Mutation of the Regulator of G Protein Signaling Crg1 Increases Virulence inCryptococcus neoformans

Abstract

The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans. A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MAT␣ strains of the fungus. Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains. In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12␣ but not on the mitogen-activated protein kinase homolog Cpk1. The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1. Interestingly, crg1 mutants of MAT␣ cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.Cryptococcus neoformans is a ubiquitous soil-borne basidiomycetous fungus that is the leading cause of fungal meningitis in high-risk individuals such as those with human immunodeficiency virus infection, high-dose chemotherapy, or solid organ and bone marrow transplants (27,31). Haploid strains of this pathogen are either of the MAT␣ or MATa mating types and, of the MAT␣ strains, the var. grubii accounts for Ͼ95% of clinical and environmental isolates, with only two MATa isolates being described thus far (33, 36). The MAT␣ mating-typeassociated virulence has been attributed to components of the MAT␣ mating-type locus, such as STE20␣, whose mutation resulted in attenuation of virulence (38). Mating, haploid differentiation, and the virulence traits of polysaccharide capsule and melanin production are regulated via two major G protein signal transduction pathways (1, 39). Crg1, a regulator of G protein signaling (RGS) protein homolog, was found to have a key role in the pheromone responsive mating of var. grubii MAT␣ and var. gattii strains (17,33). Thus, the identification of Crg1 has provided a new insight into the role of G protein signaling in the physiology of the fungus.The first RGS protein, initially identified from the baker's yeast Saccharomyces cerevisiae, acts as the GTPase-activating protein (GAP) for the G␣ subunit Gpa1 by terminating its activation (3,5,6,13,22). RGS proteins can also directly interfere with the activation of effector molecules (20, 24) or prevent spontaneous receptor-independent G protein signaling (34). RGS and RGS-like proteins are widely distributed throughout eukaryotic organisms and have an impressive array of regulatory functions affecting cellular growth and development (4,11,12,19). The present study reveals a novel role for an RGS protein in a pathogenic microorganism linking enhanced pheromone response to increased virulence. The resulting hypervirulent C. neoformans strain will be of significant importance in studying the mechanism for fungal virulence. MATERIALS AND METHODSStrains and media. C. neoformans var. grubii MAT␣ strain H99 and MATa strain KN99 and C. neoformans var. neoformans MAT␣ strain JEC21 and MATa strain JEC20 were previously de...

Transcription Factor STE12α Has Distinct Roles in Morphogenesis, Virulence, and Ecological Fitness of the Primary Pathogenic YeastCryptococcus gattii

“…Interestingly, C. neoformans var. grubii STE12␣ was later shown to be involved in reversal of hypervirulence of a crg1 mutant, which encodes the regulator of G protein signaling (89). Taken together, these findings suggested that STE12 is co-opted to perform distinct regulatory control functions between closely related C. neoformans varieties.…”

Clinical and Environmental Isolates of Cryptococcus gattii from Australia That Retain Sexual Fecundity

“…Of the MAT␣ isolates, the environmental isolates (n ϭ 51) were 92.2% VGI (21), robustly mating serotype C strains B4546 (MATa) and NIH312 (MAT␣), and their crg1 mutant derivatives JF109 (MATa crg1⌬) and JF101 (MAT␣ crg1⌬) (9). CRG1 encodes an RGS (regulator of G protein signaling) protein that attenuates pheromone signaling, and as a consequence, the crg1 mutation enhances pheromone response and mating (31,44). Mating ability for each test isolate was determined by coculturing sample isolates with tester strains on V8 medium in the dark at room temperature for 21 days.…”

Impact of Mating Type, Serotype, and Ploidy on the Virulence of Cryptococcus neoformans

“…The mating type locus of C. neoformans is far more complex than that of C. albicans and contains more than 20 genes, including genes encoding not only homeodomain sexual development regulators but also pheromones, pheromone receptors, and pheromone response pathway elements (22,43). Although the sexual regulator Sxi1␣ does not contribute to the virulence of C. neoformans (30,31), previous studies have suggested that pheromone production and sensing may occur during infection (17,21,56,74). It is possible that when both mating types are present in a single AD hybrid strain, some portion of the cells could behave as a cells, while other cells behave as ␣ cells, mimicking coinfection with ␣ and a cells.…”