Isolation and Characterization of Senescent Cryptococcus neoformans and Implications for Phenotypic Switching and Pathogenesis in Chronic Cryptococcosis

Abstract

Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo.Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.

Characterization of the virulence ofCryptococcus neoformansstrains in an insect model

“…Standard in vitro phagocytosis indices, and macrophage killing assays were done in J774.16 cells as previously described. 24 IPR and laccase activity was determined as previously described. 15 For infection with Galleria mellonella, a 10 ml suspension of 2£10 4 , 10 5 , or 10 6 C. neoformans cells was used to infect larvae (n D 20) (Vanderhorst Wholesale, Inc., OH) in the last proleg as described previously.…”

Chronological Aging Is Associated with Biophysical and Chemical Changes in the Capsule of Cryptococcus neoformans

“…Chronological aging refers to the effects of time on a cell after it has stopped growing, whereas generational age refers to the number of daughter cells produced by a given cell. Fungal cellular aging may be important in the pathogenesis of cryptococcosis, since chronicity is associated with the persistence of cells in lung (21) and senescent cells have been shown to accumulate in the course of infection (25). Aging in C. neoformans produced capsule changes that were associated with resistance to phagocytosis by macrophages and antibody (Ab) reactivity.…”

“…In this study, we investigated the effect of chronological aging under prolonged stationary-phase growth conditions on the dynamics of the PS capsule. We note that such aging in nondividing yeast cells (2) is a process fundamentally different from reproductive senescence, which has also been implicated in virulence and persistence for C. neoformans (25). Chronological aging refers to the effects of time on a cell after it has stopped growing, whereas generational age refers to the number of daughter cells produced by a given cell.…”

New surprises from within the black box of fungal melanization

“…Recent studies have begun to investigate the effects of age on C. neoformans cells to reveal other effects, like an increase in phenotypic switching frequency. 26 Nevertheless, most researchers use a standard overnight culture of cells grown in rich medium for starting inocula in experiments, a condition that is unlikely the wild or during infection are unknown, although there are candidate molecules for within the central nervous system and in the environment.…”

“…20 For melanin, the authors have previously shown that laccase is preformed in vesicles Further, an increase in phenotypic switching rates in aged cells could also provide better adaptability. 26 A prediction from these findings is that growth conditions may influence the outcome of an infection. This has been demonstrated independently, in other studies not focused on melanin, in which the culture conditions used prior to inoculating the cells into animals influences the rapidity of disease development.…”