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Principal Investigator: Praveen Rao Juvvadi
Organization: ARKANSAS CHILDREN'S HOSPITAL RES INST
Fiscal Year: 2024
Award: $251,107
Funding agency: National Institute of Allergy and Infectious Diseases
Invasive aspergillosis (IA), caused by the fungus Aspergillus fumigatus, is a leading infectious cause of death in
immunocompromised patients. A significant barrier to deciphering the mechanisms of pathogenesis and
developing effective antifungals is a lack of understanding of the regulation of A. fumigatus growth leading to
invasive disease. Cellular recycling mediated via autophagy-associated proteins is a key catabolic pathway and
critical to invasive fungal growth and virulence in the well-known nutrient-limited host environment. Protein kinase
A (PKA) is vital for growth and virulence of numerous fungal pathogens. However, the underlying basis for its
regulation of pathogenesis remains poorly understood in any species. Our A. fumigatus PKA-dependent whole
proteome and phosphoproteome studies employing advanced mass spectroscopic (MS) approaches identified
numerous previously undefined PKA-regulated proteins in catabolic pathways. Preliminary characterization of
two such novel direct PKA target proteins, Atg24 and Not4, demonstrated their requirement for A. fumigatus
virulence and also revealed functional importance of PKA-dependent phosphorylation of specific target sites. We
have identified and prioritized more than 30 autophagy-associated proteins as likely novel PKA-regulated
effectors, and will now characterize the top 6 priority proteins with regard to their roles in fungal growth and
pathogenesis, as well as their regulation by PKA. Our overall objective is to leverage our robust PKA whole
proteomic and phosphoproteomic data to define fungal-specific mechanisms of PKA control over autophagy
function and validate these PKA-regulated effectors’ novel contributions to fungal virulence. In Aim 1, we will
define PKA control over autophagy by validating PKA-dependent phosphorylation of prioritized
phosphoregulated effectors via MS analysis and expression-regulated effectors via qRT-PCR. We will
characterize the role of each PKA effector in fungal growth and stress responses through deletion and
phosphosite mutagenesis approaches. We will also perform structural elucidation of PKA-effector
phosphoregulation and function using molecular modeling and molecular dynamics simulations. In Aim 2, we
will validate the roles of PKA-regulated autophagy proteins in virulence and host-pathogen interaction using our
murine model of invasive aspergillosis to assess their impact on mortality, fungal burden and host lung tissue
damage via histological analysis. We will also assess the impact of PKA-regulated proteins on both the pathogen
and host response to infection by expression profiling of A. fumigatus autophagy marker genes, as well as
simultaneous examination of the host immune cytokine response during infection with wild-type and mutant
strains. PKA regulates catabolic processes critical to fungal growth, yet an understanding of PKA regulation of
autophagy pathways is lacking in any organism. We will define novel PKA-dependent regulation of newly-
identified fungal-specific autophagy-associated proteins and provide critical insight into future exploitation of
PKA-dependent control over A. fumigatus disease.
Terms: <A fumigatus><A. fumigatus><Address><Adenosine Cyclic Monophosphate-Dependent Protein Kinases><Aspergillosis><Aspergillus fumigatus><Autophagocytosis><Autophagosome><Biological><Biology><Body Tissues><Carbon><Catabolic Process><Catabolism><Cause of Death><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cell Wall><Cellular Function><Cellular Physiology><Cellular Process><Critical Paths><Critical Pathways><Cyclic AMP-Dependent Protein Kinases><Data><Death Rate><Defect><Disease><Disorder><Environment><Expression Profiling><Fungus Diseases><Future><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Generalized Growth><Genes><Genetic Markers><Genetics-Mutagenesis><Goals><Growth><Histologic><Histologically><Immune><Immune response><Immune system><Immunes><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunological response><Immunosuppressed Host><Infection><Intracellular Communication and Signaling><Invaded><Kinases><Knowledge><Lung Parenchyma><Lung Tissue><Mediating><Metabolic><Metabolic Control><Molecular><Molecular Dynamics Simulation><Molecular Modeling Nucleic Acid Biochemistry><Molecular Modeling Protein/Amino Acid Biochemistry><Molecular Models><Mutagenesis><Mutagenesis Molecular Biology><Mycoses><Nitrogen><Nutrient><Organism><PKA><Pathogenesis><Pathway interactions><Patients><Phenotype><Phosphopeptides><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Protein Kinase A><Protein Phosphorylation><Proteins><Proteome><Proteomics><Quantitative RTPCR><Quantitative Reverse Transcriptase PCR><Recycling><Regulation><Research><Resistance><Role><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Site><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Specificity><Structure of parenchyma of lung><Subcellular Process><Targeted DNA Modification><Targeted Modification><Testing><Therapeutic><Therapeutic Fungicides><Time><Tissue Growth><Tissues><Translations><Transphosphorylases><Virulence><anti-fungal><anti-fungal agents><anti-fungal drug><autophagy><biologic><biological adaptation to stress><biological signal transduction><cAMP-Dependent Protein Kinases><combat><comparative><cytokine><environmental stresses><environmental stressor><fungal infection><fungal pathogen><fungi pathogen><fungus><fungus infection><gene biomarker><gene expression biomarker><gene marker><gene signature biomarker><genetic biomarker><host response><immune system response><immunoresponse><immunosuppressed patient><improved><insight><living system><mimetics><molecular dynamics><molecular modeling><mortality><mortality rate><mortality ratio><mouse model><murine model><mutant><novel><ontogeny><pathogen><pathogenic fungus><pathway><phospho-proteomics><phosphoproteomics><qRTPCR><reaction; crisis><resistant><response><social role><stress response><stress; reaction><therapeutically effective><translation><translational study>