Anita Sil - US grants

Fungal infections are a growing medical threat, particularly for immunocompromised individuals, yet little is understood about the molecular basis of immune evasion by fungal pathogens. The long-term goal of this research is to understand how intracellular fungal pathogens subvert host defenses. This aim will fundamentally interface with the goal of this program project application, which is to understand how diverse pathogens such as Francisella tularensis, Listeria monocytogenes, and Mycobacterium tuberculosis manipulate macrophage responses. We hypothesize that a comparative analysis of macrophage responses to these pathogens, in addition to a fungal intracellular pathogen, is a more powerful means of dissecting host response than studying any single intracellular pathogen in isolation. Our model system is Histoplasma capsulatum, an intracellular fungal pathogen that survives and replicates in the phagosome of macrophages. How H. capsulatum colonizes a niche that is normally hostile to microbes is a mystery. We hypothesize that H. capsulatum produces gene products that inhibit acidification of the host phagosome, thereby allowing fungal cells to survive and grow intracellularly. The objective of this proposal is to use molecular genetic approaches to uncover which H. capsulatum molecules are required for intracellular survival and growth. Furthermore, functional genomics will be used to identify host genes and pathways that are targeted by H. capsulatum. Our specific aims are to (1) use well-established assays to characterize the stages of infection of murine bone-marrow derived macrophages with H. capsulatum; (2) perform a genetic screen to identify Histoplasma genes required for survival and growth in macrophages; and (3) compare the global gene expression profile of macrophages during infection with wild-type or mutant H. capsulatum and the other intracellular pathogens mentioned above. These studies will identify regulatory circuits in host cells that are manipulated by H. capsulatum and other key intracellular pathogens relevant to biodefense.

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine how environmental signals such as temperature regulate morphology and virulence in the fungal pathogen Histoplasma capsulatum. H. capsulatum grows in a filamentous form in the soil; once inhaled into a mammalian host, these cells switch their growth program to a parasitic yeast form that subverts the innate immune system to cause disease. A similar switch from soil to host form is observed for the evolutionarily related systemic dimorphic fungal pathogens, which include H. capsulatum as well as select agent pathogens such as Coccidioides species. For all of these pathogens, temperature is a key signal that regulates this morphogenetic switch, which is thought to be essential for H. capsulatum virulence. By elucidating how H. capsulatum cells sense and respond to host temperature, we will define critical molecular landmarks that promote changes in morphology as well as the expression of virulence traits. These studies will shed light on fundamental processes such as signal transduction and gene regulation, as well as uncover the role of temperature-dependent pathways in fungal pathogenesis. Over the last funding period, we identified the first transcriptional regulators required for growth in the yeast form in response to host temperature. These factors, named Ryp1, Ryp2, and Ryp3, are homologous to key developmental regulators in other fungi, and represent critical elements of the temperature-dependent regulatory circuit in H. capsulatum. Interestingly, the precise biochemical function of the orthologous regulators in other fungi is unclear, thus adding additional significance to our functional characterization in H. capsulatum. We have recently shown that the Ryp proteins associate with the upstream regions of both morphology and virulence genes, suggesting that they directly regulate essential components of the morphology and virulence programs. Furthermore, these data provide the first molecular evidence that the transcriptional regulation of morphology and virulence factors is coupled. In this proposal, our specific aims are to investigate and identify (1) the molecular mechanism of activation of the Ryp proteins by temperature; (2) the mechanisms by which the Ryp proteins regulate gene expression; and (3) the elements of the Ryp-dependent regulatory circuit that lie downstream of these factors and their effect on morphology and virulence. Taken together, the resultant data will greatly enhance our understanding of the molecular response pathway of H. capsulatum to host temperature. Additionally, we will gain valuable knowledge about individual morphology and virulence factors that are required for H. capsulatum pathogenesis in the host. PUBLIC HEALTH RELEVANCE: Histoplasma capsulatum is a primary pathogen that infects approximately 500,000 individuals per year in the U.S. and is a significant source of morbidity and mortality in immunocompromised patients. Since very little is understood about how this fungus causes disease, the identification of fungal factors that influence pathogenesis and manipulate the host immune response will significantly advance the field and allow for the development of new therapeutics.

