Variation and Regulation of Phase-Specific Virulence Factors of Histoplasma capsulatum

Date of Award

Winter 12-15-2013

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Histoplasma capsulatum is one of the classic dimorphic fungal pathogens, undergoing a temperature-dependent phenotypic conversion from a multicellular hyphal form to a unicellular yeast form that is required to cause disease. H. capsulatum is found worldwide and is endemic to the Midwestern U.S. H. capsulatum causes histoplasmosis, a significant disease with a wide range of clinical manifestations from an acute respiratory infection to a life-threatening disseminated disease. Although histoplasmosis is most often seen in immunocompromised patients, H. capsulatum is a primary pathogen that can cause serious complications in immunocompetent hosts. Additionally, disease outcome appears to be dependent on the amount and strain of fungus inhaled.

The calcium-binding protein CBP is one of the main virulence factors in H. capsulatum and the major protein secreted in yeast culture. CBP1, similar to most of the virulence genes described in dimorphic fungi, is only expressed during the yeast phase. In order to identify a regulator of CBP1, I used Agrobacterium-mediated mutagenesis to generate a library of mutants to screen for clones that failed to regulate CBP1 in a phase-specific manner. I was able to identify several potential regulators of CBP1, and I focused my work on the characterization of two of them: the putative transcription factor Nit6p, and the ribosomal protein Yml20p. I also evaluated the importance of CBP in a strain-specific manner, comparing levels of secretion, protein stability, and the requirement for virulence among three phylogenetically distinct Histoplasma strains (G186A, G217B and WU24). I was able to show that although CBP is critical to progression of infection and virulence, there are strain-specific differences for the CBP requirement during H. capsulatum infection.

I also performed a comprehensive parallel strain comparison of G186A, G217B, and WU24 that covered the complete course of infection (acute and resolution phases of disease) and included several parameters to measure virulence in addition to fungal burdens. The strain comparison led to a more detailed characterization of WU24, a recent clinical H. capsulatum isolate that was collected from an HIV-positive individual in North America. In contrast to previously tested isolates from the same lineage, WU24 infected both macrophages and wild-type mice. I also showed that WU24 virulence is dependent on the presence of cell wall α-(1,3)-glucan, another well-studied H. capsulatum virulence factor that is not required for virulence in many other North American strains. Surprisingly, comparison of WU24 with the two previously characterized isolates G186A and G217B revealed that many conclusions regarding relative strain virulence and certain hallmarks of histoplasmosis are dependent on the inoculum size.

Language

English (en)

Chair and Committee

William E Goldman

Committee Members

Tamara L Doering, Michael G Caparon, Daniel E Goldberg, Paul H Schlesinger, William E Goldman, Jennifer K Lodge

Comments

Permanent URL: https://doi.org/10.7936/K7VM4964

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