Co-Operative Biofilm Interactions between Aspergillus fumigatus and Pseudomonas aeruginosa through Secreted Galactosaminogalactan Exopolysaccharide

The mold Aspergillus fumigatus and bacterium Pseudomonas aeruginosa form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by A. fumigatus depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), while P. aeruginosa biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (A. fumigatus) or the periplasmic deacetylase PelA (P. aeruginosa). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated. P. aeruginosa were observed to adhere to A. fumigatus hyphae in a GAG-dependent manner and to GAG-coated coverslips of A. fumigatus biofilms. In biofilm adherence assays, incubation of P. aeruginosa with A. fumigatus culture supernatants containing de-N-acetylated GAG augmented the formation of adherent P. aeruginosa biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within P. aeruginosa biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent A. fumigatus biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease

Transcriptional Profiling Identifies a Role for BrlA in the Response to Nitrogen Depletion and for StuA in the Regulation of Secondary Metabolite Clusters in Aspergillus fumigatus▿ ‡

Conidiation (asexual sporulation) is a key developmental process in filamentous fungi. We examined the gene regulatory roles of the Aspergillus fumigatus developmental transcription factors StuAp and BrlAp during conidiation. Conidiation was completely abrogated in an A. fumigatus ΔbrlA mutant and was severely impaired in a ΔstuA mutant. We determined the full genome conidiation transcriptomes of wild-type and ΔbrlA and ΔstuA mutant A. fumigatus and found that BrlAp and StuAp governed overlapping but distinct transcriptional programs. Six secondary metabolite biosynthetic clusters were found to be regulated by StuAp, while only one cluster exhibited BrlAp-dependent expression. The ΔbrlA mutant, but not the ΔstuA mutant, had impaired downregulation of genes encoding ribosomal proteins under nitrogen-limiting, but not carbon-limiting, conditions. Interestingly, inhibition of the target of rapamycin (TOR) pathway also caused downregulation of ribosomal protein genes in both the wild-type strain and the ΔbrlA mutant. Downregulation of these genes by TOR inhibition was associated with conidiation in the wild-type strain but not in the ΔbrlA mutant. Therefore, BrlAp-mediated repression of ribosomal protein gene expression is not downstream of the TOR pathway. Furthermore, inhibition of ribosomal protein gene expression is not sufficient to induce conidiation in the absence of BrlAp

A conserved C-terminal domain of the Aspergillus fumigatus developmental regulator MedA is required for nuclear localization, adhesion and virulence.

MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA(346-557), that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA(346-557) alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA(346-557) domain is both necessary and sufficient to mediate MedA function

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Amino acid sequence alignment of <i>A. fumigatus</i> MedA putative NLSs 1–4 with other MedA homologues.

<p>(A) Sequence alignment and motif prediction using PSORT II identified NLS1 sequence among orthologues of MedA. (B) Sequence alignment of the MedA minimal nuclear localization domain, MedA^346–557 with other MedA orthologues. The sequences representing the putative NLSs 2, 3, and 4 are boxed. The basic amino acids within the putative NLSs of <i>A. fumigatus</i> MedA and the corresponding amino acids in MedA orthologues are highlighted in gray. The presence of an asterisk or a colon below the basic amino acids indicates a fully or strongly conserved residue, respectively. Numbers indicate the amino acid position within the primary amino acid sequence of the protein. Af_MedA: <i>A. fumigatus</i> MedA (GenBank: EAL93620.1), An_MedA: <i>A. nidulans</i> MedA (GenBank: AAC31205.1), Nc_ACON-3: <i>N. crassa</i> ACON-3 (GenBank: ADL28820.1), Mg_Acr1: <i>M. grisea</i> Acr1 (GenBank: BAC41196.1), and Fo_Ren1: <i>F. oxysporum</i> Ren1 (GenBank: BAC55015.1).</p

Reducing Aspergillus fumigatus Virulence through Targeted Dysregulation of the Conidiation Pathway

The mold Aspergillus fumigatus reproduces by the production of airborne spores (conidia), a process termed conidiation. In immunocompromised individuals, inhaled A. fumigatus conidia can germinate and form filaments that penetrate and damage lung tissues; however, conidiation does not occur during invasive infection. In this study, we demonstrate that forced activation of conidiation in filaments of A. fumigatus can arrest their growth and impair the ability of this fungus to cause disease in both an insect and a mouse model of invasive infection. Activation of conidiation was linked to profound changes in A. fumigatus metabolism, including a shift away from the synthesis of polysaccharides required for cell wall structure and virulence in favor of carbohydrates used for energy storage and stress resistance. Collectively, these findings suggest that activation of the conidiation pathway may be a promising approach for the development of new agents to prevent or treat A. fumigatus infection.Inhalation of conidia of the opportunistic mold Aspergillus fumigatus by immunocompromised hosts can lead to invasive pulmonary disease. Inhaled conidia that escape immune defenses germinate to form filamentous hyphae that invade lung tissues. Conidiation rarely occurs during invasive infection of the human host, allowing the bulk of fungal energy to be directed toward vegetative growth. We hypothesized that forced induction of conidiation during infection can suppress A. fumigatus vegetative growth, impairing the ability of this organism to cause disease. To study the effects of conidiation pathway dysregulation on A. fumigatus virulence, a key transcriptional regulator of conidiation (brlA) was expressed under the control of a doxycycline-inducible promoter. Time- and dose-dependent brlA overexpression was observed in response to doxycycline both in vitro and in vivo. Exposure of the inducible brlA overexpression strain to low doses of doxycycline under vegetative growth conditions in vitro induced conidiation, whereas high doses arrested growth. Overexpression of brlA attenuated A. fumigatus virulence in both an invertebrate and mouse model of invasive aspergillosis. RNA sequencing studies and phenotypic analysis revealed that brlA overexpression results in altered cell signaling, amino acid, and carbohydrate metabolism, including a marked upregulation of trehalose biosynthesis and a downregulation in the biosynthesis of the polysaccharide virulence factor galactosaminogalactan. This proof of concept study demonstrates that activation of the conidiation pathway in A. fumigatus can reduce virulence and suggests that brlA-inducing small molecules may hold promise as a new class of therapeutics for A. fumigatus infection