ABSTRACT
Cryptococcal meningitis (CM) causes high rates of HIV-related mortality, yet the Cryptococcus factors influencing patient outcome are not well understood. Pathogen-specific traits, such as the strain genotype and degree of antigen shedding, are associated with the clinical outcome, but the underlying biology remains elusive. In this study, we examined factors determining disease outcome in HIV-infected cryptococcal meningitis patients infected with Cryptococcus neoformans strains with the same multilocus sequence type (MLST). Both patient mortality and survival were observed during infections with the same sequence type. Disease outcome was not associated with the patient CD4 count. Patient mortality was associated with higher cryptococcal antigen levels, the cerebrospinal fluid (CSF) fungal burden by quantitative culture, and low CSF fungal clearance. The virulence of a subset of clinical strains with the same sequence type was analyzed using a mouse inhalation model of cryptococcosis. We showed a strong association between human and mouse mortality rates, demonstrating that the mouse inhalation model recapitulates human infection. Similar to human infection, the ability to multiply in vivo, demonstrated by a high fungal burden in lung and brain tissues, was associated with mouse mortality. Mouse survival time was not associated with single C. neoformans virulence factors in vitro or in vivo; rather, a trend in survival time correlated with a suite of traits. These observations show that MLST-derived genotype similarities between C. neoformans strains do not necessarily translate into similar virulence either in the mouse model or in human patients. In addition, our results show that in vitro assays do not fully reproduce in vivo conditions that influence C. neoformans virulence.

© 2018 Hommel et al. http://creativecommons.org/licenses/by/4.0/ the pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.

Candida albicans is a human opportunist pathogen that can grow as yeast, pseudohyphae, or true hyphae in vitro and in vivo, depending on environmental conditions. Reversible cellular morphogenesis is an important virulence factor that facilitates invasion of host tissues, escape from phagocytes, and dissemination in the blood stream. The innate immune system is the first line of defense against C. albicans infections and is influenced by recognition of wall components that vary in composition in different morphological forms. However, the relationship between cellular morphogenesis and immune recognition of this fungus is not fully understood. We therefore studied various vegetative cell types of C. albicans, singly and in combination, to assess the consequences of cellular morphogenesis on selected immune cytokine outputs from human monocytes. Hyphae stimulated proportionally lower levels of certain cytokines from monocytes per unit of cell surface area than yeast cells, but did not suppress cytokine response when copresented with yeast cells. Pseudohyphal cells induced intermediate cytokine responses. Yeast monomorphic mutants had elevated cytokine responses under conditions that otherwise supported filamentous growth and mutants of yeast and hyphal cells that were defective in cell wall mannosylation or lacking certain hypha-specific cell wall proteins could variably unmask or deplete the surface of immunostimulatory ligands. These observations underline the critical importance of C. albicans morphology and morphology-associated changes in the cell wall composition that affect both immune recognition and pathogenesis.

ABSTRACT
.
. Cryptococcus neoformans
. is a major life-threatening fungal pathogen. In response to the stress of the host environment,
. C. neoformans
. produces large polyploid titan cells. Titan cell production enhances the virulence of
. C. neoformans
. yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon
. in vitro
. replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions.
.
.
. IMPORTANCE
. The ability to adapt to stress is a key element for survival of pathogenic microbes in the host and thus plays an important role in pathogenesis. Here we investigated the predominantly haploid human fungal pathogen
. Cryptococcus neoformans
. which is capable of ploidy and cell size increases during infection through production of titan cells. The enlarged polyploid titan cells are then able to rapidly undergo ploidy reduction to generate progeny with reduced ploidy and/or aneuploidy. Under stressful conditions, titan cell progeny have a growth and survival advantage over typical cell progeny. Understanding how titan cells enhance the rate of cryptococcal adaptation under stress conditions may assist in the development of novel drugs aimed at blocking ploidy transitions.
.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1β in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans. © Society for Leukocyte Biology.