Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

SUMMARY Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs, and how it is deposited as extracellular granules, remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally-derived melanosomes.

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.

Cellulose nanocrystals (CNCs) have emerged as a sustainable nanomaterial for several environmental applications, including the development of novel antimicrobial agents. Although previous studies have reported antibacterial activity for CNCs, their toxicity mechanism to bacterial cells is still unknown. Here, we investigate the toxicity of CNCs dispersed in water and coated surfaces against Escherichia coli cells. CNC-coated surfaces were able to inactivate approximately 90% of the attached E. coli cells, confirming potential of CNCs to be applied as a sustainable and cost-effective antibiofouling nanomaterial. The toxicity of CNCs in a suspension was concentration-dependent, and an inhibitory concentration (IC50%) of 200 μg/mL was found. Glutathione and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFA) assays were conducted to evaluate the role of oxidative stress in the CNC toxicity mechanism. Our findings showed that oxidative stress has no significant effect on the antimicrobial activity of CNC. In contrast, scanning electron microscopy (SEM) images and a leakage assay performed with dye-encapsulated phospholipid vesicles indicated that CNCs inactivate bacteria by physically damaging their cell membrane. CNC interaction with dye-encapsulated vesicles resulted in a dye leakage corresponding to 43% of the maximum value, thus confirming that contact-mediated membrane stress is the mechanism governing the toxicity of CNCs to bacteria cells.

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In this study, we were able to impart antimicrobial properties onto the surface of a commercial thin-film composite (TFC) membrane using sustainably derived cellulose nanocrystals (CNC) extracted from elephant grass (Pennisetum purpureum) leaves. Carboxylic acid-containing CNC were chemically bound to the amine-terminated polyamide active layer of TFC membranes using a cross-linking reaction. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy were conducted to confirm the presence of CNC on the membrane surface. TFC membranes functionalized with needle-like and antimicrobial CNC nanoparticles showed robust toxicity to bacteria, inactivating ∼89% of attached Escherichia coli cells under contact. These findings establish that functionalization with CNC is a promising approach for mitigating biofouling on TFC membranes and substantiates the application of sustainable materials for the design of the next-generation membranes for water purification.

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