How host-like signals drive gene expression and gene expression drives capsule growth in Cryptococcus neoformans
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ABSTRACT:
ORGANISM(S): Cryptococcus neoformans var. grubii
PROVIDER: GSE226656 | GEO | 2023/08/02
REPOSITORIES: GEO
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Project description:Cryptococcus neoformans is a fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew cells with and without all combinations of 5 signals suspected of affecting capsule size: Tissue-culture medium (DMEM or RPMI), temperature (37°C), CO2 (5%), and the addition of HEPES buffer. For each combination of conditions, we grew cultures with or without exogenous cAMP, and sampled cells for RNA-Seq at 0, 30, 90, 180, 1440 minutes and for measurement of capsule thickness at 1440 min. We also took samples for RNA-Seq at 30, 90, 180, 1440 minutes and carried out RNA-Seq in quadruplicate.
2023-08-02 | GSE226255 | GEO
Project description:Cryptococcus neoformans is a fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew wild-type cells and cells lacking components of the cAMP pathway: Pde1, Pde2, Pkr1. We also took samples for RNA-Seq at 30, 90, 180, 1440 minutes and carried out RNA-Seq in quadruplicate. We also measured capsule thickness at 1440 min.
2023-08-02 | GSE226651 | GEO
Project description:Cryptococcus neoformans is a fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth in response to host-like conditions. To understand how individual host-like signals affect capsule size and gene expression, we grew cells wild-type KN99 cells with either no added cyclic AMP (cAMP), 1.1 mM, 1.8 mM, 3.3 mM, 11 mM, or 20 mM cAMP. capsule thickness at 1440 min. We also took samples for RNA-Seq at 30, 90, 180, 1440 minutes and carried out RNA-Seq in quadruplicate. We also measured capsule thickness at 1440 min.
2023-08-02 | GSE226637 | GEO
Project description:UnlabelledThe fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans.ImportanceCryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.
| S-EPMC4056547 | biostudies-literature
Project description:The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.
| S-EPMC174025 | biostudies-other
Project description:Microbial capsules are important virulence traits that mediate cell-host interactions and provide protection against host immune defense mechanisms. Cryptococcus neoformans is a yeast-like fungus that is capable of synthesizing a complex polysaccharide (PS) capsule that is required for causing disease. Microscopic visualization of capsule enlargement is difficult, because the capsule is a highly hydrated structure with an index of refraction that is very close to that of aqueous medium. In this study, we took advantage of the capsular reaction ("quellung" effect) produced by IgM monoclonal antibody (MAb) 13F1 to increase the refraction index difference between capsule and medium such that we visualized the capsule using differential interference contrast (DIC) microscopy. Time-lapse size measurements allowed us to quantify the growth rate of the capsule relative to that of the cell body. The increase in capsule volume per unit of time was consistent with a logistic variable slope model in which the capsule's final size was proportional to the rate of its growth. The rate of capsule growth (0.3 to 2.5 µm(3)/min) was at least 4-fold faster than the rate of cell body growth (0.1 to 0.3 µm(3)/min), and there was large cell-to-cell variation in the temporal kinetics of capsule and cellular growth. Previous to the first cellular replication event, both the capsule and cell body enlarged simultaneously, and their differences showed monotonic growth, which was affected only by its rate of volume increase per unit of time. Using these results, we provide an updated model for cryptococcal capsule biogenesis.
| S-EPMC3811331 | biostudies-literature
Project description:Microbial capsules are important for virulence, but their architecture and physical properties are poorly understood. The human pathogenic fungus Cryptococcus neoformans has a large polysaccharide capsule that is necessary for virulence and is the target of protective antibody responses. To study the C. neoformans capsule we developed what we believe is a new approach whereby we probed the capsular elastic properties by applying forces using polystyrene beads manipulated with optical tweezers. This method allowed us to determine the Young's modulus for the capsule in various conditions that affect capsule growth. The results indicate that the Young's modulus of the capsule decreases with its size and increases with the Ca(2+) concentration in solution. Also, capsular polysaccharide manifests an unexpected affinity for polystyrene beads, a property that may function in attachment to host cells and environmental structures. Bead probing with optical tweezers provides a new, nondestructive method that may have wide applicability for studying the effects of growth conditions, immune components, and drugs on capsular properties.
| S-EPMC2726329 | biostudies-literature
Project description:Cryptococcus neoformans is unusual among melanotic fungi in that it requires an exogenous supply of precursor to synthesize melanin. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. L-3,4-dihydroxyphenylalanine (L-DOPA) is a natural catecholamine that is frequently used to induce melanization in C. neoformans and L-DOPA-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents and heavy metals. Given that C. neoformans needs exogenous substrate for melanization one question in the field is the extent to which melanin-associated phenotypes reflect the presence of melanin or metabolic changes in response to substrates. In this study we analyze the response of C. neoformans to L-DOPA with respect to melanization, gene expression and metabolic incorporation. Increasing the concentration of L-DOPA promotes melanin formation up to concentrations > 1 mM, after which toxicity is apparent as manifested by reduced growth. The timing of C. neoformans cells to melanization is affected by growth phase and cell density. Remarkably, growth of C. neoformans in the presence of L-DOPA results in the induction of relatively few genes, most of which could be related to stress metabolism. We interpret these results to suggest that the biological effects associated with melanization after growth in L-DOPA are largely due to the presence of the pigment. This in turn provides strong support for the view that melanin contributes to virulence directly through its presence in the cell wall.
| S-EPMC3173677 | biostudies-literature
Project description:Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsular cap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factor STE12alpha. Deletion of STE12alpha downregulated the expression levels of CAP10 while overexpression of STE12alpha upregulated the expression levels of CAP10. Animal model studies indicate that deletion of the CAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10 restores virulence. Thus, CAP10 is required for capsule formation and virulence.
| S-EPMC94082 | biostudies-literature
Project description:The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell.
| S-EPMC3416491 | biostudies-literature