ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

Dawoon Chung,Chao Cheng,Wenjie Xu,Beatrix E Lechner,Aurélien Mazurie,Robert A Cramer,Thomas K Mitchell,Brittney Merriman,Sourabh Dhingra,Hubertus Haas,Aaron P Mitchell,Kengo Morohashi,Sara J Blosser,Ernst R Werner,Charles C Carey,Bridget M Barker,Tamara L Doering

doi:10.1371/journal.ppat.1004487

Abstract

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Highlights

Invasive fungal infections have increased in frequency due to a substantial rise in the number of immune compromised patients [1,2,3]
The Aspergillus fumigatus transcription factor SrbA is a major regulator of the fungal response to hypoxia found at sites of invasive fungal growth in vivo
New insights into how SrbA mediates hypoxia adaptation and virulence were revealed through identification of direct transcriptional targets of SrbA under hypoxic conditions

Summary

Introduction

Invasive fungal infections have increased in frequency due to a substantial rise in the number of immune compromised patients [1,2,3]. Restores Hypoxic Growth These data suggest that SrbB co-regulates a sub-set of SrbA target genes and that loss of SrbA markedly reduces SrbB levels. Quantitative real-time PCR was conducted to verify if these promoters induced transcript level increases of srbB. Compared to DsrbA, srbB transcript levels increased by 5.1- and 3.7-fold in normoxia and 2.7- and 1.4-fold in hypoxia in TDC43.18 (Figure 10A). Restoration of srbB transcript levels strongly promotes growth of DsrbA in hypoxia, growth is not fully restored to wild type levels and was dependent on srbB transcript levels (Figure 10B). As predicted, restoration of srbB transcript levels in

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Discussion

Conclusion