Toxoplasma bradyzoites reside in tissue cysts that undergo cycles of expansion, rupture, and release to foster chronic infection. The glycosylated cyst wall acts as a protective barrier, although the processes responsible for formation, remodeling, and turnover are not understood. Herein, we identify a noncanonical chitinase-like enzyme TgCLP1 that localizes to micronemes and is targeted to the cyst wall after secretion. Genetic deletion of TgCLP1 resulted in a thickened cyst wall that decreased cyst turnover, blocked the export of virulence effectors into host cells, and resulted in failure to persist during chronic infection. Genetic complementation with a series of mutants revealed that the GH19 glycosidase domain was crucial for regulating glycosylation of several glycoproteins in the cyst wall. Overall, our findings reveal that TgCLP1 is a multifunctional survival factor that modifies glycoproteins within the cyst wall to modulate export of virulence effectors and regulate turnover of tissue cysts.

Methionine aminopeptidase 2 (MetAP2) plays an important role in the regulation of protein synthesis and post-translational processing. Preclinical/clinical applications of MetAP2 inhibitors for the treatment of various diseases have been explored because of their antiangiogenic, anticancer, antiobesity, antidiabetic, and immunosuppressive properties. However, the effects of MetAP2 inhibitors on CNS diseases are rarely examined despite the abundant presence of MetAP2 in the brain. Previously, we synthesized a novel boron-containing MetAP2 inhibitor, BL6, and found that it suppressed angiogenesis and adipogenesis yet improved glucose uptake. Here, we studied the anti-inflammatory effects of BL6 in SIM-A9 microglia and in a mouse model of Alzheimer’s disease generated by the intracerebroventricular (icv) injection of streptozotocin (STZ). We found that BL6 reduced proinflammatory molecules, such as nitric oxide, iNOS, IL-1β, and IL-6, together with phospho-Akt and phospho-NF-κB p65, which were elevated in lipopolysaccharide (LPS)-activated microglial SIM-A9 cells. However, the LPS-induced reduction in Arg-1 and CD206 was attenuated by BL6, suggesting that BL6 promotes microglial M1 to M2 polarization. BL6 also decreased glial activation along with a reduction in phospho-tau and an elevation in synaptophysin in the icv-STZ mouse model. Thus, our experiments demonstrate an anti-neuroinflammatory action of BL6, suggesting possible clinical applications of MetAP2 inhibitors for brain disorders in which neuroinflammation is involved.

PURPOSE OF REVIEW:Human toxoplasmosis remains a significant, yet often underrecognized, global health concern. This review highlights emerging advances in prevention and management, offering timely updates for clinicians and researchers. RECENT FINDINGS:Recent venison-associated outbreaks in the United States have emphasized the risk of ocular toxoplasmosis and severe disease in immune competent individuals and the need for heightened clinical suspicion. Updated guidelines for hematopoietic stem cell transplant (HSCT) recipients now recommend systematic screening, qPCR monitoring, and prophylaxis to reduce mortality from Toxoplasma gondii reactivation. Emerging evidence suggests that chronic T. gondii infection may contribute to adverse pregnancy outcomes, challenging the long-held assumption that chronic infection is protective against these complications. Although the potential association between chronic T. gondii infection and neuropsychiatric disorders remains debated, its public health relevance warrants further investigation. SUMMARY:Improved clinical awareness, applied preventive strategies, and expanded research are essential to mitigate the broader health impact of chronic T. gondii infection. Future well designed studies and rigorous analyses are critical to defining maternal-fetal risks and potential neuropsychiatric effects, providing the evidence needed to update clinical guidelines and inform public health policies.

