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Maintaining appropriate levels of trace elements during infection of a host is essential for microbial pathogenicity. Here we compared the uptake of 10 trace elements from 3 commonly-used laboratory media by 3 pathogens, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, and a model yeast, Saccharomyces cerevisiae. The trace element composition of the yeasts, C. albicans, C. neoformans and S. cerevisiae, grown in rich (YPD) medium, differed primarily in P, S, Fe, Zn and Co. Speciation analysis of the intracellular fraction, which indicates the size of the organic ligands with which trace elements are complexed, showed that the ligands for S were similar in the three fungi but there were significant differences in binding partners for Fe and Zn between C. neoformans and S. cerevisiae. The profile for Cu varied across the 3 yeast species. In a comparison of C. albicans and A. fumigatus hyphae, the former showed higher Fe, Cu, Zn and Mn, while A. fumigatus contained higher P, S Ca and Mo. Washing C. albicans cells with the cell-impermeable chelator, EGTA, depleted 50–90 % of cellular Ca, suggesting that a large proportion of this cation is stored in the cell wall. Treatment with the cell wall stressor, Calcofluor White (CFW), alone had little effect on the elemental profile whilst combined Ca + CFW stress resulted in high cellular Cu and very high Ca. Together our data enhance our understanding of trace element uptake by pathogenic fungi and provide evidence for the cell wall as an important storage organelle for Ca.

Highlights • Mutants lacking GH16 chitin transferases are sensitive to cell wall perturbation reagents. • Mutants lacking GH17 β-1,3-glucan transferases are sensitive to cell wall perturbation reagents. • In N. crassa, GH17 β-1,3-glucan transferases and GH72 β-1,3-glucan/lichenin transferases are not redundant activities. • Neurospora GH72 enzymes form lichenin-enzyme intermediates. • Neurospora GH72 enzymes are lichenin transferases., GH16 chitin transferases, GH17 β-1,3-glucan transferases, and GH72 β-1,3-glucan/lichenin transferases are important fungal cell wall crosslinking enzymes. The Neurospora crassa genome encodes three genes from the GH17 gene family and five members in the GH16 subfamily 18 and 19 fungal chitin transferases. We created deletion mutants lacking all three GH17 genes and determined that they had wild type morphology and are more sensitive to cell wall perturbation reagents than the wild type. We also created deletion mutants lacking all five GH16 subfamily 18 and 19 genes and found that they had wild type morphology and are more sensitive to cell wall perturbation reagents than the wild type. We conclude that GH16 and GH17 enzymes play roles in cell wall biogenesis. In N. crassa, GH72 enzymes have been reported to be lichenin transferases, while in other fungi they have been shown to be the β-1,3-glucan transferases. Neurospora triple GH72 deletions give rise to a tight colonial morphology, sensitivity to cell wall perturbation reagents, and release of cell wall proteins into the medium. To ask if GH72 and GH17 enzymes might be redundant in N. crassa, we created sextuple mutants lacking the three GH72 genes and the three GH17 genes and found that they were indistinguishable from the GH72 triple mutant. We also found that a recombinant GH72 enzyme is able to form a lichenin-enzyme intermediate demonstrating that GH72 enzymes are lichenin transferases. The N. crassa GH72 enzymes are lichenin transferases and are not redundant with the GH17 β-1,3-glucan transferases.

This study provides new insights into the complex evolutionary history of the fungal cell wall. We analyzed fungal enzymes that convert sugars acquired from the environment into the diverse sugars that make up the fundamental building blocks of the cell wall., The fungi are an enormously successful eukaryotic lineage that has colonized every aerobic habitat on Earth. This spectacular expansion is reflected in the dynamism and diversity of the fungal cell wall, a matrix of polysaccharides and glycoproteins pivotal to fungal life history strategies and a major target in the development of antifungal compounds. Cell wall polysaccharides are typically synthesized by Leloir glycosyltransferases, enzymes that are notoriously difficult to characterize, but their nucleotide-sugar substrates are well known and provide the opportunity to inspect the monosaccharides available for incorporation into cell wall polysaccharides and glycoproteins. In this work, we have used phylogenomic analyses of the enzymatic pathways that synthesize and interconvert nucleotide-sugars to predict potential cell wall monosaccharide composition across 491 fungal taxa. The results show a complex evolutionary history of these cell wall enzyme pathways and, by association, of the fungal cell wall. In particular, we see a significant reduction in monosaccharide diversity during fungal evolution, most notably in the colonization of terrestrial habitats. However, monosaccharide distribution is also shown to be varied across later-diverging fungal lineages.

Aspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB) funded project, we effectively aimed to improve the composition of post-fermentation fungal biomass for extraction of the value-added product chitosan as a derivative of cell wall chitin (FunChi). As chitin/chitosan is not encountered in plant or human tissue, it often acts as an elicitor to plant and animal immune responses in order to fight off possible impending fungal infections. The application of both chitin and chitosan oligomers have been shown to prime plants against infection. This thesis discusses the identification of genes that are important for chitin deposition in the cell wall of A. niger. In addition, the work described here also investigates the genes that facilitate chitin cross-linking to the cell wall. The relevance of all findings are discussed in relation to both the improvement of chitin extraction from post-fermentation biomass and to the integrity of the fungal cell wall.

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches - the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish "natto." Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.

[Background] During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as β(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of β(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. [Purpose] Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. [Methods] An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms “natural antifungals” and “plant extracts” with “fungal cell wall”. [Results] The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. [Conclusion] This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs., J.C.G.C., M.A.C. and J.C.R. acknowledge Ministerio de Ciencia e Innovación (MICINN; PGC2018-098924-B-I00) and Junta de Castilla y León/FEDER (CSI150P20 and "Escalera de Excelencia" CLU-2017-03), Spain, for funds. L.A.S. acknowledges Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT2016-1833 and Universidad Nacional de Rosario (UNR) 1BIO571 for funds. E.B. acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for a postdoctoral fellowship. L.A.S. is member of the CONICET Researcher career. L.A.S. and E.B. belong to the teaching staff of Pharmacognosy area.

Fungal pathogens are the major cause of decay during postharvest handling and storage of fresh produce. Among the plethora of different fungi causing decay, necrotrophic fungi represent the main threat to major fruit crops, such as citrus, pome, or stone fruits. Classically, plant necrotrophic fungi have been considered unsophisticated pathogens that kill host cells with the use of toxins and/or cell wall-degrading enzymes (CWDE). However, studies conducted in recent years, mainly with Botrytis cinerea Pers. and Sclerotinia sclerotiorum (Lib.) de Bary are changing this simplistic perception about necrotrophic fungi. Consequently, there is an increased interest in elucidating the pathogenicity mechanisms deployed by these fungi. A recent review covered the molecular aspects of the interactions between postharvest pathogenic fungi and fruits, attending to both the pathogen and the host (Tian et al., 2016). This chapter is more focused on the current knowledge of the molecular mechanisms deployed by necrotrophic postharvest fungi.

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.

(1) Background: Mechanisms of cellular and molecular adaptation of fungi to salinity have been commonly drawn from halotolerant strains and few studies in basidiomycete fungi. These studies have been conducted in settings where cells are subjected to stress, either hypo- or hyperosmotic, which can be a confounding factor in describing physiological mechanisms related to salinity. (2) Methods: We have studied transcriptomic changes in Aspergillus sydowii, a halophilic species, when growing in three different salinity conditions (No NaCl, 0.5 M, and 2.0 M NaCl). (3) Results: In this fungus, major physiological modifications occur under high salinity (2.0 M NaCl) and not when cultured under optimal conditions (0.5 M NaCl), suggesting that most of the mechanisms described for halophilic growth are a consequence of saline stress response and not an adaptation to saline conditions. Cell wall modifications occur exclusively at extreme salinity, with an increase in cell wall thickness and lamellar structure, which seem to involve a decrease in chitin content and an augmented content of alfa and beta-glucans. Additionally, three hydrophobin genes were differentially expressed under hypo- or hyperosmotic stress but not when the fungus grows optimally. Regarding compatible solutes, glycerol is the main compound accumulated in salt stress conditions, whereas trehalose is accumulated in the absence of salt. (4) Conclusions: Physiological responses to salinity vary greatly between optimal and high salt concentrations and are not a simple graded effect as the salt concentration increases. Our results highlight the influence of stress in reshaping the response of extremophiles to environmental challenges.

