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Rac proteins control polarized growth in many organisms but the specific function of these proteins remains undefined. In this study, we describe the cloning and functional characterization of a RAC homolog, cflB, from the dimorphic fungus Penicillium marneffei. P. marneffei produces asexual spores on complex structures (conidiophores)and switches between hyphal and yeast growth. CflB colocalizes with actin at the tips of vegetative hyphal cells and at sites of cell division. Deletion of cflB results in cell division (septation) and growth defects in both vegetative hyphal and conidiophore cell types such that cells become depolarized, exhibit inappropriate septation and the actin cytoskeleton is severely disrupted. This data suggests that Rac proteins play a crucial role in actin dependent polarized growth and division. The CDC42 ortholog in P. marneffei, cflA, controls vegetative hyphal and yeast growth polarization but does not affect asexual development. By contrast, CflB affects cellular polarization during asexual development and hyphal growth but not during yeast growth. This shows that these two GTPases have both overlapping and distinct roles during growth and development. RAC orthologs are not found in less morphologically complex eukaryotes such as Saccharomyces cerevisiae, suggesting that RAC genes might have evolved with increasing cellular complexity.

The ability to adapt to a changing environment provides a selective advantage to microorganisms. In the case of many pathogens, a large change in their environment occurs when they move from a natural setting to a setting within a human host and then during the course of disease development to various locations within that host. Two clinical isolates of the human fungal pathogen Cryptococcus neoformans were identified from a collection of environmental and clinical strains that exhibited a mutator phenotype, which is a phenotype which provides the ability to change rapidly due to the accumulation of DNA mutations at high frequency. Whole-genome analysis of these strains revealed mutations in MSH2 of the mismatch repair pathway, and complementation confirmed that these mutations are responsible for the mutator phenotype. Comparison of mutation frequencies in deletion strains of eight mismatch repair pathway genes in C. neoformans showed that the loss of three of them, MSH2, MLH1, and PMS1, results in an increase in mutation rates. Increased mutation rates enable rapid microevolution to occur in these strains, generating phenotypic variations in traits associated with the ability to grow in vivo, in addition to allowing rapid generation of resistance to antifungal agents. Mutation of PMS1 reduced virulence, whereas mutation of MSH2 or MLH1 had no effect on the level of virulence. These findings thus support the hypothesis that this pathogenic fungus can take advantage of a mutator phenotype in order to cause disease but that it can do so only in specific pathways that lead to a mutator trait without a significant tradeoff in fitness.