Methods for Manipulating Cryptococcus Spores.
Ontology highlight
ABSTRACT:
SUBMITTER: Frerichs AB
PROVIDER: S-EPMC8779225 | biostudies-literature | 2021 Dec
REPOSITORIES: biostudies-literature
Publications
Methods for Manipulating <i>Cryptococcus</i> Spores.
Frerichs Anna B AB Huang Mingwei M Ortiz Sébastien C SC Hull Christina M CM
Journal of fungi (Basel, Switzerland) 20211222 1
Spores are essential for the long-term survival of many diverse organisms, due to their roles in reproduction and stress resistance. In the environmental human fungal pathogen, <i>Cryptococcus</i>, basidiospores are robust cells with the ability to cause disease in animal models of infection. Here we describe methods for producing and purifying <i>Cryptococcus</i> basidiospores in quantities sufficient for large-scale analyses. The production of high numbers of pure spores has facilitated the de ...[more]
Similar Datasets
Project description:Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (approximately 95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.
| S-EPMC2747963 | biostudies-literature
Project description:SUMMARYSpores are primary infectious propagules for the majority of human fungal pathogens; however, relatively little is known about their fundamental biology. One strategy to address this deficiency has been to develop the basidiospores of Cryptococcus into a model for pathogenic spore biology. Here, we provide an update on the state of the field with a comprehensive review of the data generated from the study of Cryptococcus basidiospores from their formation (sporulation) and differentiation (germination) to their roles in pathogenesis. Importantly, we provide support for the presence of basidiospores in nature, define the key characteristics that distinguish basidiospores from yeast cells, and clarify their likely roles as infectious particles. This review is intended to demonstrate the importance of basidiospores in the field of Cryptococcus research and provide a solid foundation from which researchers who wish to study sexual spores in any fungal system can launch their studies.
| S-EPMC10966950 | biostudies-literature
Project description:Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.
| S-EPMC4551743 | biostudies-literature
Project description:Restoring all facility operations after the 2001 Amerithrax attacks took years to complete, highlighting the need to reduce remediation time. Some of the most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite (SM-SPC): a single cellulose sponge samples multiple coupons with a single pass across each coupon; 2) single medium multi-pass composite: a single cellulose sponge samples multiple coupons with multiple passes across each coupon (SM-MPC); and 3) multi-medium post-sample composite (MM-MPC): a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155 CFU/cm2). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted dry wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p< 0.0001) and coupon material (p = 0.0006). Recovery efficiency (RE) was higher overall using the MM-MPC method compared to the SM-SPC and SM-MPC methods. RE with the MM-MPC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, dry wall, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces.
| S-EPMC5063342 | biostudies-literature
Project description:Phagocytosis by innate immune cells is an important process for protection against multiple pathologies and is particularly important for resistance to infection. However, phagocytosis has also been implicated in the progression of some diseases, including the dissemination of the human fungal pathogen, Cryptococcus neoformans. Previously, we identified Dectin-1 as a likely phagocytic receptor for C. neoformans spores through the use of soluble components in receptor-ligand blocking experiments. In this study, we used gain-of-function and loss-of-function assays with intact cells to evaluate the in vivo role of Dectin-1 and other C-type lectins in interactions with C. neoformans spores and discovered stark differences in outcome when compared with previous assays. First, we found that non-phagocytic cells expressing Dectin-1 were unable to bind spores and that highly sensitive reporter cells expressing Dectin-1 were not stimulated by spores. Second, we determined that some phagocytes from Dectin-1-/- mice interacted with spores differently than wild type (WT) cells, but the effects varied among assays and were modest overall. Third, while we detected small but statistically significant reductions in phagocytosis by primary alveolar macrophages from Dectin-1-/- mice compared to WT, we found no differences in survival between WT and Dectin-1-/- mice challenged with spores. Further analyses to assess the roles of other C-type lectins and their adapters revealed very weak stimulation of Dectin-2 reporter cells by spores and modest differences in binding and phagocytosis by Dectin-2-/- bone marrow-derived phagocytes. There were no discernable defects in binding or phagocytosis by phagocytes lacking Mannose Receptor, Mincle, Card-9, or FcRγ. Taken together, these results lead to the conclusion that Dectin-1 and other C-type lectins do not individually play a major roles in phagocytosis and innate defense by phagocytes against C. neoformans spores and highlight challenges in using soluble receptor/ligand blocking experiments to recapitulate biologically relevant interactions.
| S-EPMC5345868 | biostudies-literature
Project description:Understanding how the psychology of predators shapes the defenses of colorful aposematic prey has been a rich area of inquiry, with emphasis on hypothesis-driven experiments that independently manipulate color and palatability in prey to examine predator responses. Most of these studies focus on avian predators, despite calls to consider more taxonomically diverse predators. This taxonomic bias leaves gaps in our knowledge about the generalizability of current theory. Here we have adapted tools that have been successfully used with bird predators and scaled them down and tested them with smaller predators (Habronattus jumping spiders) and small insect prey (termites, milkweed bug nymphs, pinhead crickets, fruit flies). Specifically, we test (1) the application of denatonium benzoate (DB) to the surface of live termites, crickets, and fruit flies, and (2) the effectiveness of manipulating the palatability of milkweed bug nymphs through diet. We also test the effectiveness of combining these palatability manipulations with various color manipulations. Across several experiments, we confirm that our palatability manipulations are not detectable to the spiders before they attack (i.e., they do not produce aversive odors that spiders avoid), and show that unpalatable prey are indeed quickly rejected and spiders do not habituate to the taste with experience. We also investigate limitations of these techniques by assessing possible unintended effects on prey behavior and the risk of contact contamination when using DB-treated prey in experiments. While similar tools have been used to manipulate color and palatability with avian predators and relatively large insect prey, we show how these techniques can be effectively adapted for use with small invertebrate predators and prey.
| S-EPMC7138310 | biostudies-literature
Project description:Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40-72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20 min) and lysozyme (100 μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.
| S-EPMC9338887 | biostudies-literature
Project description:Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.
| S-EPMC92894 | biostudies-literature
Project description:BackgroundCryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples.MethodsThe 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains.Principal findingsThe 5.8S DNA-ITS PCR was more sensitive (89-100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96-100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF.ConclusionUse of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future.
| S-EPMC10019727 | biostudies-literature
Project description:Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.
| S-EPMC6056713 | biostudies-literature