Most fungi have multiple chitin synthases (CSs) that may make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. The structure-based model for CS function is for transfer of GlcNAc from UDP-GlcNAc at the cytoplasmic face of the plasma membrane to the non-reducing end of a growing chitin chain, which is concomitantly translocated through a transmembrane channel formed by the synthase. Two aspects of CS mechanism are investigated: how chains might be initiated, and what governs how long they can get. First, it is shown that CSs incorporate free GlcNAc into di-N-acetylchitobiose and into insoluble chitin in a UDP-GlcNAc-dependent manner, and therefore that GlcNAc primes chitin synthesis. Second, average lengths of insoluble chitin chains were measured by determining the molar ratio of priming GlcNAc to chain-extending, UDP-GlcNAc-derived GlcNAc, and showed dependence on UDP-GlcNAc concentration, approaching a maximum at higher concentrations of substrate. These results, together with previous findings that 2-acylamido GlcN analogues prime formation of chitin oligosaccharides and stimulate chitin synthesis are discussed in the context of the structure-based model, and lead to the following proposals. 1) CSs may “self-prime” by hydrolyzing UDP-GlcNAc to yield GlcNAc. 2) A CS’s active site is not continuously occupied by a nascent chitin chain, rather, CSs can release chitin chains, then re-initiate, and therefore synthesize chitin chains in bursts. 3) The length of chitin chains made by a given CS will impact that CS’s contribution to construction of the fungal cell wall. Keywords: Chitin synthesis, Fungal cell wall, Polysaccharide

The animal immune response to chitin is not well understood and needs to be investigated further. However, this is a challenging topic to study because of the technical difficulties in purifying chitin, and because this material usually comes associated with contaminating components that can activate the immune system. In this study, improvements to previously described purification protocols were investigated for chitin obtained from different sources, including commercial shellfish, Candida albicans yeast and hyphal cell walls, as well as cell walls of the filamentous fungi Aspergillus fumigatus and Mucor circinelloides. The immune response to these different chitin preparations was tested using human peripheral blood mononuclear cells. In agreement with previous literature, small chitin particles of an average size of 0.2 µm were not immunogenic. On the other hand, bigger chitin particles induced in some cases a pro-inflammatory response. The results of this work suggest that not only the purity and size of the chitin particles, but also their shape can influence immune recognition.

In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is absent in host mammalian cells, it stands as an important target when developing selective compounds for the treatment of fungal infections. Consequently, treatment with echinocandins, a family of antifungal agents that specifically inhibits the biosynthesis of cell wall (1-3)β-D-glucan, has been established as an alternative and effective antifungal therapy. However, the existence of many pathogenic fungi resistant to single or multiple antifungal families, together with the limited arsenal of available antifungal compounds, critically affects the effectiveness of treatments against these life-threatening infections. Thus, new antifungal therapies are required. Here we review the fungal cell wall and its relevance in biotechnology as a target for the development of new antifungal compounds, disclosing the most promising cell wall inhibitors that are currently in experimental or clinical development for the treatment of some invasive mycoses., We thank Emma Keck for language revision. J.C.G.C acknowledges support from a Postdoctoral Contract granted by the University of Salamanca, Spain. This publication was supported by the grants BIO2015-69958-P from the Ministry of Science, Innovation and Universities (MICIU) (http://www.ciencia.gob.es/) and grants CSI068P17 and Escalera de Excelencia CLU-2017-03 from the Regional Government of Castile and Leon (JCyL/FEDER) and the European Regional Development Fund (ERDF) (https://www.jcyl.es/).