Adult salmon enteritis (ASE), characterised by severe ulcerative enteritis, has been linked to prespawn mortality (PSM) in spring Chinook salmon (Oncorhynchus tshawytscha) in certain rivers in Oregon, USA. Catastrophic losses of spring Chinook salmon have resulted from PSM, a significant threat to their population stability. Understanding the causes of ASE is therefore critical for mitigating PSM and supporting conservation. This study investigates the potential infectious aetiology of ASE using a juvenile Chinook salmon model. Fish were immunocompromised with dexamethasone implants, fasted, and exposed to intestinal tissues from ASE-affected adult Chinook. Histopathology of recipient fish revealed mid-intestinal lesions consistent with ASE. The microsporidium Enterocytozoon schreckii, which is observed in ASE-affected adults from rivers, was transmitted for the first time to juvenile Chinook Salmon, making E. schreckii a potential new pathogen of juvenile salmon. Additionally, intranuclear inclusions were identified in enterocytes by histopathology and viral particles were detected by electron microscopy in recipient fish. The study demonstrates that intestinal lesions consistent with ASE can be experimentally induced in juvenile Chinook salmon through oral exposure to infected tissues, supporting an infectious aetiology. Further research is needed to isolate specific pathogens, including viruses and E. schreckii, and to elucidate their roles in ASE development.

Abstract Background Previous literature has demonstrated an inverse relationship between the prevalence of parasitic infections and chronic inflammatory diseases such as allergies, inflammatory bowel diseases, and autoimmunity. The “Old Friends” and “Hygiene” Hypotheses posit that in the sanitized society of high-income countries, the lack of allergens and microbiota is causing immune dysregulation. This research has led some individuals to pursue unregulated "helminth therapies" - ingesting parasites to relieve autoimmune symptoms - despite the lack of FDA approval or controlled clinical trials for the marketed products. The growth of an unregulated market, with transactions often conducted via untraceable Bitcoin and without transparency of origin, poses a public health risk that requires awareness among infectious disease specialists. Methods The researchers investigated 9 different helminth distributors to determine helminth species offered, origin, price, and “recommended dosage" of helminth therapy through the website Helminth Therapy Wiki. Helminth distributors analyzed include Symmbio, Au NAturel, Nathural, Tanawisa, Helminth Therapy NZ, Biome Restoration Ltd, The Llamas Clinic, YourSymbionts, and Autoimmune Therapies (AIT). Results 78% of the helminth providers sold Necator americanus, i.e. hookworms (NA), 22% sold Trichuris trichiura, i.e. whipworms (TTO), 22% sold Hymenolepis diminuta, i.e. rat tapeworms (HDC), and 11% sold Trichuris suis, i.e. pig whipworms (TSO). The price of one dose of these various parasites ranged from $120 USD to $1,789 USD, depending on species and provider. “Doses” were highly variable; the dose of NA ranged from 1 - 25 larvae, while TSO ranged from 500 - 2,500 larvae. Many companies used untraceable payment methods and contact preferences to maintain secrecy. Statements on several provider websites used inaccurate marketing, over-promised benefits, and under-emphasized risks. Conclusion The unregulated market for helminth therapies poses significant public health and consumer protection issues, given the variability in doses, high costs, and dubious marketing claims. The lack of transparency and medical provider oversight further exacerbates the safety concerns in this market. Disclosures All Authors: No reported disclosures

T cells have been reported to play critical roles in preventing of microsporidia dissemination. However, there roles and functions of each subset remain unclear. Here in the study, we performed a thorough analysis of murine splenic T-cell response analysis via single-cell RNA sequencing during microsporidia E. cuniculi infection. We demonstrated that Type I T helper (Th1) cells, T follicular helper (Tfh) cells, effector CD8 + T cells and proliferating CD8 + T cells were activated and expanded after infection. Activated Th1 cells and Tfh cells presented significantly upregulated gene expression of Ifng and Il21, respectively. A subcluster of Th1 cells with high Csf1 expression was detected after infection. Subsets of activated CD8 + T cells were markedly enriched with high expression of cytotoxic-function related genes such as Gzma and Gzmb, whereas some active CD8 T cells were enriched with proliferation-function related genes Mki67 and Stmn1. Other subsets of T cells including NK T cells, Myb+ T cells, γδ T cells and Cxcr6+ T cells, were also analyzed in this study yet no expansion was observed. In summary, our findings provide in-depth and comprehensive insights into T-cell responses during microsporidia infection, which will be valuable for further investigations.

