed many long PVM invaginations containing CHMP4B filaments. These invaginations were shorter in IVN-deficient parasites, suggesting cooperation between the IVN and ESCRT. In infected cells expressing Vps4A-DN, enlarged intra-PV structures containing host endolysosomes accumulated, reflecting defects in PVM scission. Parasite mutants lacking T. gondii (Tg)GRA14 or TgGRA64, which interact with ESCRT, reduced CHMP4B-DN-induced PVM invaginations and intra-PV host organelles, with greater defects in a double knockout, revealing the exploitation of ESCRT to scavenge host organelles by Toxoplasma.

A Case Report and Literature Review of Babesiosis-Induced Acute Respiratory Distress Syndrome.

Babesiosis, a tick-borne protozoan disease, has been increasing in frequency in recent years. Familiarity with presentations of babesiosis is important for clinicians. Acute respiratory distress syndrome (ARDS) is a rarely seen complication of severe babesiosis. In most cases, the patients with babesiosis developed ARDS several days after initiation of antibabesia therapy. We present a unique case of babesiosis without any respiratory symptoms on presentation who developed ARDS within 24 hours of babesiosis treatment initiation. Furthermore, we reviewed published cases of ARDS in babesiosis.

Dense Granule Protein GRA64 Interacts with Host Cell ESCRT Proteins during Toxoplasma gondii Infection.

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In an attempt to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a transmembrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrated that GRA64 appears to interact with components of the host endosomal sorting complexes required for transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence or encystation in mice, as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of Δgra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCE Toxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immunocompromised individuals. Part of this parasite's success lies in its ability to infect diverse organisms and host cells and to persist as a latent infection within parasite-constructed structures called tissue cysts. In this study, we characterized a protein that is secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuolar membrane, this protein is exposed to the host cell cytosol and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

The Function and Structure of the Microsporidia Polar Tube.

Microsporidia are obligate intracellular pathogens that were initially identified about 160 years ago. Current phylogenetic analysis suggests that they are grouped with Cryptomycota as a basal branch or sister group to the fungi. Microsporidia are found worldwide and can infect a wide range of animals from invertebrates to vertebrates, including humans. They are responsible for a variety of diseases once thought to be restricted to immunocompromised patients but also occur in immunocompetent individuals. The small oval spore containing a coiled polar filament, which is part of the extrusion and invasion apparatus that transfers the infective sporoplasm to a new host, is a defining characteristic of all microsporidia. When the spore becomes activated, the polar filament uncoils and undergoes a rapid transition into a hollow tube that will transport the sporoplasm into a new cell. The polar tube has the ability to increase its diameter from approximately 100 nm to over 600 nm to accommodate the passage of an intact sporoplasm and penetrate the plasmalemma of the new host cell. During this process, various polar tube proteins appear to be involved in polar tube attachment to host cell and can interact with host proteins. These various interactions act to promote host cell infection.

Boron Chemicals in Drug Discovery and Development: Synthesis and Medicinal Perspective.

A standard goal of medicinal chemists has been to discover efficient and potent drug candidates with specific enzyme-inhibitor abilities. In this regard, boron-based bioactive compounds have provided amphiphilic properties to facilitate interaction with protein targets. Indeed, the spectrum of boron-based entities as drug candidates against many diseases has grown tremendously since the first clinically tested boron-based drug, Velcade. In this review, we collectively represent the current boron-containing drug candidates, boron-containing retinoids, benzoxaboroles, aminoboronic acid, carboranes, and BODIPY, for the treatment of different human diseases.In addition, we also describe the synthesis, key structure-activity relationship, and associated biological activities, such as antimicrobial, antituberculosis, antitumor, antiparasitic, antiprotozoal, anti-inflammatory, antifolate, antidepressant, antiallergic, anesthetic, and anti-Alzheimer's agents, as well as proteasome and lipogenic inhibitors. This compilation could be very useful in the exploration of novel boron-derived compounds against different diseases, with promising efficacy and lesser side effects.

Boron-Containing heterocycles as promising pharmacological agents.

The incorporation of the "magic" boron atom has been established as an important new strategy in the field of medicinal chemistry as boron compounds have been shown to form various bonds with their biological targets. Currently, a number of boron-based drugs (e.g. bortezomib, crisaborole, and tavaborole) have been FDA approved and are in the clinic, and several other boron-containing compounds are in clinical trials. Boron-based heterocycles have an incredible potential in the ongoing quest for new therapeutic agents owing to their plethora of biological activities and useful pharmacokinetic profiles. The present perspective is intended to review the pharmacological applications of boron-based heterocycles that have been published. We have classified these compounds into groups exhibiting shared pharmacological activities and discussed their corresponding biological targets focusing mainly on the most potent therapeutic compounds.

Strongyloides stercoralis.

Strongyloidiasis has been estimated to affect over 600 million people worldwide. It is caused by Strongyloides stercoralis, a roundworm endemic to the tropics and subtropics, especially areas where sanitation is suboptimal Autochthonous transmission has been documented in rural areas of the USA and Europe. Humans are infected when larvae penetrate the skin or are ingested. Autoinfection, in which larvae generated in the host go on to re-infect the host, leads to a state of chronic asymptomatic infection often with eosinophilia. Hyperinfection syndrome may develop when patients develop immune suppression, due to medications such as corticosteroids or following solid-organ transplantation. Hyperinfection is characterized by exponential increase in parasitic burden, leading to tissue invasion and life-threatening disease and associated bloodstream infections due to enteric organisms. Cases following use of corticosteroids for COVID-19 pneumonia have been described. Strongyloidiasis can be diagnosed by direct visualization of larvae in stool or other body fluids, or by serology. Ivermectin is highly effective in treating the disease. Patients with exposure to endemic areas and those expected to become immune suppressed should be screened and treated before starting immune suppressive agents. Empiric treatment should be considered when timely testing is not readily available.

Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondiiBAG1+/SAG1-, whereas this downregulation effect was reversed in case of T. gondiiBAG1-/SAG1+. We also identified two distinct host cell subsets that contained T. gondiiBAG1+/SAG1-, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

Current Therapy and Therapeutic Targets for Microsporidiosis.

Microsporidia are obligate intracellular, spore-forming parasitic fungi which are grouped with the Cryptomycota. They are both opportunistic pathogens in humans and emerging veterinary pathogens. In humans, they cause chronic diarrhea in immune-compromised patients and infection is associated with increased mortality. Besides their role in pébrine in sericulture, which was described in 1865, the prevalence and severity of microsporidiosis in beekeeping and aquaculture has increased markedly in recent decades. Therapy for these pathogens in medicine, veterinary, and agriculture has become a recent focus of attention. Currently, there are only a few commercially available antimicrosporidial drugs. New therapeutic agents are needed for these infections and this is an active area of investigation. In this article we provide a comprehensive summary of the current as well as several promising new agents for the treatment of microsporidiosis including: albendazole, fumagillin, nikkomycin, orlistat, synthetic polyamines, and quinolones. Therapeutic targets which could be utilized for the design of new drugs are also discussed including: tubulin, type 2 methionine aminopeptidase, polyamines, chitin synthases, topoisomerase IV, triosephosphate isomerase, and lipase. We also summarize reports on the utility of complementary and alternative medicine strategies including herbal extracts, propolis, and probiotics. This review should help facilitate drug development for combating microsporidiosis.

Enterocytozoon schreckii n. sp. Infects the Enterocytes of Adult Chinook Salmon (Oncorhynchus tshawytscha) and May Be a Sentinel of Immunosenescence.

A novel Enterocytozoon infection was identified in the intestines of sexually mature Chinook salmon. While microsporidian parasites are common across a diverse range of animal hosts, this novel species is remarkable because it demonstrates biological, pathological, and genetic similarity with Enterocytozoon bieneusi, the most common causative agent of microsporidiosis in AIDS patients. There are similarities in the immune and endocrine processes of sexually mature Pacific salmon and immunocompromised humans, suggesting possible common mechanisms of susceptibility in these two highly divergent host species. The discovery of Enterocytozoon schreckii n. sp. contributes to clarifying the phylogenetic relationships within family Enterocytozoonidae. The phylogenetic and morphological features of this species support the redescription of Enterocytozoon to include Enterospora as a junior synonym. Furthermore, the discovery of this novel parasite may have important implications for conservation, as it could be a sentinel of immune suppression, disease, and prespawning mortality in threatened populations of salmonids. IMPORTANCE In this work, we describe a new microsporidian species that infects the enterocytes of Chinook salmon. This novel pathogen is closely related to Enterocytozoon bieneusi, an opportunistic pathogen commonly found in AIDS patients and other severely immunocompromised humans. The discovery of this novel pathogen is of interest because it has only been found in sexually mature Chinook salmon, which have compromised immune systems due to the stresses of migration and maturation and which share similar pathological features with immunocompromised and senescent humans. The discovery of this novel pathogen could lead to new insights regarding how microsporidiosis relates to immunosuppression across animal hosts.

