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Principal Investigator: Sinem Beyhan
Organization: J. CRAIG VENTER INSTITUTE, INC.
Fiscal Year: 2024
Award: $97,500
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
Histoplasma capsulatum causes pulmonary and systemic infections in both healthy and immunocompromised
individuals, and is the most common cause of fungal respiratory infections in healthy hosts. It is a dimorphic
fungal pathogen that can sense and respond to human body temperature by changing its growth program from
a filamentous (mold) form to a parasitic yeast form. Infection occurs when the soil is disrupted, facilitating
dispersion of hyphal fragments or spores that are inhaled by humans. Once introduced into the host, the
pathogen converts to a budding-yeast form, which survives and replicates within host macrophages. In the
laboratory, the switch between the infectious and parasitic states is modeled by changing the temperature: cells
grow in the filamentous form at room temperature, whereas growth at 37ºC is sufficient to trigger growth in the yeast
form and expression of virulence factors. In previous studies, we and others have identified four transcriptional
regulators, Ryp1,2,3,4, which are required for yeast-phase growth and virulence gene expression. Ryps directly
regulate expression of a set of yeast-phase specific genes that encode proteins with putative secretion signals
and transmembrane helices; thus, form the core of the yeast-phase specific transcriptional network. In
preliminary studies, we have found that two major players of the heat shock response, the heat shock factor
Hsf1 and a chaperone Hsp90, also regulate the yeast-phase growth under in vitro conditions. However, these
genes are essential and their function cannot be studied by conventional knockout strategies in Histoplasma.
There is limited research done in Histoplasma, in part due to the limitation of the genetic tools available. In
particular, there is no controllable gene expression systems that can be used to study gene function in vivo. In
this project, we aim to develop controllable (Tet-inducible and Tet-repressible) gene expression systems and
optimize episomal DNA maintenance. Using the Tet-inducible system, we will induce expression of knockdown
cassettes for Ryp1,2,3,4, Hsf1 and Hsp90 under in vivo conditions and assess their role in maintenance of the
parasitic growth in vivo. In addition, we will identify centromeric sequences and/or DNA elements that promote
autonomous replication or segregation; and utilize them in episomal vectors to ensure proper segregation of the
episomal DNA during cell division. We will generate knockdown and overexpression mutants of yeast- or hyphal-
phase specific transcription factors using improved episomal vectors. Ultimately, results of the experiments
proposed in this project will further advance our ability to study gene function in Histoplasma, and will expand
our knowledge of the transcriptional network that governs the parasitic yeast-phase growth.
Terms: <A fumigatus><A. fumigatus><Address><Airway infections><Aspergillus fumigatus><Assay><Autonomous Replication><Basal Transcription Factor><Basal transcription factor genes><Bioassay><Biological Assay><Body Temperature Changes><Budding Yeast><C albicans><C. albicans><C.albicans><Candida albicans><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell division><Cells><Centromere><Chaperone><Chimera Protein><Chimeric Proteins><DNA><DNA Maintenance><DNA Stability><Defect><Deoxyribonucleic Acid><Development><Disease><Disorder><E coli><E. coli><Electroporation><Elements><Endomycetales><Ensure><Episome><Escherichia coli><Essential Genes><Filamentous Fungi><Fusion Protein><Gene Expression><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Genes><Genetic><Genetic Transcription><Goals><Growth><H capsulatum><H. capsulatum><HSF1><HSP-90><HSP90><Health><Heat Shock><Heat shock factor><Heat-Shock Proteins 90><Heat-Shock Reaction><Heat-Shock Response><Histoplasma><Histoplasma capsulatum><Histoplasmosis><Human><Human Figure><Human body><Hyphae><Immunocompetent><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><In Vitro><Individual><Infection><Inhalation><Inhaling><Intracellular Communication and Signaling><Knock-out><Knockout><Knowledge><Laboratories><Lung><Lung Respiratory System><Lung infections><Macrophage><Maintenance><Membrane><Methods><Mice><Mice Mammals><Mississippi><Modeling><Modern Man><Molds><Molecular Chaperones><Morbidity><Morbidity - disease rate><Morphology><Murine><Mus><Mφ><Ohio><Organism><Pathogenicity Factors><Phase><Phenotype><Plasmids><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Proteins><RNA Expression><RNA Interference><RNA Silencing><RNAi><Repressor Proteins><Reproduction spores><Research><Respiratory Infections><Respiratory Tract Infections><Role><Saccharomycetales><Sequence-Specific Posttranscriptional Gene Silencing><Signal Transduction><Signal Transduction Systems><Signaling><Soil><Source><Spores><System><Systemic infection><Techniques><Technology><Temperature><Tet><Tetanus Helper Peptide><Tetracyclines><Time><Tissue Growth><Transcription><Transcription Activator><Transcription Coactivator><Transcription Factor Coactivator><Transcription Factor Proto-Oncogene><Transcription Repressor><Transcription factor genes><Transcriptional Activator><Transcriptional Activator/Coactivator><Transcriptional Coactivator><Transcriptional Repressor><Virulence><Virulence Factors><Work><Yeasts><biological signal transduction><developmental><electroporative delivery><experience><experiment><experimental research><experimental study><experiments><fungal pathogen><fungi pathogen><fungus><gene electrotransfer><gene function><gene manipulation><genetic manipulation><genetic repressor><genetically manipulate><genetically perturb><hsp90 Family><human disease><immune competent><immunosuppressed patient><improved><in vivo><knock-down><knockdown><living system><membrane structure><mortality><mutant><ontogeny><overexpress><overexpression><pathogen><pathogenic fungus><pathogenicity gene><pleiotropic effect><pleiotropism><pleiotropy><programs><pulmonary><pulmonary infections><repressor complex><segregation><social role><telomere><tool><transcription co-activator><transcription factor><transcriptional co-activator><vector><virulence gene><virulent gene>