Defining the Aspergillus fumigatus Calcineurin-Dependent Regulatory Network through Whole Phosphoproteome Analysis
Defining the Aspergillus fumigatus Calcineurin-Dependent Regulatory Network through Whole Phosphoproteome Analysis
批准号:
9214478
负责人:
Praveen Rao Juvvadi
金额:
$23.85万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2016
资助国家:
美国
项目状态:
已结题
起止时间:
2016-12-15 至 2018-11-30
关键词:
Alpha CellAntifungal AgentsAspergillosisAspergillus fumigatusBindingCalcineurinCause of DeathDefectDiseaseExhibitsFutureGeneticGoalsGrowthImmune systemImmunocompromised HostIn VitroInvadedKnowledgeMapsMeasuresMediatingModelingMultienzyme ComplexesMutagenesisMutationMycosesPPP3CA genePathogenesisPatientsPharmacologyPhenotypePhosphoric Monoester HydrolasesPhosphorylationPhosphotransferasesPositioning AttributeProtein DephosphorylationProteinsProteomeProteomicsRegulationReportingRoleSignal PathwaySignal TransductionTherapeuticTimeValidationVirulenceWorkcalcineurin phosphatasefungusgenetic approachimprovedimproved outcomemortalitymouse modelmultiple reaction monitoringmutantnovelpathogenphosphoproteomicsprotein expressionscreeningtranscription factortrend
中文摘要
由烟曲霉引起的侵袭性曲霉病是免疫功能低下患者的主要杀手。一个
英文摘要
Invasive aspergillosis due to Aspergillus fumigatus is a leading killer in immunocompromised patients. A
significant barrier to developing effective antifungal therapeutics is the lack of understanding of the regulation
of A. fumigatus hyphal growth and disease. We have established that calcineurin is required for A. fumigatus
growth and virulence. We also showed that calcineurin is dynamically localized at active points of growth, the
hyphal tip and septum, and that calcineurin phosphatase activity is required at these active points to regulate
hyphal growth and virulence. Calcineurin (CnaA) functions as a phosphatase to dephosphorylate substrates
and through the transcription factor, CrzA. However, in contrast to our CnaA deletion strain, our ΔcrzA strain
exhibited a minimal hyphal growth defect and, surprisingly, CrzA did not localize at the active points of growth.
An extensive targeted mutagenesis screen revealed that the calcineurin substrate binding PxIxIT motif, and a
novel FxDxF motif, are indispensable for CnaA localization. These new findings indicate that the major effects
on calcineurin-mediated growth and virulence are likely due to calcineurin interactions with as yet undefined
key effectors at these critical growth points. As a logical next step, we now seek to define calcineurin’s main
function as a phosphatase to orchestrate growth and virulence via its interaction with these unknown effectors.
Our overall goal is to identify the calcineurin downstream effectors that control A. fumigatus hyphal growth
and virulence. Our hypothesis is that calcineurin, as a critical phosphatase, interacts with phosphorylated
effectors to dephosphorylate them to control growth and virulence. To define these interactions and the
mechanism of regulation of effectors facilitating hyphal growth and virulence, we will utilize holistic ultra-
sensitive proteomic strategies to define the complete CnaA phosphoproteome.
In Aim 1, we will define the A. fumigatus native proteome and calcineurin-dependent phosphoproteome
using two independent quantitative global phosphoproteomic approaches paired with quantitative analysis of
the native proteome. This will define downstream effector substrates dephosphorylated in the wild-type strain,
due to phosphatase activity, but still phosphorylated in the calcineurin phosphatase-deficient mutant. We will
then employ Multiple Reaction Monitoring to measure protein expression trends as well as validate effectors
from the phosphoproteome. In Aim 2, we will prioritize the identified CnaA effectors to validate their role in
calcineurin-mediated downstream effects in growth and disease via an iterative approach. We will use genetic
deletion (single, multiple), in vitro growth screening, targeted mutations, and a murine model validation for
virulence defects. This multi-faceted approach will identify key phosphorylation-dependent downstream
effectors of calcineurin and develop a model for fungal-specific control of hyphal growth and virulence. We will
generate a map of previously undescribed downstream effectors regulated by calcineurin in a pathogen, which
will have important biologic ramifications for broader calcineurin, fungal growth, and pathogenesis studies.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
Calcineurin regulatory network control of Aspergillus fumigatus hyphal septation
-
批准号:10540607
-
项目类别:
-
资助金额:$16.35万
-
财政年份:2021
-
负责人:Praveen Rao Juvvadi
-
依托单位:
Calcineurin regulatory network control of Aspergillus fumigatus hyphal septation
-
批准号:10283247
-
项目类别:
-
资助金额:$24.15万
-
财政年份:2021
-
负责人:Praveen Rao Juvvadi
-
依托单位:
海外基金