Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.

Miltefosine Has a Postantifungal Effect and Induces Apoptosis in Cryptococcus Yeasts.

Cryptococcus spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in Cryptococcus is poorly understood. Here, we examined the effects of MFS on C. neoformans and C. gattii yeasts (planktonic and biofilm lifestyles) to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic Cryptococcus cells, with similar activities against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had postantifungal effect on Cryptococcus, with a proliferation delay of up to 8.15 h after a short exposure to fungicidal doses. MFS at fungicidal concentrations increased the plasma membrane permeability, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry with light microscopy), plasma membrane irregularities, mitochondrial swelling, and a less conspicuous cell wall. Our results suggest that MFS increases the plasma membrane permeability in Cryptococcus via an interaction with ergosterol and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against C. neoformans and C. gattii and warrant further studies to establish clinical protocols for MFS use against cryptococcosis.

[Ultrastructural Patterns of Interactions between Murine Lung Macrophages and Yeast Cells of Cryptococcus neoformans Strains with Different Virulence].

This article presents the ultrastructural patterns of interactions between the murine lung macrophages and cells of low- (RKPGY-881, -1165, -1178) and high-virulence (RKPGY-1090, -1095, -1106) strains of Cryptococcus neoformans at the seventh post-experimental day. It was found that if macrophages ingest living yeast cells, the latter can: 1) become completely free from polysaccharide capsules, after that their contents undergo lysis, and cell wall debris are extruded from the macrophage (first scenario); 2) become partly free from their capsules, destroy the phagosomal plasma membrane and induce destructive processes inside the macrophage causing their death (second scenario); or 3) not lose their capsules and localize inside macrophage in latent state (third scenario). Macrophages can also ingest senescent and dead C. neoformans cells surrounded by capsules that are lost at the ingesting and phagosome stages (fourth scenario). The study revealed the dependence of cell-mediated immunity on the stage of development of ingested C. neoformans yeast cells. Here we describe a new mechanism of capsular polysaccharide elimination of C. neoformans yeast cells by murine macrophages.

Xylose donor transport is critical for fungal virulence.

Cryptococcus neoformans, an AIDS-defining opportunistic pathogen, is the leading cause of fungal meningitis worldwide and is responsible for hundreds of thousands of deaths annually. Cryptococcal glycans are required for fungal survival in the host and for pathogenesis. Most glycans are made in the secretory pathway, although the activated precursors for their synthesis, nucleotide sugars, are made primarily in the cytosol. Nucleotide sugar transporters are membrane proteins that solve this topological problem, by exchanging nucleotide sugars for the corresponding nucleoside phosphates. The major virulence factor of C. neoformans is an anti-phagocytic polysaccharide capsule that is displayed on the cell surface; capsule polysaccharides are also shed from the cell and impede the host immune response. Xylose, a neutral monosaccharide that is absent from model yeast, is a significant capsule component. Here we show that Uxt1 and Uxt2 are both transporters specific for the xylose donor, UDP-xylose, although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Both proteins also transport the galactofuranose donor, UDP-galactofuranose. We further show that Uxt1 and Uxt2 are required for xylose incorporation into capsule and protein; they are also necessary for C. neoformans to cause disease in mice, although surprisingly not for fungal viability in the context of infection. These findings provide a starting point for deciphering the substrate specificity of an important class of transporters, elucidate a synthetic pathway that may be productively targeted for therapy, and contribute to our understanding of fundamental glycobiology.

Aging: an emergent phenotypic trait that contributes to the virulence of Cryptococcus neoformans.

The pathogenic fungus, Cryptococcus neoformans, is known to undergo phenotypic variation, which affects its virulence in the host. Recent investigations on C. neoformans cells in humans have validated the concept that phenotypic variation is present and relevant for the outcome of chronic cryptococcosis. The C. neoformans capsule is not the only trait that varies among strains. An emerging variant is the "old cell phenotype" generated when C. neoformans undergoes replicative aging. This phenotype, which other than larger size also exhibits a thickened cell wall, inhibits phagocytosis and killing by antifungals in vitro. In concert with the finding that old cells accumulate in vivo, this emergent trait could have significant impact on cryptococcal virulence and infection, and contribute to treatment failure.

A role for LHC1 in higher order structure and complement binding of the Cryptococcus neoformans capsule.

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.

Neurovirulence of Cryptococcus neoformans determined by time course of capsule accumulation and total volume of capsule in the brain.

Cryptococcal meningitis remains a significant opportunistic infection in HIV-infected individuals worldwide, despite availability of antiretroviral therapies in developed nations. Current therapy with amphotericin B is difficult to administer and only partially effective. Mechanisms of cryptococcal neuropathogenesis are still not clearly defined. In the present study, we used a C57Bl/6 mouse model with intravenous inoculation of three isogenic strains of Cryptococcus neoformans: H99, Cap59, and Pkr1-33. These strains differ in their capsule production and are normocapsular, hypocapsular, and hypercapsular, respectively. We studied the role of capsule in the morbidity and mortality of our host animal. Surprisingly, we found that the hypercapsular strain was least virulent while the strains that produced less capsule were more virulent and had higher concentrations of organism in the brain. These results suggest that neurovirulence is related to total capsule volume and rate of capsule accumulation in the brain, rather than the amount of capsule produced per organism. Therapies which decrease central nervous system dissemination and inhibit replication rates in the brain may be more effective than therapies which target capsule production.