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 the hollow polar tube that transports the sporoplasm components including nucleus into the host cell to achieve the invasion. Due to the complicated topological changes occurring in this process, the formation of sporoplasm is unclear. Here, electron microscopy observation and DiI staining confirmed that during spore germination, a large number of vesicles derived from the polaroplast, nucleus and other cytoplasm were transported out via the polar tube. Meanwhile, the posterior vacuole and plasma membrane remained in the empty spore coat. In addition, there was no DiI-labeled membrane around the nucleus in mature spores, whereas a DiI-labeled limit membrane wrapping nucleus was found at the tip of the extruded polar tube, suggesting that the membrane of sporoplasm was formed outside the mature spore. Two Nosema bombycis sporoplasm surface proteins (NbTMP1 and NoboABCG1.1) were located at the polaroplast in mature spores, in the extruded polar tube and on the sporoplasm membrane, which indicated that the polaroplast transported via the polar tube finally became the limiting membrane of the sporoplasm. Golgi-tracker green and Golgi marker protein syntaxin 6 were also found the same model, which was consistent with the transported polaroplast derived from Golgi transformed into the novel sporoplasm membrane during spore germination. Importance Microsporidia, obligate intracellular pathogenic organisms, cause huge economic losses in agriculture and even threaten human health. The key to successful infection of microsporidia is its unique invasion apparatus which includes the polar filament, polaroplast and posterior vacuole. When the spore is activated to geminate, the polar filament uncoils and undergoes a rapid transition into the hollow polar tube that will transport the sporoplasm components including nucleus into a host cell to achieve the invasion. Knowledge of structure difference between 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 elongated nucleus and other sporoplasm components. And we confirm that the transported polaroplast finally transforms into the novel sporoplasm membrane during spore germination. Our study provides new insights into the cargo transportation process of polar tube and origin of the sporoplasm membrane, which serve as foundations for clarifying the microsporidian infection mechanism.

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. The Journal of Immunology, 2024, 212: 617-631.

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC–MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization–Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC–MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot’s Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

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.

The hetero-multimeric protein machinery known as Endosomal Sorting Complex Required for Transport (ESCRT) operates across a spectrum of major cellular pathways including endosomal sorting, intralumenal vesicle biogenesis, and repair of endo-lysosomal and plasma membranes. Because of its pivotal roles in membrane remodeling, the ESCRT machinery is also exploited by many viral and bacterial pathogens for replication and intracellular survival. We recently reported that the protozoan pathogen Toxoplasma gondii (Tg) also subverts the ESCRT machinery, at least in one case for the uptake and degradation of host cytosolic proteins to support of its metabolic needs. Although apoptosis-linked gene 2 (ALG-2) is among the various ESCRT-related proteins that Tg recruits to its intracellular niche, the parasitophorous vacuole (PV), no other pathogens are known to exploit ALG-2; hence, the mechanism behind recruitment of ALG-2 to the PV remained unknown. ALG-2, an EF hand calcium-binding ESCRT accessory protein involved in membrane repair and other functions, binds to other proteins bearing certain short linear amino acid motifs. By searching for Tg secretory proteins containing such motifs, we identified TgGRA8 as a candidate with multiple putative ALG-2 binding elements. Reciprocal immunoprecipitation-mass spectrometry experiments suggested an interaction between TgGRA8 and ALG-2 in infected cells, along with other known ALG-2 binding proteins including ALG-2 interacting protein X (ALIX) and TSG101. Accordingly, by knocking out TgGRA8 we found that it is necessary for ALG-2 and ALIX recruitment to the PV. TgGRA8 also contributes to TSG101 recruitment. Biochemical experiments confirmed a direct and unexpectedly Ca2+-independent interaction between ALG-2 and a peptide from TgGRA8 encompassing one of its three conserved ALG-2 binding elements. Also unexpectedly, binding of GRA8 peptide to ALG-2 generated large oligomers suggestive of the peptide's ability to link ALG-2 molecules together. Collectively, our work identifies TgGRA8 as the first microbial effector targeting host ALG-2. Ongoing studies aim to unravel the biochemical mechanisms of TgGRA8 binding to ALG-2 under native cellular conditions along with defining the specific purpose for which Tg recruits ALG-2 to its PV. We acknowledge the funding support from NIH-NIAID (R01 AI120607) for this research work.

