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Background & Aims: Schistosomiasis is a parasitic infection which affects more than 200 million people globally. Schistosome eggs, but not the adult worms, are mainly responsible for schistosomiasis-specific morbidity in the liver. It is unclear if S. mansoni eggs consume host metabolites, and how this compromises the host parenchyma. Methods: Metabolic reprogramming was analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging, liquid chromatography with high-resolution mass spectrometry, metabolite quantification, confocal laser scanning microscopy, live cell imaging, quantitative real-time PCR, western blotting, assessment of DNA damage, and immunohistology in hamster models and functional experiments in human cell lines. Major results were validated in human biopsies. Results: The infection with S. mansoni provokes hepatic exhaustion of neutral lipids and glycogen. Furthermore, the distribution of distinct lipid species and the regulation of rate-limiting metabolic enzymes is disrupted in the liver of S. mansoni infected animals. Notably, eggs mobilize, incorporate, and store host lipids, while the associated metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes. Administration of reactive oxygen species scavengers ameliorates these deleterious effects. Conclusions: Our findings indicate that S. mansoni eggs completely reprogram lipid and carbohydrate metabolism via soluble factors, which results in oxidative stress-induced cell damage in the host parenchyma. Impact and implications: The authors demonstrate that soluble egg products of the parasite S. mansoni induce hepatocellular reprogramming, causing metabolic exhaustion and a strong redox imbalance. Notably, eggs mobilize, incorporate, and store host lipids, while the metabolic reprogramming causes oxidative stress-induced DNA damage in hepatocytes, independent of the host's immune response. S. mansoni eggs take advantage of the host environment through metabolic reprogramming of hepatocytes and enterocytes. By inducing DNA damage, this neglected tropical disease might promote hepatocellular damage and thus influence international health efforts.
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The snake venom gland is the place for the synthesis, storage, and secretion of a complex mixture of proteins and peptides, i.e., the venom. The morphology of the gland has been revealed by classical histology and microscopic studies. However, knowledge about the gland's cellular secretory and functional processes is still incomplete and has so far been neglected by the omics disciplines. We used autofocusing atmospheric-pressure matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular distributions in the venom glands of the saw-scaled viper, Echis carinatus sochureki, employing different sample preparation methods. Fresh-freezing and formalin-fixation were tested for the gland to obtain intact tissue sections. Subsequently, MSI was conducted with 12 µm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC, SM, PE, PS, PA, and TG are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (m/z 444.2233) was identified in the venom by top-down LC-MS/MS and localized by MALDI-MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom's enzymatic activity during long-term storage within the venom gland. With a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.
Assuntos
Glândulas Exócrinas/ultraestrutura , Imageamento Hiperespectral/métodos , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Venenos de Víboras/análise , Viperidae/anatomia & histologia , Animais , Cromatografia Líquida/métodos , Glândulas Exócrinas/química , Formaldeído , Congelamento , Metaloproteases/análise , Metaloproteases/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Fixação de Tecidos/métodos , Venenos de Víboras/enzimologiaRESUMO
The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 µm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.
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Coccidiose , Espaço Intracelular/parasitologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Toxoplasmose Animal , Animais , Bovinos , Células Cultivadas , Coccidiose/diagnóstico por imagem , Coccidiose/parasitologia , Células Endoteliais/citologia , Células Endoteliais/parasitologia , Imagem Molecular , Sarcocystidae/patogenicidade , Análise de Célula Única , Toxoplasma/patogenicidade , Toxoplasmose Animal/diagnóstico por imagem , Toxoplasmose Animal/parasitologiaRESUMO
Schistosomes are human pathogens causing the neglected tropical disease schistosomiasis, which occurs worldwide in (sub-)tropical regions. This infectious disease is often associated with poverty, and more than 700 million people are at risk of infection. Exploitation of novel habitats and limited therapeutic options brought schistosomes into research focus. Schistosomes are the only trematodes that have evolved separate sexes. They are covered by their metabolically active tegument, a surface area representing the interface between male and female in their permanent mating contact but also between parasite and host. The tegument comprises, besides others, numerous specific lipid compounds. Limited information is available on the exact lipid composition and its spatial distribution. We used atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to characterize the Schistosoma mansoni tegument surface in comparison to tissue sections of whole worms or couples. We found that phosphatidylcholines (PC) and specific phosphatidylethanolamines (PE) are significantly more abundant inside the worm body compared to the tegument. On the other hand, the latter was found to be enriched in sphingomyelins (SM), phosphatidylserines (PS), lysophosphatidylcholines (LPC), and specific PE species. We further investigated lipid classes concerning number of carbon atoms in fatty acyl chains as well as the degree of unsaturation and found pronounced differences between the tegument and whole-worm body. Furthermore, differences between male and female teguments were found. The lipid composition of S. mansoni tissues has been investigated in an untargeted, spatially resolved manner for the first time.
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Lipidômica/métodos , Lipídeos/química , Schistosoma mansoni/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Feminino , Metabolismo dos Lipídeos , Masculino , Schistosoma mansoni/metabolismoRESUMO
Schistosomes are parasitic platyhelminthes that cause schistosomiasis, which is a life-threatening infectious disease for humans in the tropics and subtropics worldwide. Within the human host, female and male schistosomes develop and pair as a prerequisite for egg production. Part of the eggs get lodged in organs such as the gut, spleen, and liver, where they cause severe inflammatory processes, including liver fibrosis, which is one of the most serious pathological symptoms. High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) has been used as a powerful tool to investigate adult schistosomes at the topographic molecular level. An MSI-compatible protocol was developed, covering critical sample preparation steps and focusing on obtaining artifact-free, longitudinal cryosections. Planar, consecutive sections were prepared from â¼400 µm thick S. mansoni worm couples, comparing several microembedding approaches. High-resolution MSI at both, 10 and 5 µm lateral resolution unraveled anatomical structures and differential abundances of glycerophospholipids and saccharides in females and males. In addition, glycerophospholipids occurred differentially abundant in worm tissues of the female, such as the gut, which is essential for nutrient uptake and subsequent metabolism. Fragment ions of isobaric phospholipids were investigated by on-tissue MS2 imaging experiments, unambiguously showing isomer-specific ion signals. This study provides a solid basis for investigating schistosome parasites in chemical detail at the whole-worm level by MSI.