Trichinella spiralis cystatin, TsCstN, modulates STAT4/IL-12 to specifically suppress IFN-γ production.

We have previously identified a cystatin, TsCstN, derived from the L1 stage of Trichinella spiralis and have shown that this protein is internalised in macrophages. Here we sought to address if this macrophage-TsCstN interaction could alter downstream T-cell priming. Using LPS-primed macrophages to stimulate T-cells in a co-culture system with or without TsCstN we assessed the resultant T-cell outcomes. IFN-γ, both protein and mRNA, but not IL-17A was negatively regulated by inclusion of TsCstN during macrophage priming. We identified a cell-cell contact independent change in the levels of IL-12 that led to altered phosphorylated STAT4 levels and translocation. TsCstN also negatively regulated the autonomous response in the myotubule cell line, C2C12. This work identifies a potential pathyway for L1 larvae to evade protective Th1 based immune responses and establish muscle-stage T. spiralis infection.

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes.

Artemisinin combination therapy remains effective for the treatment of falciparum malaria. However, Plasmodium falciparum can escape the effects of artemisinin by arresting their growth. The growth-arrested parasites cannot be distinguished from nonviable parasites with standard microscopy techniques due to their morphological similarities. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. As a result of the differential cell permeabilities of two DNA-binding fluorophores, growth-arrested P. falciparum can be distinguished from parasites killed by artemisinin, since the latter lose cell membrane permeability. This fluorescence-based assay increased the sensitivity and specificity of the ring survival assay in the assessment of artemisinin susceptibility. When combined with a third fluorophore-conjugated anti-human leukocyte antibody, this trio fluorophore assay became more useful in identifying growth-arrested parasites in mock human blood samples. This novel assay is a simple and rapid technique for monitoring artemisinin resistance with greater sensitivity and accuracy compared with morphology-based observations under a light microscope.

Modelling amoebic brain infection caused by Balamuthia mandrillaris using a human cerebral organoid.

The lack of disease models adequately resembling human tissue has hindered our understanding of amoebic brain infection. Three-dimensional structured organoids provide a microenvironment similar to human tissue. This study demonstrates the use of cerebral organoids to model a rare brain infection caused by the highly lethal amoeba Balamuthia mandrillaris. Cerebral organoids were generated from human pluripotent stem cells and infected with clinically isolated B. mandrillaris trophozoites. Histological examination showed amoebic invasion and neuron damage following coculture with the trophozoites. The transcript profile suggested an alteration in neuron growth and a proinflammatory response. The release of intracellular proteins specific to neuronal bodies and astrocytes was detected at higher levels postinfection. The amoebicidal effect of the repurposed drug nitroxoline was examined using the human cerebral organoids. Overall, the use of human cerebral organoids was important for understanding the mechanism of amoeba pathogenicity, identify biomarkers for brain injury, and in the testing of a potential amoebicidal drug in a context similar to the human brain.

Serine protease inhibitor derived from Trichinella spiralis (TsSERP) inhibits neutrophil elastase and impairs human neutrophil functions.

During early infection with Trichinella spiralis, host neutrophils destroy newborn larvae migrating in the bloodstream, preventing infection. However, parasites secrete various immunomodulatory molecules to escape the host's defense mechanisms, allowing them to infect the host and live for long periods. T. spiralis secretes serine protease inhibitors (TsSERPs), which are key inhibitory molecules that regulate serine proteases involved in digestion and inflammation. However, the modulatory roles of TsSERP in the inhibition of neutrophil serine proteases (NSPs) and neutrophil functions are unknown. Therefore, the immunomodulatory properties of recombinant TsSERP1 (rTsSERP1) on NSPs and neutrophil functions were investigated in this study. rTsSERP1 preferentially inhibited human neutrophil elastase (hNE). In addition, incubation of rTsSERP1 with fMLP-induced neutrophils impaired their phagocytic ability. The formation of neutrophil extracellular traps (NETs) was activated with phorbol myristate acetate (PMA), and NETs were dramatically reduced when treated with rTsSERP1. Furthermore, rTsSERP1 suppressed the production of proinflammatory cytokines and chemokines during neutrophil activation, which are essential for neutrophil-mediated local or systemic inflammation regulation. In conclusion, T. spiralis immune evasion mechanisms are promoted by the inhibitory properties of TsSERP1 against neutrophil elastase and neutrophil defense functions, and these might be promising alternative treatment targets for inflammatory disorders.