The environmental fungal pathogens Coccidioides and Histoplasma have been proposed as agents of warfare because of their easy availability, stability of spores, and infection by aerosolization (Casadevall and Pirofski, 2006); thus more knowledge about the pathogenesis of these closely related organisms is critical to biodefense. Each of these organisms converts to a parasitic form in mammalian hosts after inhalation of infectious spores from the soil. We aim to understand pathogenesis of these fungi by identifying fungal genes that promote disease and manipulate the host. To do so, we propose a collaboration between two biologists who study human pathogenic fungi, one experienced in fungal evolutionary biology (John Taylor, UC Berkeley) and one experienced in fungal molecular and developmental molecular biology (Anita Sil, UCSF). Use of a comparative genomics approach to analyze Coccidioides and Histoplasma is likely to be fruitful because of their close evolutionary relationship and the similarities of their lifestyles in the soil and the host. We will take advantage of information inherent in the genomes of these pathogens and related fungi to generate a set of genes most likely to influence disease. This comparative approach will reveal genes that have undergone strong positive selection in these fungal pathogens. Additionally, we will identify conserved genes in Coccidioides and Histoplasma that are implicated in the conversion to the parasitic form of each organism by virtue of their role in Histoplasma. These studies will allow us to prioritize a testable number of candidate virulence genes that will be assessed for their role in pathogenicity in the mouse models of Coccidioides and Histoplasma infection. In addition, these studies will identify organism-specific targets for diagnosis, therapy and vaccination, and thus are designed to interface closely with Coccidioides projects proposed by Marc Orbach and John Galgiani.

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand how the fungal pathogen Histoplasma capsulatum causes disease. H. capsulatum is a highly virulent pathogen that causes significant morbidity in both immunocompromised and immunocompetent individuals, with approximately 500,000 Histoplasma infections estimated to occur every year in the U.S. H. capsulatum is a pathogen of macrophages, which phagocytose microbes and digest them via an arsenal of microbicidal mechanisms. In contrast to most microbes, H. capsulatum replicates to high levels in the macrophage phagosome. Colonization of the macrophage is followed by host-cell death and release of live yeast cells, but the mechanism that triggers host-cell death is unknown. We have determined that the previously identified Histoplasma factor Cbp1 is dispensable for high intracellular fungal burden, but required for host-cell lysis. Additionally, our preliminary studies uncovered that Cbp1 is required to induce transcription of a specific and limited set of macrophage genes during infection, which we have named the Histoplasma response cluster (HRC). We hypothesize that Cbp1 interacts with unknown host factors, resulting in the induction of this unique transcriptional signature as well as host-cell death. Here we will (1) investigate whether Cbp1 triggers host-cell death by established or novel pathways, (2) determine which molecular characteristics of Cpb1 are important for host cell death, (3) establish if Cbp1 is required for pathogenesis of human macrophages, and (4) assess the role of Cbp1 in host-cell death and inflammation in the mouse model of histoplasmosis. These studies will generate new paradigms of virulence strategies used by human fungal pathogens during infection. Additionally, identifying host pathways that are potential targets of Cbp1, as well as understanding how Cbp1-modulated host-cell death contributes to disease progression, will significantly enrich our understanding of how eukaryotic pathogens have evolved to manipulate their mammalian hosts.

DESCRIPTION (provided by applicant): Natural products are usually isolated from free-living terrestrial or aquatic organisms that have no connection with human biology. In contrast, microbes that have evolved to live in close association with humans are especially likely to synthesize natural products that inhibit key human targets, and remain underexplored as a source of novel molecules. We propose a systematic approach to identify natural products from the fungal pathogen Histoplasma capsulatum, which is a soil organism that spends much of its time in the mammalian lung. In terms of identifying new natural products that bind novel human targets, Histoplasma is a particularly compelling choice for the following reasons: (1) It has evolved to live within human immune cells. Although other fungi are capable of colonizing immunocompromised hosts, very few can colonize immunocompetent hosts (a more difficult task that indicates that H. capsulatum can manipulate the normal innate immune response). (2) It is straightforward to grow production-scale cultures of Histoplasma for natural product isolation. (3) Histoplasma molecular genetics allows the straightforward construction of mutants that lack putative biosynthetic enzymes. (4) Although analysis of the genome predicts that each strain of Histoplasma produces 10-20 natural products, neither Histoplasma nor any of its close relatives have ever been mined for novel molecules by an academic or industrial natural product discovery effort. In sum, since Histoplasma is an intracellular pathogen that subverts the innate immune response to colonize host immune cells by unknown mechanisms, we hypothesize that Histoplasma natural products may be enriched for novel compounds that modulate targets in mammalian hosts. We will combine the expertise of the Sil lab in Histoplasma biology and molecular genetics with the expertise of the Fischbach lab in natural product discovery to comprehensively identify and characterize natural products secreted by Histoplasma. We propose a three-pronged approach: (1) Based on our preliminary data, we will take a microbial approach, growing large quantities of Histoplasma under laboratory conditions that recapitulate its free-living and host-associated environments. Utilizing standard expertise in the Fischbach lab, we will purify milligram quantities of 4-6 abundant natural products by preparative HPLC, and use high-resolution MS and NMR to solve their structures. (2) Taking advantage of expertise in the Sil lab, we will take a complementary genetic approach by using RNA interference to deplete Histoplasma cells of individual natural product biosynthetic genes. Both predictive algorithms from the Fischbach lab and the resultant changes in the HPLC/MS profile in supernatant extracts from the mutant strains will allow us to correlate particular molecules with their biosynthetic genes. (3) We will execute a series of in vitro and in vivo assays to determine the bioactivity of natural products identified by this project, with an emphasis on the identification of biosynthetic genes and natural products that contribute to disease pathogenesis. These exploratory studies of natural product biology in a ubiquitous, human-associated microbe have strong potential for uncovering new molecules that modulate communication at the host-pathogen interface.

? DESCRIPTION (provided by applicant): The long-term goal of our research is to elucidate the mechanism of action of key fungal molecules that mediate pathogenesis. In recent years, proteomics technologies and analyses have advanced to the point where comprehensive, genome-wide studies of host-pathogen interactions can be performed. We propose to apply these technologies to identify mammalian host proteins that interact with secreted fungal proteins from the intracellular fungal pathogen Histoplasma capsulatum. This work is a collaboration between a fungal biologist experienced in Histoplasma molecular biology and genetics (Anita Sil, UCSF) and a premiere expert in using proteomics to identify host-pathogen interactions (Nevan Krogan, UCSF). After inhalation into mammalian hosts, Histoplasma senses the temperature of the host and undergoes a morphologic transition to yield yeast-form cells. Yeast cells colonize macrophages and must evade anti-microbial defenses to replicate to high levels within these immune cells. Based on precedent from viral and bacterial pathogens, we hypothesize that secreted factors from Histoplasma yeast cells are likely to interact with intracellular host proteins to manipulate the outcome of infection. We have used experimental data and bioinformatics analyses to identify secreted fungal proteins with preferential expression in the yeast form of Histoplasma. We will use a robust proteomics pipeline developed by the Krogan laboratory to define the network of host proteins that interact with these putative fungal virulence factors. We will then exploit host and pathogen genetics to define the role of these host-pathogen protein-protein interactions during infection. Analysis of the resultant dataset will generate specific hypotheses about mechanisms of pathogenesis that will fuel current and future advances in our understanding of how fungi manipulate host cells. Ultimately, these studies will lead to the development of anti-fungal therapeutics that will target key molecules in ubiquitous fungal pathogen.

Project Summary Histoplasma capsulatum (Hc) is an understudied fungal pathogen that causes fatal disease in immunocompromised individuals with AIDS. Our long-term research goal is to gain insight into the pathogenic mechanisms used by Hc to kill infected immune cells, ultimately resulting in improved understanding and treatment of Hc infections in HIV-infected individuals. Hc is a soil fungus that is endemic in the Midwestern United States, Central and South America, Africa, and other regions of the world. It is introduced into mammalian hosts by inhalation and is subsequently phagocytosed by macrophages. Unlike most microbes, Hc survives and replicates within the macrophage phagosome. Robust proliferation of Hc within the phagosome is followed by host-cell death, thus allowing live fungal cells to escape from the macrophage and undergo subsequent rounds of phagocytosis and intracellular proliferation. Individuals who lack a cell-mediated immune response are more likely to develop severe disseminated disease, and AIDS patients with Hc infection are subjected to prolonged, sometimes lifelong, anti-fungal therapy. We recently discovered that Hc utilizes the secreted effector Cbp1 to trigger an integrated stress response (ISR) in host macrophages, resulting in host cell death after intracellular fungal replication. The ISR is an intracellular signaling cascade that triggers phosphorylation of the ?-subunit of the translation elongation factor eIF2 as well as induction of the pro-apoptotic transcription factor CHOP in response to a variety of stresses. We have shown that CHOP is required for host sensitivity to Hc infection in the mouse model of infection. These data are now in press at PLoS Pathogens. To our knowledge, these are the first data that implicate the ISR in the host response to fungal pathogens. However, we still have very little understanding of how Cbp1 induces the ISR. Here we propose to build on robust preliminary data to define the mechanism and consequences of Cbp1-dependent ISR induction during Hc infection. We will: (1) investigate the mechanism of how Cbp1 induces the ISR; (2) elucidate if Cbp1 acts alone during Hc infection to induce the ISR and/or host- cell death, or whether other Hc effectors are involved; and (3) determine the mechanism and role of eIF2? phosphorylation, the central signaling event that initiates the ISR, in response to Hc infection. These studies will explore new strategies used by eukaryotic pathogens to control the viability of macrophages, ultimately developing our understanding of general principles deployed by intracellular microbial pathogens to trigger cellular stress and cause disease. Given the susceptibility of AIDS patients to a variety of intracellular pathogens, the establishment of these general principles may be particular relevant in the context of developing therapeutic strategies (such as ISR inhibition) for AIDS-related opportunistic infections with Hc and other like pathogens.