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins.

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the host Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

Secreted Effectors Modulating Immune Responses to Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that chronically infects a third of humans. It can cause life-threatening encephalitis in immune-compromised individuals. Congenital infection also results in blindness and intellectual disabilities. In the intracellular milieu, parasites encounter various immunological effectors that have been shaped to limit parasite infection. Parasites not only have to suppress these anti-parasitic inflammatory responses but also ensure the host organism's survival until their subsequent transmission. Recent advancements in T. gondii research have revealed a plethora of parasite-secreted proteins that suppress as well as activate immune responses. This mini-review will comprehensively examine each secreted immunomodulatory effector based on the location of their actions. The first section is focused on secreted effectors that localize to the parasitophorous vacuole membrane, the interface between the parasites and the host cytoplasm. Murine hosts are equipped with potent IFNγ-induced immune-related GTPases, and various parasite effectors subvert these to prevent parasite elimination. The second section examines several cytoplasmic and ER effectors, including a recently described function for matrix antigen 1 (MAG1) as a secreted effector. The third section covers the repertoire of nuclear effectors that hijack transcription factors and epigenetic repressors that alter gene expression. The last section focuses on the translocation of dense-granule effectors and effectors in the setting of T. gondii tissue cysts (the bradyzoite parasitophorous vacuole).

Approaching the Interpretation of Discordances in SARS-CoV-2 Testing.

The coronavirus disease 2019 pandemic has upended life throughout the globe. Appropriate emphasis has been placed on developing effective therapies and vaccines to curb the pandemic. While awaiting such countermeasures, mitigation efforts coupled with robust testing remain essential to controlling spread of the disease. In particular, serological testing plays a critical role in providing important diagnostic, prognostic, and therapeutic information. However, this information is only useful if the results can be accurately interpreted. This pandemic placed clinical testing laboratories and requesting physicians in a precarious position because we are actively learning about the disease and how to interpret serological results. Having developed robust assays to detect antibodies generated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and serving the hardest-hit areas within the New York City epicenter, we found 3 types of discordances in SARS-CoV-2 test results that challenge interpretation. Using representative clinical vignettes, these interpretation dilemmas are highlighted, along with suggested approaches to resolve such cases.

Microsporidiosis in Humans.

Microsporidia are obligate intracellular pathogens identified ∼150 years ago as the cause of pébrine, an economically important infection in silkworms. There are about 220 genera and 1,700 species of microsporidia, which are classified based on their ultrastructural features, developmental cycle, host-parasite relationship, and molecular analysis. Phylogenetic analysis suggests that microsporidia are related to the fungi, being grouped with the Cryptomycota as a basal branch or sister group to the fungi. Microsporidia can be transmitted by food and water and are likely zoonotic, as they parasitize a wide range of invertebrate and vertebrate hosts. Infection in humans occurs in both immunocompetent and immunodeficient hosts, e.g., in patients with organ transplantation, patients with advanced human immunodeficiency virus (HIV) infection, and patients receiving immune modulatory therapy such as anti-tumor necrosis factor alpha antibody. Clusters of infections due to latent infection in transplanted organs have also been demonstrated. Gastrointestinal infection is the most common manifestation; however, microsporidia can infect virtually any organ system, and infection has resulted in keratitis, myositis, cholecystitis, sinusitis, and encephalitis. Both albendazole and fumagillin have efficacy for the treatment of various species of microsporidia; however, albendazole has limited efficacy for the treatment of Enterocytozoon bieneusi. In addition, immune restoration can lead to resolution of infection. While the prevalence rate of microsporidiosis in patients with AIDS has fallen in the United States, due to the widespread use of combination antiretroviral therapy (cART), infection continues to occur throughout the world and is still seen in the United States in the setting of cART if a low CD4 count persists.