Molecular characterization and functional analysis of Schistosoma mekongi neuroglobin homolog.

Schistosomes are blood-dwelling parasites that are constantly exposed to high-level oxidative stress arising from parasite-intrinsic and host defense mechanisms. To survive in their hosts, schistosomes require an antioxidant system to minimize with oxidative stress. Several schistosome antioxidant enzymes have been identified and have been suggested to play indispensable antioxidant roles for the parasite. In addition to antioxidant enzymes, non-enzymatic antioxidants including small molecules, peptides, and proteins have been identified and characterized. Neuroglobin (Ngb), a nervous system-specific heme-binding protein, has been classified as a non-enzymatic antioxidant and is capable of scavenging a variety of free radical species. The antioxidant activity of Ngb has been well-studied in humans. Ngb is involved in cellular oxygen homeostasis and reactive oxygen/nitrogen scavenging in the central and peripheral nervous systems, but its functions in schistosome parasites have not yet been characterized. In this study, we aimed to characterize the molecular properties and functions of Schistosoma mekongi Ngb (SmeNgb) using bioinformatic, biochemical, and molecular biology approaches. The amino acid sequence of Ngb was highly conserved among schistosomes as well as closely related trematodes. SmeNgb was abundantly localized in the gastrodermis, vitelline, and ovary of adult female S. mekongi worms as well as in the tegument of adult male worms. Assessment of antioxidant activity demonstrated that recombinant SmeNgb had Fe2+ chelating and hydrogen peroxide scavenging activities. Intriguingly, siRNA silencing of SmeNgb gene expression resulted in tegument pathology. Understanding the properties and functions of SmNgb will help in future development of effective treatments and vaccines against S. mekongi, other schistosome parasites, and other platyhelminths.

Molecular cloning and characterization of serine protease inhibitor from food-borne nematode, Gnathostoma spinigerum.

Gnathostoma spinigerum is a causative agent of human gnathostomiasis and infects people residing in endemic areas as well as travelers. Cutaneous and visceral larval migrants cause clinical manifestations, resulting in severe morbidity and mortality. To survive in hosts, these parasites have evolved various immune evasion mechanisms, including the release of regulatory molecules. Serine protease inhibitors (serpins) that are present in many parasitic helminths are proteins suspected of suppressing host serine protease-related digestion and immune responses. In this study, the serpin secreted by G. spinigerum (GsSerp) was characterized using bioinformatics and molecular biology techniques. The bioinformatics revealed that GsSerp contains 9 helices, 3 ß-sheets, and a reactive central loop, which are conserved structures of the serpin superfamily. Recombinant GsSerp (rGsSerp) was expressed in E. coli (molecular weight, 39 kDa) and could inhibit chymotrypsin. Mouse polyclonal antibody against GsSerp could detect the native GsSerp in crude worm antigen but not the excretory-secretory product (ES) of infective-stage larva (aL3Gs). Moreover, the expression of GsSerp in the aL3Gs tissue was located in the hemolymph and intestinal tissue, indicating its role in parasite homeostasis. Our findings may help develop effective strategies for preventing and controlling gnathostomiasis.

A novel cystatin derived from Trichinella spiralis suppresses macrophage-mediated inflammatory responses.

Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory-secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine protease inhibitor. The protein elicits an anti-inflammatory property by suppressing pro-inflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression.