The Cryptococcus neoformans transcriptome at the site of human meningitis

As an investigator in John Perfect's laboratory, I have continued to study the molecular pathogenesis of Cryptococcus neoformans. We are interested in identifying virulence genes expressed by the fungus during infection using our rabbit model of cryptococcal meningitis and during in vitro exposure to the pathological temperature of 37 degrees C. By PCR differential display, we are identifying genes associated with pathogenesis of the organism and assessing their effects at the molecular level using a 2-hybrid system. In addition, we are disrupting targeted virulence genes using biolistic transformation and assessing their role in the virulence of C neoformans in rabbit and murine models of cryptococcal disease.

Key words: Cryptococcus neoformans
Virulence
Molecular Pathogenesis
Transformation

Among patients with AIDS, leukemia or other cancers, organ or bone marrow transplants, and similar immunocompromising risk factors, the incidence of opportunistic mycoses and the number of different fungal pathogens are increasing dramatically. For many of these fungi, the definition of a species and the recognition of pathogen are highly problematic. Conventional methods of identification are based on morphological and physiological characteristics and are often time-consuming, difficult to interpret, and inconsistent. This laboratory is using DNA-based methods to (i) identify fungal pathogens, (ii) resolve taxonomic issues, (iii) facilitate epidemiological studies, (iv) recognize strains with clinically relevant phenotypes, such as resistance to antifungal drugs, (v) elucidate the origin(s) of diversity and the population genetics of the major pathogens, and (vi) explore functional genomics to identify virulence factors. We have developed reliable methods to genotype strains and are analyzing gene sequences to clarify the phylogeny of controversial taxa.

To conduct rigorous population studies of Candida albicans, we developed single-locus markers based on polymorphisms of PCR products. Genotypic frequencies and segregation patterns at these loci have confirmed that C. albicans is diploid and suggest that some form of recombination occurs in this "asexual" yeast. To investigate whether separate populations of C. albicans exist in disparate geographical locations, we compared strains collected from healthy and HIV-infected persons in U.S. and Brazil. Although a number of different genotypes were recognized at each location, the same multilocus genotype was prevalent among the clinical isolates, indicating a remarkable homogeneity among these populations.

We are using DNA-based methods to compare global isolates of Cryptococcus neoformans from patients with AIDS and other sources, to analyze the distribution and relatedness of strains, to identify genotypes of clinical importance, and to create linkage map of this pathogen. To determine the source of C. neoformans in patients, we developed a genetic markers to investigate the structure of clinical and environmental populations. With analysis of quantitative trait loci, specific genotypes will be identified that represent clones that have significantly diverged with respect to clinically relevant phenotypes, including susceptibility to antifungal drugs and the expression of virulence factors. We are investigating genomic evolution and phenotypic variation in natural populations of C. neoformans. These approaches will correlate genotypes with pathobiological phenotypes, leading to beneficial and predictive information about the epidemiology, diagnosis and prognosis of cryptococcosis in patients with AIDS.

Research in my laboratory focuses around several aspects of medical mycology. We are investigating antifungal agents (new and old) in animal models of candida and cryptococcal infections. We have examined clinical correlation of in vitro antifungal susceptibility testing and with in vivo outcome. Our basic science project examines the molecular pathogenesis of cryptococcal infections. We have developed a molecular foundation for C. neoformans, including transformation systems, gene disruptions, differential gene expression screens, and cloning pathogenesis genes. The goal of this work is to use C. neoformans as a model yeast system to identify molecular targets for antifungal drug development. There are a series of clinical trials in fungal infections which are being coordinated through this laboratory and my work also includes a series of antibiotic trials in various aspects of infections. Finally, we have now been awarded a NIH sponsored Mycology Unit for 5 years with 6 senior investigators which is focused on C. neoformans as a pathogenic model system, but will include multiple areas of medical mycology from diagnosis to treatment.