Regulation of cell shape and virulence in microbes by temperature
Progress in biology is driven both by medical necessity and scientific curiosity, and studies of fungal pathogenesis lie at the intersection of these two forces. As the numbers of immunocompromised patients rise, fungal infections continue to be a growing threat, yet our knowledge about how these eukaryotic organisms manipulate their host is far from adequate. Additionally, fungi display beautiful and intricate biology that can be explored on a molecular basis using genetics and genomics.
In our lab, we have built a number of fundamental tools to study the molecular biology of Histoplasma capsulatum (which is thought to be the number one cause of fungal respiratory infections in the world). We constructed the first shotgun microarray for this organism (before the genome was sequenced), used this microarray to perform the first large-scale analysis of temperature-regulated genes (Hwang et al., 2003) , obtained funding to sequence the genome of this organism in collaboration with William Goldman and the Genome Sequencing Center at Washington University, St. Louis, designed whole-genome oligonucleotide microarrays for use by all Histoplasma researchers, built a web-accessible open access database to integrate gene annotation and expression data (http://histo.ucsf.edu), developed a conditional promoter to study the consequences of mis-regulating genes (Gebhart et al.,
2006), and performed the first large-scale genetic screens to identify both genes that regulate the response to temperature (Nguyen and Sil, 2008, Webster and Sil, 2008) and genes that are required for H. capsulatum to colonize macrophages (Isaac et al, unpublished).
Research Focus and Rationale
Our research is driven by two key questions. First, how do cells sense temperature and make a developmental switch from the soil to the host program? We focus on temperature because it is a sufficient signal to recapitulate the morphologic switch between Histoplasma filaments (the soil form) and yeast (the host form) in culture (Maresca et al., 1994; Maresca and Kobayashi, 1989) . This question is critical to understanding the basic biology of Histoplasma as well as a number of closely related fungi such as Blastomyces , Coccidioides , and Paracoccidioides , each of which is a ubiquitous pathogen of immunocompetent hosts in endemic areas. In fact, one of the fascinating evolutionary questions about these environmental fungi is how regulatory circuits have evolved to link morphology and virulence programs with growth at host temperature. Additionally, studying the regulation of Histoplasma development by temperature will be an entry point to broader studies of host-fungal interactions, since it will define critical developmental changes that promote the expression of virulence traits, as well as delineate molecular landmarks that will allow us to stage the interactions of the fungus with host cells. We have identified three genes that are required for yeast-phase growth (Nguyen and Sil, 2008; Webster and Sil, 2008), and are currently studying how these genes are activated by temperature as well as their molecular function.
Fig. 1: Whereas wild-type cells grow in the yeast form at 37ºC, ryp mutants are unable to grow as yeast, and instead grow constitutively as filaments independent of temperature. A complemented ryp1 mutant, where a copy of the wild-type gene has been restored, is also shown.
Second, how does H. capsulatum defy the innate immune response to take up residence, often permanent, in immunocompetent hosts? The past ten years have witnessed an exponential increase in our understanding of the innate immune response to microbes, and yet, in the case of fungi, our insight is rudimentary at best. Our studies explore the molecular communication at the host-pathogen interface between H. capsulatum and the macrophage. H. capsulatum displays extremely robust macrophage colonization, so it is currently the best fungal candidate to probe the Achilles' heel of these powerful innate immune cells and determine novel mechanisms of virulence that have evolved in eukaryotic pathogens.
Fig. 2: Wild-type Histoplasma enters macrophages, grows intracellularly within the macrophage, and then lyses these host cells. Intact (uninfected) monolayers stain purple when exposed to the dye crystal violet, where monolayers infected with wild-type (WT) Histoplasma are cleared and do not stain efficiently with crystal violet. Macrophages infected with an auxotropic mutant (ura5), or a Histoplasma mutant that is unable to lyse macrophages (ldf) remain intact as indicated by crystal violet staining.
Maresca B, Carratł L, Kobayashi GS.(1994) Morphological transition in the human fungal pathogen Histoplasma capsulatum. Trends Microbiol.( 4):110-4.