Aspergillus fumigatus flbB Encodes Two Basic Leucine Zipper Domain (bZIP) Proteins Required for Proper Asexual Development and Gliotoxin Production

Abstract

The opportunistic human pathogen Aspergillus fumigatus reproduces asexually by forming a massive number of mitospores called conidia. In this study, we characterize the upstream developmental regulator A. fumigatus flbB (AfuflbB). Northern blotting and cDNA analyses reveal that AfuflbB produces two transcripts predicted to encode two basic leucine zipper domain (bZIP) polypeptides, AfuFlbB␤ (420 amino acids [aa]) and AfuFlbB␣ (390 aa). The deletion of AfuflbB results in delayed/reduced sporulation, precocious cell death, the lack of conidiophore development in liquid submerged culture, altered expression of AfubrlA and AfuabaA, and blocked production of gliotoxin. While introduction of the wild-type (WT) AfuflbB allele fully complemented these defects, disruption of the ATG start codon for either one of the AfuFlbB polypeptides leads to a partial complementation, indicating the need of both polypeptides for WT levels of asexual development and gliotoxin biogenesis. Consistent with this, Aspergillus nidulans flbB ؉ encoding one polypeptide (426 aa) partially complements the AfuflbB null mutation. The presence of 0.6 M KCl in liquid submerged culture suppresses the defects caused by the lack of one, but not both, of the AfuFlbB polypeptides, suggesting a genetic prerequisite for AfuFlbB in A. fumigatus development. Finally, Northern blot analyses reveal that both AfuflbB and AfuflbE are necessary for expression of AfuflbD, suggesting that FlbD functions downstream of FlbB/FlbE in aspergilli.The opportunistic human pathogen Aspergillus fumigatus (reviewed in reference 9) propagates in the environment by producing a massive number of mitotic spores called conidia (19). Under appropriate conditions, the conidium germinates and undergoes vegetative growth that leads to the formation of a network of undifferentiated interconnected hyphae known as the mycelium. After a certain period of hyphal proliferation, in response to appropriate cues (e.g., exposure to air, nutrient deprivation, or osmotic stress) some of the vegetative cells initiate asexual development (conidiation) and go through a series of morphological changes, which result in the formation of conidiophores consisting of foot cell, stalk, vesicle, phialides, and (up to 50,000) conidia (19,26). Conidia (2 to 3 m in diameter) are then released into the environment and are small enough to reach the alveoli after being inhaled by humans (31). In immunocompromised hosts the conidia are able to germinate into invasive hyphae, which penetrate the vasculature and migrate to distal sites (reviewed in reference 26).We have been investigating the mechanisms underlying asexual development and gliotoxin biosynthesis in A. fumigatus, primarily focusing on understanding the functions of a number of developmental regulators identified in the model fungus Aspergillus nidulans (21,30,40). A key step for conidiation in A. nidulans is the activation of brlA encoding a C 2 H 2 zinc finger transcription factor (TF) (1). Together with abaA and wetA, these three elements define a c...

AbaA and WetA govern distinct stages of Aspergillus fumigatus development

“…In this model, upstream regulator AfuFluG is proposed to activate Afuflb (e.g. AfuflbB and AfuflbE; Yu et al, 2006;Kwon et al, 2010;Xiao et al, 2010;Yu, 2011) and AfuBrlA, thereby triggering the central regulatory pathway (AfubrlAAAfuabaAAAfuwetA) of conidiation. Subsequently, AfuvosA (Ni & Yu, 2007) is activated by AfuAbaA with the assistance of AfuWetA.…”

Regulation of Development inAspergillus nidulansandAspergillus fumigatus

“…In a series of recent studies, we characterized the functions of FlbE and FlbB in Afu [39, 40]. The predicted Afu FlbE protein is composed of 222 aa in length.…”

“…Xiao et al [40] conducted a series of molecular analyses and found that AfuflbB produces two transcripts predicted to encode two b-zip polypeptides, Afu FlbBb (420 aa) and Afu FlbBa (390 aa). AfuflbB deletion results in delayed and reduced conidiation, precocious cell death, the absence of conidiophore development in liquid submerged culture, altered expression of AfubrlA and AfuabaA , and the lack (or a reduction) in gliotoxin production.…”

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

“…To date a number of key asexual developmental regulators such as AbaA, BrlA, WetA, and Velvets have been identified and characterized in A. fumigatus [6,13,15,27]. AbaA plays a crucial role in the differentiation of phialides, the conidiogenous cells, which are required for formation of conidiospores.…”

“…First of all, brlA mRNA is clearly detectable during a certain period of vegetative growth (~18~24 h) [6,13,15]. Moreover, the absence of abaA causes delayed autolysis and cell death, whereas the overexpression of abaA accelerates these processes [13].…”