Molecular characterization of Aspergillus fumigatus Crz A targets by Chromatin Immunoprecipitation – DNA sequence (ChIP-seq )

Ref ID: 19528

Author:

PA Castro1, VLP Bom1, C Chen3, MC Bertolini4, FZ Freitas4, TK Mitchell3, GH Goldman1,2*

Author address:

1Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brazil
2National Laboratory of Science and Technology of Bioethanol (CTBE), Campinas, Brazil
3Universidade Estadual de Londrina, Londrina, Brazil
4D

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
Aspergillus fumigatus is an important primary and opportunistic pathogen as well as a major allergen.
The most important genetic components for the infection establishment are transcriptionfactors
that can activate different programs for virulence regulation and pathogenicity. Calcineurin is a
central signaling molecule in most eukaryotic cells and many of these activities are dependent
on the transcription factor CrzA that becomes transcriptionally active after dephosphorylation
by calcineurin. In A. fumigatus, several signaling pathways are dependent on CrzA and must be
activated during the adaptive process into the mammalian host. The identification of A. fumigatus
genes regulated by CrzA may provide greater insight into this important signal transduction pathway.
Methods:
Chromatin Immunoprecipitation technique followed byDNA sequencing (ChIP-seq) for CrzA.
ChIP-seq is a powerful tool to evaluate the location of DNA-binding proteins in vivo and this assay
provides a wide base for the identification of genes which have their mRNA accumulation modulated
by CrzA.
Results:
Bioinformatics analysis of the fragments sequenced identified putative promoter regions that
control 103 adjacent genes. Some of these genes are involved in the cAMP-dependent signaling
during conidial germination (for instance schA), in the activation of MAPKK activity involved
in osmosensory signaling pathway (for instance ssk1 and ssk2), in calcium ion homeostasis, and
also related to endoplasmic reticulum stress. The analyses of these promoter regions by MEME
and MDScan identified two common motifs as calcineurin-dependent response elements (CDRE)
possibly involved in the regulation by CrzA: 5’- AGGCTG -3’ or 5’- CAGCCT -3’. We validated
these data by performing RT-PCR for 10 selected genes and Eletrophoretic Mobility Shift assays
(EMSA) for two of them.
Conclusion:
Our results showed that mRNA accumulation of 9 of these genes is significantly decreased in the
delta crzA mutant while these two promoters regions contain as CDREs 5’- CACAGCCC -3’ and
5’-CAACCT -3’. Moreover, we constructed null mutants for the genes schA (Afu1g06400), ssk1
(Afu3g12530), ssk2 (Afu1g10940) and ckb2 (Afu1g09950), and studied their phenotypes. The
delta schA mutants strain showed attenuated virulence in a murine model of invasive pulmonary
aspergillosis.
Supported by: FAPESP and CNPq, Brazil

Abstract Number: 55

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

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