Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Abstract

Cryptococcus neoformans is a basidiomycetous, the etiologic agent of cryptococcosis, yeast-like fungus which, following inhalation from an environmental source causes respiratory and neurological disease in humans and animals. Cryptococcal infections can be fatal if the host does not have adequate T-cell-dependent immune function. Most important predisposing factors for infection with C. neoformans are virus immunodeficiency infection, use of immunosuppressive drugs, organ and bone marrow transplantation, chronic leukemia, and lymphomas (Dismukes 1988, Velegraki, et al. 2001, Horta et al. 2002. Kwon-Chung et al. (1982) established two varieties that were recognized: var. neoformans and var. gattii. Currently we recognize 3 varieties: grubii -serotype A-, gattiiserotype B, C, -and neoformans -serotype D (Franzot et al. 1999). Most likely, var. gattii will be renamed as a novel species and called C. bacillisporus (Diaz et al. 2000).This fungus appears in yeast form, presenting spherical cells (5-100 µm in diameter) with single or multiple buds. The colonies are cream-colored, glossy, viscous, and moist, the amount of mucous being directly related to the size of the capsule. Among the various virulence factors, the most significant are capsular polysaccharides such as glucuronoxylan, galactoxilamanane, and mannoproteins (Kurtzman & Fell 1998, Lacaz et al. 2002, Chiapello et al. 2003, Ellerbroek et al. 2004.The samples of C. neoformans maintained in our Fungus Collection are especially useful for biotechnology projects, scientific research related to antifungal therapy, determination of molecular characteristics, production antigen, and instructional programs, as well as functional as reference cultures. Various preservation techniques are used: successive subculture on solid medium (with or without the addition of mineral oil); spore cultivation (in soil, sand, or silica gel); lyophilized; liquid nitrogen storage; and suspension in distilled water (Castellani 1963, 1967, Urdaneta et al. 1965, Bosmans 1974, Rodrigues et al. 1992, Cavalcanti & Cavalcanti 1994, Spencer & Spencer 1996.With the advent of molecular biology, various techniques have come to be used to analyze the genotype of the potential polymorphism of fungus samples. These include randomly amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis, (PFGE), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP). The cost of performing the RFLP, PFGE, and AFLP techniques is prohibitive. However, the RAPD method can be performed at a relatively low cost. This technique suits our needs perfectly, not only due to its cost-effectiveness but also to its reproducibility (Willians 1990).In our Fungus Collection, samples of C. neoformans have been maintained in successive subcultures since 1930. In the 1970s, some of these samples were selected to be stored using the lyophilization technique. The preservation method of choice in our laboratory is still successive subculture since it is considered a simple method...

Molecular diversity of serial Cryptococcus neoformans isolates from AIDS patients in the city of São Paulo, Brazil

“…These fungal mutations may occur in humans or in the environment. Our results are similar to those obtained by other authors analyzing colonies immediately isolated from patients (Sullivan et al 1996, Jain et al 2005, Almeida et al 2007, and in vitro colonies maintained for a long time in laboratory (Franzot et al 1998, Cavalcante et al 2007). In addition, it has been recently shown that the genome sequence of C. neoformans has a significant quantity of transposon elements (Loftus et al 2005) that may have participated in the yeast microevolution.…”

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

“…This phenomenon, called microevolution, has been reported by several authors (Franzot et al 1998, Blasi et al 2001, Cavalcante et al 2007). Key words: Cryptococcus neoformans -virulence factors -pathogenicity -molecular typing C. neoformans can cause life-threatening fungal infections in immunocompromised individuals.…”

“…Although this result demonstrated that animal passage did not change the features involved, it is possible that the time period (7 days) was insufficient for microevolution. According to several studies, microevolution occurred over longer periods of time and after several passages, as an adaptation to the host (Currie et al 1995, Franzot et al 1998, Blasi et al 2001, McClelland et al 2005, Clancy et al 2006, Cavalcante et al 2007. One exception occurred with urease.…”

Viability ofHanseniaspora uvarumyeast preserved by lyophilization and cryopreservation

“…The lyophilization process has been applied to yeast and filamentous fungi with good recovery results, preserving the phenotypic and genotypic characteristics of micro‐organisms for prolonged periods (Cavalcante et al, ; McGinnis et al, ). However, some authors have reported morphological changes after lyophilization, or even the non‐viability of the fungus in freezing prior to lyophilization (Voyron et al, ; Paoli, ), a fact that was not observed in the H. uvarum strain preserved for 12 months in the present study.…”