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Abstract This study investigates the chemical inhibition of asexual development in Aspergillus nidulans to enhance the mechanical properties of mycelial materials. We hypothesized that suppressing conidiation using the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO) would increase material strength by inhibiting asexual development, promoting denser hyphal packing. Mycelial materials were grown in DFMO concentrations (0, 0.05, 0.5, and 5 mM), and conidiation and ultimate tensile strength (UTS) were measured. Results showed a dose-dependent reduction in conidiation, with significant decreases at all DFMO levels (P ≤ 0.05). While lower DFMO concentrations (0.05 and 0.5 mM) did not significantly alter UTS, 5 mM DFMO treatment doubled the material’s tensile strength compared to controls (P ≤ 0.05). Scanning electron microscopy confirmed reduced developmental structures in DFMO-treated samples, supporting the hypothesis. The non-linear relationship between conidiation suppression and strength improvement suggests additional mechanisms, such as hyphal morphology or cell wall changes, may contribute. These findings demonstrate that chemical modulation of fungal development can rationally tune mycelial material properties, offering a systematic approach for biomaterial engineering. 

JF2 08-2F Crusty is a novel melanized polyextremotolerant fungus isolated from a biological soil crust, which we believe harborsMethylobacteriumspp. endosymbionts, called Pinky. Crusty is capable of utilizing many sources of carbon and nitrogen and is resistant to multiple metals and UV-C due to its melanized cell wall. We were unable to recover a Pinky-free culture of Crusty via usage of antibiotics. However, when exposed to antibiotics that kill or stop the growth of the Pinky, growth of Crusty is significantly stunted, implying that actively growing Pinky symbionts are needed for Crusty’s optimal growth. The Crusty-Pinky symbiosis also seems to be able to perform active metabolism in carbonless and nitrogenless medium, which we believe is due to Pinky’s ability to perform aerobic anoxygenic photosynthesis. Finally, Pinky was identified as being capable of growth stimulation of the algaeChlorella sorokiniana, indicating that Pinky likely produces cytokinins or auxins whichMethylobacteriumare known for. Features of this symbiosis provide us insight into the ecological roles of these microbes within the biological soil crust. 

ABSTRACT This study investigates a previously unreported stress signal transduced as crosstalk between the cell wall integrity (CWI) pathway and the septation initiation network (SIN). Echinocandins, which target cell wall synthesis, are widely used to treat mycoses. Their efficacy, however, is species specific. Our findings suggest that this is due largely to CWI–SIN crosstalk and the ability of filamentous species to fortify with septa in response to echinocandin stress. To better understand this crosstalk, we used a microscopy-based assay to measure septum density, aiming to understand the septation response to cell wall stress. The echinocandin micafungin, an inhibitor of β-(1,3)-glucan synthase, was employed to induce this stress. We observed a strong positive correlation between micafungin treatment and septum density in wild-type strains. This finding suggests that CWI activates SIN under cell wall stress, increasing septum density to protect against cell wall failure. More detailed investigations, with targeted knockouts of CWI and SIN signaling proteins, enabled us to identify crosstalk occurring between the CWI kinase, MpkA, and the SIN kinase, SepH. This discovery of the previously unknown crosstalk between the CWI and SIN pathways not only reshapes our understanding of fungal stress responses, but also unveils a promising new target pathway for the development of novel antifungal strategies. IMPORTANCEEchinocandin-resistant species pose a major challenge in clinical mycology by rendering one of only four lines of treatment, notably one of the two that are well-tolerated, ineffective in treating systemic mycoses of these species. Previous studies have demonstrated that echinocandins fail against highly polarized fungi because they target only apical septal compartments. It is known that many filamentous species respond to cell wall stress with hyperseptation. In this work, we show that echinocandin resistance hinges on this dynamic response, rather than on innate septation alone. We also describe, for the first time, the signaling pathway used to deploy the hyperseptation response. By disabling this pathway, we were able to render mycelia susceptible to echinocandin stress. This work enhances our microbiological understanding of filamentous fungi and introduces a potential target to overcome echinocandin-resistant species. 

Abstract Under synchronized conidiation, over 2500 gene products show differential expression, including transcripts for bothbrlAandabaA, which increase steadily over time. In contrast, during wall-stress induced by the echinocandin micafungin, thebrlAtranscript is upregulated while theabaAtranscript is not. In addition, whenmpkA(last protein kinase in the cell wall integrity signaling pathway) is deleted,brlAexpression is not upregulated in response to wall stress. Together, these data imply BrlA may play a role in a cellular stress-response which is independent of the canonical BrlA-mediated conidiation pathway. To test this hypothesis, we performed a genome-wide search and found 332 genes with a putative BrlA response element (BRE) in their promoter region. From this set, we identified 28 genes which were differentially expressed in response to wall-stress, but not during synchronized conidiation. This set included seven gene products whose homologues are involved in transmembrane transport and 14 likely to be involved in secondary metabolite biosynthesis. We selected six of these genes for further examination and find that they all show altered expression behavior in thebrlAdeletion strain. Together, these data support the idea that BrlA plays a role in various biological processes outside asexual development. ImportanceTheAspergillus nidulanstranscription factor BrlA is widely accepted as a master regulator of conidiation. Here, we show that in addition to this function BrlA appears to play a role in responding to cell-wall stress. We note that this has not been observed outsideA. nidulans. Further, BrlA-mediated conidiation is highly conserved acrossAspergillusspecies, so this new functionality is likely relevant in otherAspergilli. We identified several transmembrane transporters that have altered transcriptional responses to cell-wall stress in abrlAdeletion mutant. Based on our observation, together with what is known about thebrlAgene locus’ regulation, we identifybrlAβas the likely intermediary in function ofbrlAin the response to cell-wall stress. 

Abstract Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primar yeast form. These fungi grow in xeric, nutrient depletes environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community are not well understood. We have isolated 2 novel black yeasts from the genus Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e. E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niche within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excretes melanin which can potentially provide ultraviolet resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi. 

ABSTRACT Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (Δ sepH , Δ bud3 , Δ bud4 , and Δ rho4 ) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens. 

Fungal cell wall receptors relay messages about the state of the cell wall to the nucleus through the Cell Wall Integrity Signaling (CWIS) pathway. The ultimate role of the CWIS pathway is to coordinate repair of cell wall damage and to restore normal hyphal growth. Echinocandins such as micafungin represent a class of antifungals that trigger cell wall damage by affecting synthesis of β-glucans. To obtain a better understanding of the dynamics of the CWIS response and its multiple effects, we have coupled dynamic transcriptome analysis with morphological studies of Aspergillus nidulans hyphae in responds to micafungin. Our results reveal that expression of the master regulator of asexual development, BrlA, is induced by micafungin exposure. Further study showed that micafungin elicits morphological changes consistent with microcycle conidiation and that this effect is abolished in the absence of MpkA. Our results suggest that microcycle conidiation may be a general response to cell wall perturbation which in some cases would enable fungi to tolerate or survive otherwise lethal damage.