A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen.

A Temperature-Responsive Network Links Cell Shape and Virulence Traits in a Primary Fungal Pathogen Sinem Beyhan1, Matias Gutierrez1¤, Mark Voorhies1, Anita Sil1,2* 1 Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America, 2 Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, United States of America Abstract Survival at host temperature is a critical trait for pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are soil fungi that undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. How these organisms link changes in temperature to both morphologic development and expression of virulence traits is unknown. Here we elucidate a temperature-responsive transcriptional network in H. capsulatum, which switches from a filamentous form in the environment to a pathogenic yeast form at body temperature. The circuit is driven by three highly conserved factors, Ryp1, Ryp2, and Ryp3, that are required for yeast-phase growth at 37uC. Ryp factors belong to distinct families of proteins that control developmental transitions in fungi: Ryp1 is a member of the WOPR family of transcription factors, and Ryp2 and Ryp3 are both members of the Velvet family of proteins whose molecular function is unknown. Here we provide the first evidence that these WOPR and Velvet proteins interact, and that Velvet proteins associate with DNA to drive gene expression. Using genome-wide chromatin immunoprecipitation studies, we determine that Ryp1, Ryp2, and Ryp3 associate with a large common set of genomic loci that includes known virulence genes, indicating that the Ryp factors directly control genes required for pathogenicity in addition to their role in regulating cell morphology. We further dissect the Ryp regulatory circuit by determining that a fourth transcription factor, which we name Ryp4, is required for yeast-phase growth and gene expression, associates with DNA, and displays interdependent regulation with Ryp1, Ryp2, and Ryp3. Finally, we define cis-acting motifs that recruit the Ryp factors to their interwoven network of temperature-responsive target genes. Taken together, our results reveal a positive feedback circuit that directs a broad transcriptional switch between environmental and pathogenic states in response to temperature. Citation: Beyhan S, Gutierrez M, Voorhies M, Sil A (2013) A Temperature-Responsive Network Links Cell Shape and Virulence Traits in a Primary Fungal Pathogen. PLoS Biol 11(7): e1001614. doi:10.1371/journal.pbio.1001614 Academic Editor: Joseph Heitman, Duke University Medical Center, United States of America Received February 1, 2013; Accepted June 12, 2013; Published July 23, 2013 Copyright: ß 2013 Beyhan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH (grant R01AI066224 and R01AI093640), University of California Laboratory Fees Research Program Grant (118833), and HHMI Early Career Scientist Award to A.S.; and NIH (grant T32AI060537-06), American Lung Association Senior Research Training Fellowship, and an American Cancer Society Postdoctoral Fellowship to S.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ChIP, chromatin immunoprecipitation; FPS, filamentous-phase–specific; YPS, yeast-phase–specific. * E-mail: ¤ Current address: BioImaging Lab, Bioquimica.cl S.A., Bustos 2365, Providencia, Santiago, Chile and Facultad de Ciencias Biológicas, Universidad Andrés Bello, Av. República 237, Santiago, Chile disease in the host [3]. For all thermally dimorphic fungi, host temperature is the key signal that triggers this developmental switch, but little is known about the coordinated induction of morphologic changes and virulence gene expression by temperature. Histoplasma capsulatum, which is endemic to the Ohio and Mississippi River Valleys of the United States, can cause lifethreatening respiratory and/or systemic disease (histoplasmosis) [2,4]. It is estimated that up to 25,000 people develop lifethreatening infections in endemic regions each year, with at least 10-fold more mild or asymptomatic infections [2,4]. Although the pathogen propagates as spores and in a filamentous form in the environment, H. capsulatum is found almost exclusively in the yeast form within mammalian hosts. Despite the prevalence of H. capsulatum and its threat to human health, we have a limited understanding of the transcriptional regulatory network that governs pathogenic yeast-phase growth. Previously, we identified three regulators, Ryp1, Ryp2, and Ryp3, and showed that they are Introduction Cells adapt to their environment by responding to specific environmental stimuli such as light, temperature, and nutrients. For microbial pathogens, mammalian body temperature can signal the induction of pathways required for host colonization and pathogenesis [1]. One such group of organisms is the thermally dimorphic fungal pathogens, which include Coccidioides, Paracoccidioides, Blastomyces, and Histoplasma species. These evolutionarily related fungi are notable among fungal pathogens in that they all cause disease in healthy individuals [2]. Each of these organisms grows in a mold form in the soil, forming long, connected filaments that produce vegetative spores [3]. When the soil is aerosolized, filamentous cells and spores can be inhaled by mammalian hosts and converted into a parasitic form within the host lung. Conversion entails a dramatic change in cell shape to a budding yeast form for the majority of these pathogens, as well as the transcriptional induction of virulence genes required to cause PLOS Biology | www.plosbiology.org 1 July 2013 | Volume 11 | Issue 7 | e1001614 Regulation of Fungal Virulence by Temperature ortholog VosA, the Ryp3 ortholog VelB, and VeA itself) act together to regulate asexual and sexual development and secondary metabolism [19]. Notably, since Velvet family proteins do not contain canonical DNA binding domains or other domains of known function, their mechanistic role in regulation of developmental processes is unclear. As noted above, both WOPR and Velvet family proteins are widely distributed among fungi, although the Hemiascomycetes, including Saccharomyces and Candida species, lack Velvet family proteins. Since both families of proteins are required for yeastphase growth in H. capsulatum, we explored if and how these two distinct classes of fungal regulators work together to govern temperature-responsive traits b (...truncated)