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

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.

The mouse lung early cellular innate immune response is not sufficient to control fungal infection with Cryptococcus neoformans

“…Titan cells, a common clinical observation (37), are difficult to generate in vitro. We did not observe titan cell formation in our model, but as the recently established in vitro protocol for titan cell generation takes 3 days, this suggests this phenotype does not occur this early in infection (23). This is supported by in vivo observations, where mice are only examined in vivo for titan cell formation at 3dpi (24).…”

“…Another of C. neoformans pathogenic strategies is the ability to form cells with massively increased size in vivo (>30μm), described as 'titan cells' (13). As this phenotype has only just been recapitulated in vitro, our understanding of this phenotype in vivo is still poorly understood (23). In our model, a variety of cryptococcal cell sizes were observed, but no consistent differences in size were observed between 0hpi and 24hpi.…”

Intranasal inoculation ofCryptococcus neoformansin mice produces nasal infection with rapid brain dissemination

“…We noted at 24 h post infection there were rare enlarged yeast cells within the nose turbinates and ears (Fig. 4C-G) whose cell body diameter is above 10 μm, and therefore can be consider titan cells (30–32). The finding of titan cells in the nose of mice is concordant with Lima et al ., (33) which found enlarged cells in noses of guinea pigs after some days of infection.…”

Pseudohyphal growth of the emerging pathogenCandida aurisis triggered by genotoxic stress through the S phase checkpoint

“…S1). In other fungi, the presence of enlarged round cells has been described as Titan cells in Cryptococcus neoformans (57,58), or Goliath cells in C. albicans (59). Further studies will be necessary to elucidate, whether these large cells in C. auris resemble Titan or Goliath cells or are something completely different.…”