Peroxisome Function Regulates Growth on Glucose in the Basidiomycete Fungus Cryptococcus neoformans

Abstract

The function of the peroxisomes was examined in the pathogenic basidiomycete Cryptococcus neoformans. Recent studies reveal the glyoxylate pathway is required for virulence of diverse microbial pathogens of plants and animals. One exception is C. neoformans, in which isocitrate lyase (encoded by ICL1) was previously shown not to be required for virulence, and here this was extended to exclude also a role for malate synthase (encoded by MLS1). The role of peroxisomes, in which the glyoxylate pathway enzymes are localized in many organisms, was examined by mutation of two genes (PEX1 and PEX6) encoding AAA (ATPases associated with various cellular activities)-type proteins required for peroxisome formation. The pex1 and pex6 deletion mutants were unable to localize the fluorescent DsRED-SKL protein to peroxisomal punctate structures, in contrast to wild-type cells. pex1 and pex6 single mutants and a pex1 pex6 double mutant exhibit identical phenotypes, including abolished growth on fatty acids but no growth difference on acetate. Because both icl1 and mls1 mutants are unable to grow on acetate as the sole carbon source, these findings demonstrate that the glyoxylate pathway can function efficiently outside the peroxisome in C. neoformans. The pex1 mutant exhibits wild-type virulence in a murine inhalation model and in an insect host, demonstrating that peroxisomes are not required for virulence under these conditions. An unusual phenotype of the pex1 and pex6 mutants was that they grew poorly with glucose as the carbon source, but nearly wild type with galactose, which suggested impaired hexokinase function and that C. neoformans peroxisomes might function analogously to the glycosomes of the trypanosomid parasites. Deletion of the hexokinase HXK2 gene reduced growth in the presence of glucose and suppressed the growth defect of the pex1 mutant on glucose. The hexokinase 2 protein of C. neoformans contains a predicted peroxisome targeting signal (type 2) motif; however, Hxk2 fused to fluorescent proteins was not localized to peroxisomes. Thus, we hypothesize that glucose or glycolytic metabolites are utilized in the peroxisome by an as yet unidentified enzyme or regulate a pathway required by the fungus in the absence of peroxisomes.

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

“…An understanding of the operation and regulation of the glyoxylate cycle and its integration with cellular metabolism will require further investigation of the participating metabolite transporters in the peroxisomal membrane. C. neoformans is clearly different in its metabolic capabilities because mutants lacking isocitrate lyase or malate synthase also fail to grow on acetate but retain full virulence (27,64). A difference for C. neoformans is also illustrated by the finding that pex1⌬ and pex6⌬ mutants with defects in peroxisomal function grew poorly on glucose, leading to speculation that peroxisomes might function in glycolysis or the regulation of glucose utilization (27).…”

“…C. neoformans is clearly different in its metabolic capabilities because mutants lacking isocitrate lyase or malate synthase also fail to grow on acetate but retain full virulence (27,64). A difference for C. neoformans is also illustrated by the finding that pex1⌬ and pex6⌬ mutants with defects in peroxisomal function grew poorly on glucose, leading to speculation that peroxisomes might function in glycolysis or the regulation of glucose utilization (27). However, C. neoformans mutants that are impaired in glucose utilization are attenuated for virulence and show decreased persistence in the central nervous systems of rabbits and mice (57).…”

Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

“…Similarly, the importance of ␤-oxidation for virulence is just now being investigated in C. neoformans, which, like U. maydis, is a basidiomycete pathogen with both the peroxisomal and mitochondrial pathways. In contrast to C. albicans, loss of isocitrate lyase and defects in pex genes do not reduce the virulence of C. neoformans in mice (24,48). In addition, there is evidence that the glyoxylate pathway may function outside the peroxisome in C. neoformans and that the peroxisome may function in glucose utilization (24).…”

“…In contrast to C. albicans, loss of isocitrate lyase and defects in pex genes do not reduce the virulence of C. neoformans in mice (24,48). In addition, there is evidence that the glyoxylate pathway may function outside the peroxisome in C. neoformans and that the peroxisome may function in glucose utilization (24). In a study performed in parallel with the work reported here and described in the accompanying article (28a), we recently showed that loss of ␤-oxidation functions reduced the virulence of C. neoformans, thus revealing this similarity between basidiomycete pathogens of plants and animals.…”

The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

“…We created a green fluorescent protein (GFP)-Cdc42 fusion to evaluate the interaction of Cdc42 and Rdi1 (47). A histone H3 promoter-GFP fusion (30) was cloned into the nourseothricin resistance genecontaining plasmid pCH233 (a gift from Christina Hull), utilizing EcoICRI and BamHI restriction sites to create the plasmid pCN19. The CDC42 gene from C. neoformans H99 was then amplified by PCR using primers modified with BamHI restriction sites on their 5Ј and 3Ј ends and subsequently cloned into pCN19 at the BamHI site to create the plasmid pCN20.…”