Project Summary Fungal pathogens of humans are prevalent in the environment, and commonly come into contact with hosts via dispersal of vegetative spores. Although spore germination and subsequent development are recognized as critical to the initiation of fungal-host interactions, little is known about the fungal genes that govern these events. Our goal is to leverage comparative genomics, evolutionary biology and fungal pathogenesis to define genes that are essential for spore germination, subsequent growth, and host colonization in an evolutionarily diverse group of fungi. We have chosen fungi with a range of abilities to cause disease upon interaction of spores with mammalian hosts, including primary, opportunistic, and nonpathogenic species. We will take advantage of a highly effective and innovative pipeline that reveals genes whose evolving roles have led to phenotypic differences among these species. PIs Trail and Townsend have defined a paradigm that brings together comparative genomics, developmental biology, and transcriptomics into a single, unified phylogenetic framework that will identify key genes that govern spore germination and outgrowth in these fungi. The linchpin of our approach is use of the evolutionary relationship between the fungi to infer genes whose expression has been altered during evolution from their ancestral state to each present-day lineage, thus allowing specific traits (such as pathogenesis) to evolve. In our recently published work and preliminary data, this approach was immensely powerful for identifying genes whose evolving role led to developmental and phenotypic differences among species during (1) fungal sexual development and (2) spore germination and early infection during fungal pathogenesis of plants. We will use a common medium to germinate spores from the following fungi: the primary pathogens Histoplasma capsulatum and Coccidioides posadasii, the opportunistic pathogens Aspergillus fumigatus, Fusarium oxysporum, and Chaetomium elatum, the infrequent opportunistic pathogen Aspergillus nidulans, and the non-pathogenic Neurospora crassa. We will subject these fungi to transcriptomics over a time-course of germination and subsequent development under temperature conditions relevant to germination in the environment vs. in a mammalian host. We will reconstruct evolutionary changes of gene expression across these multiple species to identify genes that have undergone recent shifts in gene expression, in particular shifts that occurred along the shared ancestral branches where key traits (such as the ability to colonize mammals) have evolved. These experiments will yield a high-confidence set of candidate genes whose function is expected to be critical for spore germination and development in each organism. We will use gene knock-out technology to interrogate the function of these candidate genes in spore germination and development. These studies will identify potential targets for diagnostic, prophylactic, and vaccine interventions for ubiquitous fungal infections of humans.

Project Summary Coccidioides spp. are major fungal pathogens endemic to Southern California, Arizona, Central America, and South America. In recent years, the incidence of coccidioidomycosis has continued to rise, resulting in hospitalization costs greater than $2 billion. Coccidioides infects, colonizes, and kills immunocompetent individuals when they inhale spores from soils. The ability of Coccidioides to cause disease depends on an elaborate developmental transition from saprophytic soil form to host form, which can be triggered in the laboratory by incubating fungal spores at elevated temperature and carbon dioxide conditions. Specifically, the hyphal form of the organism produces arthroconidia, which disperse easily and can be inhaled by mammalian hosts. Once inside the host lung, arthroconidia germinate, enlarge, and undergo nuclear division and segmentation to form large spherules filled with vegetative endospores. Rupture of the spherules allows release of endospores and dissemination of the fungus to other sites. Given the critical role of spherule development in disease progression, the focus of our proposal is the genomic and genetic dissection of this process, also known as spherulation. We will take advantage of two complementary approaches, high-resolution transcriptomics and genome-wide association studies (GWAS), to perform an innovative molecular dissection of spherulation in Coccidioides. Principal Investigator Sil has extensive experience working with Biosafety Level 3 pathogens and is well equipped to apply her expertise in transcriptional profiling of thermally dimorphic fungi to Coccidioides. PI Brem is an evolutionary and statistical geneticist with a track record of applying GWAS to fungi to identify genes that play a critical role in biologically important traits. Together, we will harness the tools of systems genetics to discover new gene functions on a genomic scale in Coccidioides. In Aim 1, we will identify a core set of spherulation-enriched transcripts by performing a high-resolution time-course analysis of the transcriptome of three Coccidioides strains undergoing spherulation. In Aim 2, we will apply GWAS analysis to identify genes that underlie variance of spherulation phenotypes, using 150 clinical isolates of Coccidioides posadasii. Taken together, these approaches will provide a rich dataset of spherulation-associated genes that will allow us to begin to elucidate critical molecular events that take place during spherule development in the context of infection.