Methionine aminopeptidases (MetAPs) have emerged as a target for medicinal chemists in the quest for novel therapeutic agents for treating cancer, obesity, and other disorders. Methionine aminopeptidase is a metalloenzyme with two structurally distinct forms in humans, MetAP-1 and MetAP-2. The MetAP2 inhibitor fumagillin, which was used as an amebicide in the 1950s, has been used for the successful treatment of microsporidiosis in humans; however, it is no longer commercially available. Despite significant efforts and investments by many pharmaceutical companies, no new MetAP inhibitors have been approved for the clinic. Several lead compounds have been designed and synthesized by researchers as potential inhibitors of MetAP and evaluated for their potential activity in a wide range of diseases. MetAP inhibitors such as fumagillin, TNP-470, beloranib, and reversible inhibitors and their analogs guide new prospects for MetAP inhibitor development in the ongoing quest for new pharmacological indications. This perspective provides insights into recent advances related to MetAP, as a potential therapeutic target in drug discovery, bioactive small molecule MetAP2 inhibitors, and data on the role of MetAP-2 as a therapeutic target for microsporidiosis.

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1’s translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell’s resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Intracellular pathogens exploit cellular resources through host cell manipulation. Within its nonfusogenic parasitophorous vacuole (PV), Toxoplasma 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 fusing with the PVM. Here, we examine the parasite usurpation of host ESCRT-III and Vps4 to create PVM buds and vesicles. CHMP4B associates with the PVM/IVN and dominant negative (DN) CHMP4B forms many long PVM invaginations containing CHMP4B filaments; the invaginations are shorter in IVN-deficient parasites, suggesting cooperation between IVN and ESCRT. In infected cells expressing Vps4-DN, enlarged intra-PV structures containing host endo-lysosomes accumulate, reflecting defects in PVM scission. Parasite mutants lacking TgGRA14 or TgGRA64 that interact with ESCRT have 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 . Summary The parasite Toxoplasma sequesters host nutrient-filled organelles into its parasitophorous vacuole through its exploitation of host ESCRT-III and Vps4 for vacuolar membrane-remodeling and fission processes utilizing the parasite proteins TgGRA14 and TgGRA64 that interact with ESCRT.

Abstract Microsporidia are found in invertebrates and vertebrates, infecting every major animal group. Microsporidia produce distinctive, environmentally resistant spores, and the shape and size of these spores vary depending on the species. This chapter summarizes microsporidia associated with human infection and related clinical manifestations. Encephalitozoon species including E. intestinalis , E. hellem ,and E. cuniculi are widely distributed in mammals and have been identified in both immune‐competent and immune‐compromised humans. The successful sequencing of various microsporidia genomes has led to the application of molecular‐based techniques for the diagnosis of microsporidia infections in humans. Serologic assays such as carbon immunoassay, indirect immunofluorescence test, enzyme‐linked immunosorbent assay, and immunoblot assays have been used to demonstrate microsporidian‐specific antibodies in several species of animals.

ABSTRACTAutophagy contributes to innate immunity by targeting intracellular pathogens for elimination. Previous studies identified a non-canonical autophagy pathway that controls Toxoplasma gondii infection in a strain-dependent manner in interferon gamma-activated human cells. Ubiquitination of unknown targets recruits adaptors and LC3 to the parasitophorous vacuole, leading to membrane envelopment and stunted growth. Vacuoles containing strain types II and III are susceptible, while type I strains of T. gondii are largely resistant and hence avoid autophagy-mediated growth restriction. Here we interrogated the genetic differences in LC3 recruitment between a resistant type I (GT1) strain and a susceptible type III (CTG) strain of T. gondii. We took advantage of a previous genetic cross between these two strains to determine the LC3 recruitment phenotype of 34 unique progeny clones. Genetic linkage mapping revealed that LC3 recruitment was highly multigenic, depending on two major quantitative trait loci (QTLs) on chromosome II and VIII, as well as three minor contributing loci. Ubiquitin affinity capture followed by mass spectrometry identified several potential targets exposed at the surface of the parasitophorous vacuole, including several candidates within the major QTLs. We tested several candidates and identified the dense granule proteins MAF1 on chromosome II, and MAG1 and PSD1 on chromosome VIII, as being partially responsible for susceptibility to LC3 recruitment. Differential susceptibility is likely due to strain-specific differences in recognition of parasite molecules, rather than actively blocking recognition, thus revealing a new mechanism for cell-autonomous restriction of intracellular pathogens.IMPORTANCEAutophagy 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.