Abstract:The 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 arres… Show more
“…When we compare our data including buds to those of García-Rodas et al [6], at first glance they seem to be in agreement: while the mother cell is budding actively, and presumably spending only brief intervals in G1/S, its own capsule does not become thicker. Our data would seem to suggest that this is less because of an intrinsic impairment of capsule growth during G2/M, as suggested by the authors of that study, and more because a mechanism exists whereby capsule polysaccharide might be destined for the capsule of the daughter cell.10.1080/21505594.2018.1515610-F0004Figure 4.Capsule thicknesses of four mother cells and their buds.…”
supporting
confidence: 87%
“…The time-lapse images show that the capsule growth is more pronounced in Sab-MOPS, although it was readily observable in both capsule induction conditions (Figure 2(a, b) and Supplemental videos 1–3). We measured the capsule size for several cells and found that the capsule growth follows sigmoidal kinetics with different rates for different cells (Figure 2(c)), as previously observed using measurements based on the Quellung effect [5,6]. 10.1080/21505594.2018.1515610-F0002Figure 2.Time-lapse imaging of C. neoformans capsule growth in inducing medium (Sab-MOPS).…”
supporting
confidence: 67%
“…C. neoformans capsules labelled with monoclonal antibodies [1] or complement [2] were incubated in capsule-inducing conditions and at specific time points during induction the probes were detected using fluorescence microscopy. More recently, the Quellung reaction observed on differential interference microscopy (DIC), which produces a change in refractive index when antibodies bind to the capsule [3,4], was used to monitor capsule thickening in C. neoformans cells in real time [5,6]. These reports yielded important insights into the kinetics of capsule synthesis, but all suffered from an important caveat: because antibodies and complement proteins interact directly with capsule components, they may change its physical properties, especially at the reasonably high concentrations needed for the observations.…”
“…When we compare our data including buds to those of García-Rodas et al [6], at first glance they seem to be in agreement: while the mother cell is budding actively, and presumably spending only brief intervals in G1/S, its own capsule does not become thicker. Our data would seem to suggest that this is less because of an intrinsic impairment of capsule growth during G2/M, as suggested by the authors of that study, and more because a mechanism exists whereby capsule polysaccharide might be destined for the capsule of the daughter cell.10.1080/21505594.2018.1515610-F0004Figure 4.Capsule thicknesses of four mother cells and their buds.…”
supporting
confidence: 87%
“…The time-lapse images show that the capsule growth is more pronounced in Sab-MOPS, although it was readily observable in both capsule induction conditions (Figure 2(a, b) and Supplemental videos 1–3). We measured the capsule size for several cells and found that the capsule growth follows sigmoidal kinetics with different rates for different cells (Figure 2(c)), as previously observed using measurements based on the Quellung effect [5,6]. 10.1080/21505594.2018.1515610-F0002Figure 2.Time-lapse imaging of C. neoformans capsule growth in inducing medium (Sab-MOPS).…”
supporting
confidence: 67%
“…C. neoformans capsules labelled with monoclonal antibodies [1] or complement [2] were incubated in capsule-inducing conditions and at specific time points during induction the probes were detected using fluorescence microscopy. More recently, the Quellung reaction observed on differential interference microscopy (DIC), which produces a change in refractive index when antibodies bind to the capsule [3,4], was used to monitor capsule thickening in C. neoformans cells in real time [5,6]. These reports yielded important insights into the kinetics of capsule synthesis, but all suffered from an important caveat: because antibodies and complement proteins interact directly with capsule components, they may change its physical properties, especially at the reasonably high concentrations needed for the observations.…”
“…García-Rodas et al 53 recently demonstrated that benomil, a drug which arrests C. neoformans cell cycle in G2/M, inhibited the capsule growth. We have shown that NaBut arrests cell cycle in G2/M as well (Fig.…”
Section: The Effect Of Hdaci On Melanin Formationmentioning
Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.
“…We propose that the function of cell-cycle regulators, which are essential for proliferation in the host, merit further investigation as virulence factors. Furthermore, there is evidence that virulence pathways are perturbed when cell-cycle progression is slowed, which suggests direct connections between cell-cycle regulators and virulence pathways [10,11]. …”
The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation.
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