Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

Abstract

Fungal morphology significantly impacts the host response. Filamentation and tissue penetration by Candida and Aspergillus species are essential for virulence, while growth as a yeast allows the thermal dimorphic fungi Coccidiodes, Histoplasma, and Talaromyces to reside inside phagocytes and disseminate. The basidiomycete Cryptococcus neoformans exhibits an unusual yeast-to-titan transition thought to enhance pathogenicity by increasing fungal survival in the host lung and dissemination to the central nervous system. In a common laboratory strain (H99), in vitro and in vivo titan induction yields a heterogenous population including >10 μm titan cells, 5-7 μm yeast cells and 2-4 μm titanides. Previous reports have shown that titan cells are associated with enhanced virulence and the generation of aneuploid cells that facilitate stress adaptation and drug resistance, while small (>10 μm) cells are associated with increased dissemination. However, the relationship between titan cells, small cells, and titanides remains unclear. Here, we characterize titanides and small cells in H99 and three clinical isolates and show that titanides share the lipid membrane order of their titan mothers and the G0 quiescent-like DNA staining of mating spores. In addition, we show that both titanizing and non-titanizing isolates exhibit altered capsule structure and PAMP exposure over time during in vitro culture, and generate aneuploidy in vivo.Author summaryThe human fungal pathogen Cryptococcus neoformans causes 200,000 HIV-associated deaths each year. In the lung, Cryptococcus makes an unusual yeast-to-titan morphological switch that contributes to disease development by altering immune polarization and introducing aneuploidy underlying host stress and drug resistance. Specifically, a proportion of 5 um haploid yeast endoreduplicate and swell, converting to large (> 10 um) polyploid titan cells that can then produce genetically distinct daughter cells. We recently developed an in vitro protocol for inducing large titan cells and additionally observed a novel small “titanide” cell type. Here we investigate the nature and origin of these small cells, demonstrating that they emerge during both in vitro and in vivo mouse-passaged titan induction in the well characterised lab strain H99 and are also apparent in a titanizing clinical isolate, Zc8. We show that these titanide cells share features with titan mothers (lipid order) and with spores produced during heterothalic mating. Finally, we show that the capacity of clinical isolates to produce both titan and titanide cells impacts aneuploidy and the emergence of drug resistance in vivo.

Host-derived Reactive Nitrogen Species mediate theCryptococcus neoformansyeast-to-titan switch via fungal-derived superoxide

“…Consistent with a defect in ROS detoxification, examination of yap1Δ titan-induced cultures revealed two key changes ( Figure 7A, B): 1) we did not detect highly polyploid cells in the yap1Δ mutant (>16C) and 2) there was a reduction in the percentage of titanides, cells smaller than 5 μm with "sub-1C" DNA content that originate from titan cells [44]. We hypothesized that this might be due to failure of yap1Δ cells to detoxify high endogenous superoxide levels observed over the course of titanization ( Fig 4A).…”

“…Co-incubation of the clinical isolates with human blood monocytes revealed that the impact of phagocytes on fungal cell size also correlates with the capacity to titanize. After 24hr co-incubation with monocytes, H99 and Zc8 increased in maximum cell size up to >25 μm (H99) and >15μm (Zc8) ( Fig 5D), sizes which were only detected in vivo previously [44]. In contrast, Zc1 and Zc12 did not exceed the 10 μm threshold even after co-incubation with human monocytes ( Fig 5D).…”

“…Here, we examined the in vitro interaction of host phagocytes with pretitanized cells [11,44]. Upon co-culture, we observed a significant further increase in cryptococcal cell size and an increase in endogenous superoxide, the generation of which is required for titanization.…”

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii

“…We also demonstrate that R265 titan cells produce daughter cells with a polyploid DNA content similar to their mother cells. In contrast, C. neoformans titan cells produce both haploid and aneuploid progeny [20], with sizes ranging between 5-7μm and 2-4μm respectively [13, 45]. Given that population heterogeneity in C. neoformans is associated with preferential dissemination to the CNS [46, 47], it is possible that this lack of heterogeneity in C. gattii may contribute to the differences in disease etiology in this species.…”

A dissemination-prone morphotype enhances extrapulmonary organ entry by the fungusCryptococcus neoformans

“…Since pho4 mutants cannot form either large or small cells in vivo, it leads to the question of whether small cells are daughters of large cells such as titans. Zhou et al (Zhou et al, 2020) reported small cells, termed "titanides," that are titan daughter cells. We can obtain small cells in vitro without inducing titan cells beforehand (Fig.…”