Phenotypic switching of Cryptococcus neoformans occurs in vivo and influences the outcome of infection

Abstract

IntroductionCryptococcosis affects 5-30% of patients with AIDS. It is caused by Cryptococcus neoformans, an encapsulated yeast that is acquired by inhalation and ultimately results in a chronic meningoencephalitis with a high tendency to relapse despite effective antifungal therapy (1). Several virulence factors (2-5) including a polysaccharide capsule (6, 7) are described for C. neoformans. Most virulence factors are invariably present (8) in clinical strains. Genetic inactivation of their regulatory proteins, which are part of signaling pathways, renders the yeast avirulent (9, 10). It is, however, unclear whether regulation of virulence factors occurs in vivo during chronic infection in order to enhance the virulence of an infecting C. neoformans strain. Therefore, it is often assumed that failure to eradicate the fungus is a function of a diminished cellmediated immune response (CMI). Several observations, however, indicate that pathogen-specific factors contribute to the pathogenesis of cryptococcosis. First, prolonged in vitro and in vivo passage of C. neoformans isolates can result in phenotypic changes associated with differences in virulence (11)(12)(13)(14). Second, serial isolates from chronically infected patients can exhibit differences in virulence (15). Third, C. neoformans variety gattii strains cause symptomatic infections primarily in immunocompetent hosts even in areas where AIDS is endemic (16).Reversible phenotypic switching can result in microevolution, defined as rapid changing of an organism to a stable, inherited phenotype (17). Microevolution and phenotypic switching can be achieved by several mechanisms, some of which involve changes of genes and follow a mendelian inheritance pattern whereas others (e.g., silencing) are epigenetic and inherited in a nonmendelian fashion. For certain pathogens, the emergence of phenotype variants is associated with invasion of mucosal barriers and immune evasion (18). For fungi, phenotypic switching is an in vitro phenomenon defined as the spontaneous emergence of colonies with altered colony morphology at rates higher than the somatic mutation rates (19-21). Phenotypic switching was demonstrated for C. neoformans in vitro in three different clinical strains and resulted in enhanced virulence (22,23). Despite an association with virulence, phenotypic switching in vivo has been demonstrated only in pathogens with complex life cycles that require the switch for survival in the host (24). Most infections require high doses, and because of the expected in vitro switching rates (10 -2 to 10 -5 ), these inocula are likely to be contaminated with in vitro switch variants. Therefore, it is difficult to demonstrate that the emergence of new phenotypes in the infected host are the result of in vivo switching events and do not represent in vivo selection of variants from an initially heterogeneous pathogen population (25). Phenotypic switching has been linked to the virulence of many pathogens, including fungi. However, it has not been conclusively shown to occur...

Characterization of Phenotypic Switching in Cryptococcus neoformans Biofilms

“…High-frequency phenotypic switching is a phenomenon widely investigated for planktonic yeasts, including C. neoformans [4]. C. neoformans can undergo phenotypic switching in vitro to colony types that differ in their virulence in mice [14]. It is reasonable to speculate that phenotypic switching can also occur during cryptococcal biofilm formation.…”

“…Therefore, the characteristics of C. neoformans phenotypic switching during biofilm formation were investigated. These phenotypes show variations in their capsular polysaccharide [14][15][16].…”

Phenotypic Switching of Cryptococcus neoformans and Cryptococcus gattii

“…grubii isolates were smooth in colony morphology whereas 100% of C. gattii were mucoid [25]. Switching in a smooth (SM) parent strain (RC2, serotype D) that switches to mucoid (MC) variants and reversibly in vitro and in vivo has been thoroughly investigated [30]. However, other colony morphologies in switch variants such as serrated, wrinkled, and pseudohyphal are also described but these rare colony phenotypes are not usually seen in clinical isolates.…”

Aislamiento de Cryptococcus gattii serotipo B a partir de detritos de Eucalyptus spp. en Colombia.

“…Se evaluó el crecimiento de los aislamientos en agar glucosado de Sabouraud a 37°C (19) y se estudió la virulencia en un modelo animal (23). Se usaron ratones BALB/c machos de 3 a 5 semanas, obtenidos en el bioterio del INS.…”

“…Los sobrevivientes se sacrificaron el día 70 posinoculación, empleando eutanasia con CO 2 . Se obtuvieron el cerebro, el bazo y el pulmón de cada animal, los cuales se procesaron para determinar el número de UFC/g de órgano en agar glucosado de Sabouraud (23,24).…”