Progress in biology is driven both by medical necessity and by scientific curiosity. Fungal pathogenesis is at the intersection of these two forces. As the numbers of immunocompromised patients rise, fungal infections are a growing threat, yet our knowledge about how these eukaryotic organisms manipulate their host is inadequate. Our laboratory studies the human pathogen Histoplasma capsulatum, which provides a ripe opportunity to delve into the cell biology of both the microbe and the relevant host cells. Our goal is to use functional genomics and genetics to generate a molecular understanding of how cell-cell interaction, signal transduction, gene regulation, and other fundamental biological processes influence pathogenesis.

Though H. capsulatum was first identified as a lethal fungal pathogen in 1906, both its biological mysteries and pathogenic ingenuity remain largely unexplored. An ongoing genome sequencing project (a collaboration between our lab, the laboratory of William Goldman, and Elaine Mardis at the Washington University Genome Sequencing Center) is now providing a wealth of information about H. capsulatum. In our laboratory, we have developed functional genomic tools for H. capsulatum (Hwang et al., Molecular Biology of the Cell, 14, 2314-2326, 2003). We are also launching genetic approaches so that we can use a combination of genomics and genetics to ask the following questions:

1. How does H. capsulatum establish and maintain two morphologic forms, one of which is important for initial infection, the other of which is important for disease?

H. capsulatum is a dimorphic fungus, meaning that it grows in two forms or morphologies. H. capsulatum grows in the soil in a filamentous, or mycelial, form. These long chains of cells produce asexual spores. Both these spores and mycelial fragments can aerosolize if the soil is disturbed. Once introduced into the host via inhalation, H. capsulatum converts to a budding yeast form. The ability of H. capsulatum to grow in both a mycelial form in the environment and a yeast form once it is in the host is critical for the establishment and progression of disease. Mycelia and yeast have obvious morphologic differences, but little is understood about the molecular differences between them. We have completed a large-scale gene expression analysis of the two forms to identify genes specific to one form or the other (Hwang et al., Molecular Biology of the Cell, 14, 2314-2326, 2003). Genes specific to the mycelial phase include orthologs of genes involved in conidiation, cell polarity, and melanin production in other organisms. Genes specific to the yeast phase include several involved in sulfur metabolism, extending previous observations that sulfur metabolism influences morphology in H. capsulatum. Other yeast-phase induced genes included several implicated in nutrient acquisition and cell-cycle regulation. We are currently using molecular genetic approaches to determine the precise function of these genes in the establishment and maintenance of the yeast and mycelial forms. We are also developing a library of insertion mutants with the goal of identifying genes that are required for establishment or maintenance of each form.

2. What is the genetic program used by Histoplasma to survive and replicate within macrophages and within the host?

Once inside the host, H. capsulatum enters and grows within macrophages. Macrophages kill most microbes, but H. capsulatum interferes with the microbicidal powers of the macrophage. We are using genomic and genetic approaches to understand how H. capsulatum subverts macrophages. We identified 30 H. capsulatum genes that are induced when the fungus is inside a macrophage. We are currently determining their identity and function. We are also building molecular genetic tools that will allow us to identify H. capsulatum genes required for survival in macrophages.

3. How does Histoplasma persist in the host in a latent form for many years?

H. capsulatum survives in the host in a latent form that can reactivate if there is a decline in the host immune response. H. capsulatum persists in the host despite exposure to nitric oxide (NO), an antimicrobial effector produced by host cells. To understand how H. capsulatum copes with NO, we have examined its transcriptional response to an NO donor by microarray. We identified an ortholog of a nitric oxide reductase that may play a role in NO detoxification. We are characterizing the regulation and function of this gene, and others, in infection and persistence.

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