The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis
Fig 5
Titan cell induction is mediated by host relevant ligands and pre-culture condition.
A) H99 cells pre-cultured in YNB were induced for Titans following 3 days growth in 10% FCS, 10% BAL, or 10 μM or 20 μM N-Acetylmuramyl-L-alanyl-D-isoglutamine (NMAiGn) as indicated. Representative micrographs are shown for live cells (CFW, SytoxGreen). Scale bar = 10 μm. B) Daughters arising from Titan mothers induced using BAL (left, T7) or NMAiGn (right, T1, T5, T6) were isolated and allowed to proliferate for 72 hr at 30°C on YPD agar prior to fixation and staining for DNA content (DAPI). Populations were analysed by flow cytometry. C) Quantification of cells induced by various conditions (FCS, BAL, MNAiGn, E. coli, S. pneumonia) (n>300). D) Lung homogenates from mice unexposed or exposed to Pen/Strep for 7 days prior to C. neoformans inhalation infection. Representative micrographs (CFW, scale bar = 20 μm) and quantification are shown. n>500 for each condition. E,F) FCS was fractionated by size exclusion chromatography (E) and the resulting fractions were tested for capacity to induce Titan cells. F) Analysis by 1H NMR and 1H-13C HSQC revealed peaks consistent with sugar and amino acid structures.