S5.3c Investigating the link between pleomorphism and virulence in <i>Cryptococcus</i>

Abstract S5.3 Cellular pleomorphism and fungal virulence, September 22, 2022, 3:00 PM - 4:30 PM   Objectives Fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are responsible for hundreds of thousands of annual deaths in immunocompromised individuals. Considerable phenotypic variation is exhibited by strains in response to stresses encountered during host infection, including increased capsule and cell size, the release of shed capsule, and the production of giant (> 15 μm), micro (< 1 μm), and irregular cells. We aimed to investigate whether the production of these morphological variants is associated with virulence using two sets of strains. The first is a collection of diverse clinical isolates obtained from HIV/AIDS patients in Botswana with accompanying clinical data. The second is a collection of lineages derived from the C. neoformans type strain H99 with high genetic similarity but differing levels of virulence. Some lineages in this set possess a mutation in SGF29, which encodes a component of the SAGA histone acetylation complex that has previously been implicated in their hypervirulence. Methods Isolates were cultured under conditions that simulate stresses encountered in vivo (DMEM, 5% CO2, 37°C) as these are known to enhance capsule production and induce cell size changes. Cells were counterstained with india ink, visualized by light microscopy, and phenotypes were scored. For clinical isolates, MLST analysis was performed to determine their relatedness. For H99 strains, Galleria mellonella larval infection assays, growth curves, and antifungal susceptibility testing was performed to confirm their relative virulence and growth profiles. Serial block face and regular scanning electron microscopy were used to investigate the internal morphology of the giant, micro, and irregular cells to confirm that they possess attributes of functional cells. Results Substantial pleomorphism was seen across both collections. In the clinical strain set, phenotypic variables fell into two groups associated with differing symptoms. The production of ‘large’ phenotypes was associated with a higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early-stage infection. ‘Small’ phenotypes were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may promote proliferation and dissemination. Isolates possessing giant cells, microcells, and shed capsules were rare, but strikingly, they were associated with patient death. In the H99 set, strains from hypervirulent lineages had larger average capsule size, greater variation in cell size, and increased production of microcells and shed capsules. Deletion of SGF29 in an intermediate virulence lineage substantially increased its production of microcells and released capsule, consistent with a switch to hypervirulence. SGF29 loss-of-function mutations were subsequently identified in clinical isolates and were found to be significantly correlated with patient death. Expansion of a TA repeat in the second intron of SGF29 in clinical isolates was positively correlated with cell and capsule size, suggesting it also affects Sgf29 function. Conclusion Our results extend the evidence for a link between pleomorphism and virulence, with a likely role for epigenetic mechanisms mediated by SAGA-induced histone acetylation.