Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on Aspergillus fumigatus

Abstract

Inhibition of fungal growth by Congo red (CR) has been putatively associated with specific binding to β-1,3-glucans, which blocks cell wall polysaccharide synthesis. In this study, we searched for transcription factors (TFs) that regulate the response to CR and interrogated their regulon. During the investigation of the susceptibility to CR of the TF mutant library, several CR-resistant and -hypersensitive mutants were discovered and further studied. Abnormal distorted swollen conidia called Quasimodo cells were seen in the presence of CR. Quasimodo cells in the resistant mutants were larger than the ones in the sensitive and parental strains; consequently, the conidia of the resistant mutants absorbed more CR than the germinating conidia of the sensitive or parental strains. Accordingly, this higher absorption rate by Quasimodo cells resulted in the removal of CR from the culture medium, allowing a subset of conidia to germinate and grow. In contrast, all resting conidia of the sensitive mutants and the parental strain were killed. This result indicated that the heterogeneity of the conidial population is essential to promote the survival of Aspergillus fumigatus in the presence of CR. Moreover, amorphous surface cell wall polysaccharides such as galactosaminogalactan control the influx of CR inside the cells and, accordingly, resistance to the drug. Finally, long-term incubation with CR led to the discovery of a new CR-induced growth effect, called drug-induced growth stimulation (DIGS), since the growth of one of them could be stimulated after recovery from CR stress. IMPORTANCE The compound Congo red (CR) has been historically used for coloring treatment and histological examination as well to inhibit the growth of yeast and filamentous fungi. It has been thought that CR binds to β-1,3-glucans in the fungal cell wall, disrupting the organization of the cell wall structure. However, other processes have been implicated in affecting CR sensitivity. Here, we explore CR susceptibility through screening a library of genetic null mutants. We find several previously uncharacterized genetic regulators important for CR susceptibility. Through biochemical and molecular characterization, we find cell membrane permeability to be important. Additionally, we characterize a novel cell type, Quasimodo cells, that occurs upon CR exposure. These cells take up CR, allowing the growth of the remaining fungi. Finally, we find that priming with CR can enhance long-term growth in one mutant.

Aspergillus fumigatus ffmA Encodes a C 2 H 2 -Containing Transcriptional Regulator That Modulates Azole Resistance and Is Required for Normal Growth

“…We found clear and reproducible reduction in the level of AbcG1 in strains lacking either the atrR or AFUB_026340 ( Afu2g10550 ) gene. The latter gene has recently been characterized as a locus enhancing expression of genes involved in fermentation (favors fermentative metabolism, ffmA [ 19 ]), and we will use this designation here. The ffmA gene has also been characterized as rfeC (regulator of FLO11 ) on the basis of activating the FLO11 promoter when expressed as a cDNA clone in Saccharomyces cerevisiae ( 22 ).…”

“…More detailed analysis of the phenotype of a ffmAΔ strain clearly indicated that loss of this gene had a modest azole drug phenotype compared to its pronounced defect in growth. Other screenings of this transcription factor deletion mutant library led to the detection of ffmA as a determinant required for resistance to cell wall stress agents but also showed a very similar general growth defect ( 19 ). Based on both this and the previous study of the phenotype of the ffmAΔ strain, it seems that the primary phenotype caused by loss of ffmA is a severe growth deficit.…”

“…We counterscreened these itraconazole-susceptible mutant strains for effects on expression of the AbcG1 ABC transporter using an antibody directed against this membrane protein ( 14 ). A poorly characterized gene called rfeC (recently renamed ffmA ) ( 19 ) was identified that caused a large decrease in AbcG1 expression. The ffmA gene encodes a protein containing a C 2 H 2 -containing sequence-specific DNA-binding domain ( 20 ).…”

The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans

“…We therefore spotted cells of the WT strain, the grx4Δ mutants and the complemented strain onto YPD media supplemented with SDS, Congo Red, caffeine, or CFW. The detergent SDS disrupts the plasma membrane, whereas CFW and Congo Red interfere with the synthesis of cell wall polysaccharides ( Elorza et al 1983 ; Wood and Fulcher 1983 ; Dichtl et al 2016 ; Liu et al 2021 ). Caffeine has a variety of targets in fungi including target of rapamycin (TOR) signaling and components of the CWI pathway ( Ruta and Farcasanu 2020 ).…”

Cell Wall Integrity and Its Industrial Applications in Filamentous Fungi

“…Overall, CFW and CR are thought to act by binding to chitin and β-linked-glucan chains, thereby inhibiting the assembly of chitin and β-linked-glucans and weakening the cell wall [ 114 ]. Recently, several transcription factors involved in CR sensitivity and CR dynamics in fungal cells have been analyzed [ 116 ]. The amorphous cell surface polysaccharide, galactosaminogalactan (GAG), interferes with the uptake of CR into the fungal cell [ 116 ].…”