Distinct Cohorts of Aspergillus fumigatus Transcription Factors Are Required for Epithelial Damage Occurring via Contact- or Soluble Effector-Mediated Mechanisms

Abstract

Damage to the lung epithelium is a unifying feature of disease caused by the saprophytic fungus Aspergillus fumigatus. However, the mechanistic basis and the regulatory control of such damage is poorly characterized. Previous studies have identified A. fumigatus mediated pathogenesis as occurring at early (≤ 16 hours) or late (>16 hours) phases of the fungal interaction with epithelial cells, and respectively involve direct contact with the host cell or the action of soluble factors produced by mature fungal hyphae. Both early and late phases of epithelial damage have been shown to be subject to genetic regulation by the pH-responsive transcription factor PacC. This study sought to determine whether other transcriptional regulators play a role in modulating epithelial damage. In particular, whether the early and late phases of epithelial damage are governed by same or distinct regulators. Furthermore, whether processes such as spore uptake and hyphal adhesion, that have previously been documented to promote epithelial damage, are governed by the same cohorts of epithelial regulators. Using 479 strains from the recently constructed library of A. fumigatus transcription factor null mutants, two high-throughput screens assessing epithelial cell detachment and epithelial cell lysis were conducted. A total of 17 transcription factor mutants were found to exhibit reproducible deficits in epithelial damage causation. Of these, 10 mutants were defective in causing early phase damage via epithelial detachment and 8 mutants were defective in causing late phase damage via epithelial lysis. Remarkably only one transcription factor, PacC, was required for causation of both phases of epithelial damage. The 17 mutants exhibited varied and often unique phenotypic profiles with respect to fitness, epithelial adhesion, cell wall defects, and rates of spore uptake by epithelial cells. Strikingly, 9 out of 10 mutants deficient in causing early phase damage also exhibited reduced rates of hyphal extension, and culture supernatants of 7 out of 8 mutants deficient in late phase damage were significantly less cytotoxic. Our study delivers the first high-level overview of A. fumigatus regulatory genes governing lung epithelial damage, suggesting highly coordinated genetic orchestration of host-damaging activities that govern epithelial damage in both space and time.

Virulence Attributes in Aspergillus fumigatus

“…Other vital functions of hyphae-secreted GAG are mediating neutrophil apoptosis and resistance to neutrophil extracellular traps (NETs), modulating host immune responses through platelet activation, inducing secretion of IL-1 receptor antagonists, and inflammasome activation [31,73,75]. Therefore, GAG is critical for host damage and fungal virulence, as studies in mouse models of invasive pulmonary aspergillosis (IPA) have shown, where GAG-deficient strains exhibit reduced adherence to lung epithelial cells, do not form biofilms, and are less virulent [7,32]. The multiple and important functions of GAG in virulence have prompted the study of the mechanisms involved in its biosynthesis.…”

“…The pathogenesis of the disease mediated by A. fumigatus occurs in a multi-step manner and involves the morphological transition of the inhaled fungal spore to a hyphae form. Epithelial damage can be considered to occur in the early (conidia) or late (hyphae) phase of the fungal interaction with epithelial cells [7]. In healthy immunocompetent individuals, the growth of hyphae is impeded by immune mechanisms, whereas, in people with immune deterioration, such as those who have leukemia or who have undergone bone marrow or solid organ transplantation, mycelial development leads to severe disease, which can be lifethreatening (Figure 2) [8][9][10][11].…”

Inferring fungal growth rates from optical density data

“…Growth rates specific to a fungal morphology (morphology-specific growth rates), such as germination rates of fungal spores or proliferation rates of hyphae, can be estimated from morphology-specific growth data to evaluate morphology-specific changes in fungal growth over time. For example, hyphal extension rates have been derived from hyphal microscopy data to investigate hyphae-specific growth defects in mutant strains of Aspergillus fumigatus [ 22 ]. Such morphology-specific growth rates can also be estimated from mechanistic models of fungal growth fit to directly measured time-course data.…”

“…Several previous studies have calculated fungal growth rates to quantitatively evaluate temporal changes in fungal growth in different experimental conditions [22][23][24][25]. Growth rates specific to a fungal morphology (morphology-specific growth rates), such as germination rates of fungal spores or proliferation rates of hyphae, can be estimated from morphology-specific growth data to evaluate morphology-specific changes in fungal growth over time.…”

The C 2 H 2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus

“…In this study, we identify that the zinc-finger C 2 H 2 type transcription factor (TF) SltA is functionally linked to the control of germination as it has a non-redundant role in controlling emergence from dormancy, germ tube formation and temporal and spacial control of hyphal branching. The ∆ sltA strain was recently studied for virulence and showed a reduced capacity to adhere to, invade, and damage pulmonary epithelial cells ( 16 , 17 ). Additionally, ∆ sltA showed increased susceptibility to cell membrane stressors ( 16 ).…”