The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Ivy M Dambuza,Ambre Chapuis,Matthew C Fisher,Raif Yuecel,Donna M Maccallum,Marcel Jaspars,Thomas S Harrison,Elizabeth R Ballou,Robin C May,Xin Zhou,Joao Correia,Thomas Drake,Leanne Taylor-Smith,Tihana Bicanic,Gordon D Brown,Nathalie Legrave,Tim Rasmussen,Aaron P Mitchell

doi:10.1371/journal.ppat.1006978

Abstract

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Highlights

Fungi change shape in response to environmental stimuli
We show that Titan cells are a regulated morphotype analogous to the yeast-to-hyphal transition and establish new ways to study Titans outside the host lung
While investigating the impact of nutrient starvation on virulence factor production, we observed that when C. neoformans cells grown in Yeast Nitrogen Base (YNB) with 2% glucose were transferred to 10% heat inactivated (HI)-Fetal Calf Serum (FCS) at 5% CO2, 37 ̊C, large cells formed after several days (Fig 1A, 3 days; S1A Fig, 7 days)

Summary

Introduction

Fungi change shape in response to environmental stimuli. These morphogenic transitions drive pathogenesis and allow fungi to occupy different environmental niches. There are early clinical reports of Titan cells, in which large encapsulated yeast were isolated from the lung and brain of infected patients [17, 18]. In both cases, cell size was dependent on growth condition, shifting from >40 μm in patient samples to 40 μm in murine infection. Cruickshank et al report distinct capsule and cell wall structure of enlarged cells[18] Despite this clear morphological transition, both early reports concluded that the patient samples represented atypical isolates

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