Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P . falciparum- infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P . falciparum , as well as in the plasma and infected RBCs of P . berghei -infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.
Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co-culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.
Common variable immunodeficiency (CVID) is a clinically and genetically heterogeneous disorder with inadequate antibody responses and low levels of immunoglobulins including IgA that is involved in the maintenance of the intestinal homeostasis. In this study, we analyzed the taxonomical and functional metagenome of the fecal microbiota and stool metabolome in a cohort of six CVID patients without gastroenterological symptomatology and their healthy housemates. The fecal microbiome of CVID patients contained higher numbers of bacterial species and altered abundance of thirty-four species. Hungatella hathewayi was frequent in CVID microbiome and absent in controls. Moreover, the CVID metagenome was enriched for low-abundance genes likely encoding nonessential functions, such as bacterial motility and metabolism of aromatic compounds. Metabolomics revealed dysregulation in several metabolic pathways, mostly associated with decreased levels of adenosine in CVID patients. Identified features have been consistently associated with CVID diagnosis across the patients with various immunological characteristics, length of treatment, and age. Taken together, this initial study revealed expansion of bacterial diversity in the host immunodeficient conditions and suggested several bacterial species and metabolites, which have potential to be diagnostic and/or prognostic CVID markers in the future.
Background Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer’s Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. Methods Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. Results We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. Conclusions Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing. Graphic Abstract
Cerebral organoids are a prolific research topic and an emerging model system for neurological diseases in human neurobiology. However, the batch-to-batch reproducibility of current cultivation protocols is challenging and thus requires a high-throughput methodology to comprehensively characterize cerebral organoid cytoarchitecture and neural development. We report a mass spectrometry-based protocol to quantify neural tissue cell markers, cell surface lipids, and housekeeping proteins in a single organoid. Profiled traits probe the development of neural stem cells, radial glial cells, neurons, and astrocytes. We assessed the cell population heterogeneity in individually profiled organoids in the early and late neurogenesis stages. Here, we present a unifying view of cell-type specificity of profiled protein and lipid traits in neural tissue. Our workflow characterizes the cytoarchitecture, differentiation stage, and batch cultivation variation on an individual cerebral organoid level.
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