Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

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Abstract
We investigated the phenomenon of cell–cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC‐2). Cell aggregation into clumps of 5–40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump– cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host–pathogen interactions. Adherence of clump+ cells to murine macrophage‐like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump– cells. Furthermore, complement‐mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump– cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (104) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump– cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump– cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation‐like phenotype. We hypothesize that this cell–cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement‐mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.