Larvae of the stag beetle, Dorcus rectus, feed on decaying wood, which they digest with the aid of symbiotic yeasts; however, they can be successfully reared on artificial diets containing only fungal tissue. In this study we tested whether D. rectus larvae can utilize fungal cell walls, which are an insoluble component of mycelium. Lyophilized Bjerkandera adusta mycelium cultured in potato-dextrose liquid medium consisted of a 47.6% hot-water insoluble fraction by mass, which contains 53.7% of the total nitrogen in the mycelium. D. rectus larvae that hatched from surface-sterilized eggs were reared for 14 days on agar-based diets containing either the soluble fraction, insoluble fraction or both, extracted from 100 mg of mycelium. The larvae increased in mass most on the mixed diet, and there was no difference in their growth on the mixed and positive control diets. Both the soluble and insoluble fractions improved larval growth compared to the negative control diet; however, the growth rates were much lower than those expected from the nitrogen dose-growth response curve obtained in a previous study. Addition of b-chitin to the soluble fraction did not positively affect larval growth. Therefore, we conclude that (1) D. rectus larvae need both the soluble and insoluble fractions of mycelium and (2) the larvae digest the insoluble fraction using their own enzymes.

Fungi cause more than a billion skin infections, more than 100 million mucosal infections, 10 million serious allergies and more than a million deaths each year. Global mortality owing to fungal infections is greater than for malaria and breast cancer and is equivalent to that owing to tuberculosis (TB) and HIV. These statistics evidence fungal infections as a major threat to human health and a major burden to healthcare budgets worldwide. Those patients who are at greatest risk of life-threatening fungal infections include those who have weakened immunity or have suffered trauma or other predisposing infections such as HIV. To address these global threats to human health, more research is urgently needed to understand the immunopathology of fungal disease and human disease susceptibility in order to augment the advances being made in fungal diagnostics and drug development. Here, we highlight some recent advances in basic research in medical mycology and fungal immunology that are beginning to inform clinical decisions and options for personalized medicine, vaccine development and adjunct immunotherapies. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.

[Background]: The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: β-1,3 glucanase and chitinase activities (zymolyase), inhibition of β-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red). [Results]: Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common >signature of cell wall maintenance (CWM)>. This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, wsc1δ, elo2δ and elo3δ, showed a significant decrease in β-1,3-glucan synthase activity. [Conclusions]: This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms., This work was supported by grants BIO2010-22146, BIO2013-48136-P (MINECO, Spain) and S2010/BMD-2414 (Comunidad de Madrid) to J.A, and grant BIO2012-35372 (MINECO, Spain) to JCR.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:00Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:48:22Z : No. of bitstreams: 1 2-s2.0-84876989266.pdf: 2281619 bytes, checksum: fe1f0ca3dc04edbed781a1dcdd1a6925 (MD5) Made available in DSpace on 2014-05-27T11:29:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-30 Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis. © 2013 Silva et al. Department of Clinical Analyses Faculty of Pharmaceutical Sciences São Paulo State University - University Estadual Paulista Araraquara, São Paulo Department of Immunology Biomedical Institute São Paulo University, São Paulo Department of Clinical Analyses Faculty of Pharmaceutical Sciences São Paulo State University - University Estadual Paulista Araraquara, São Paulo

With the exception of the unicellular yeasts, fungi typically grow by means of hyphae that extend only at their apices and ramify into a mycelium. This mode of growth provides fungi with a certain mobility and the ability to invade dead and living organic substrata. They are thus the main decomposers of plant residues but they also have established intricate symbiotic relationships with plants, both mutualistic and parasitic. The process of apical growth of a hypha requires the controlled expansion of the apical wall which must be transformed subsequently into a wall that resists turgor pressure and maintains the tubular shape of the hypha. Although the driving force for hyphal extension is probably the turgor pressure, a subtle interplay between wall extension and cytoplasmic activity is necessary because only a precise gradient of wall-synthetic activity can maintain uniform wall thickness during expansion. Possibly, the presence in the plasma membrane of mechanico-sensitive proteins plays a role in conjunction with the cytoskeleton at the apex, particularly actin. Although the major structural wall polysaccharides are probably manufactured directly on the expanding apical plasma membrane, proteins (and probably some wall components) are delivered to the growing surface by a continuous stream of exocytotic vesicles that fuse with the plasma membrane, at the same time extending its surface. Our analyses of the chemistry of the fungal wall and its biosynthesis and assemblage have disclosed a simple mechanism (though complex in detail) that may explain the transition from a newly formed expandable wall at the apex to a more rigid wall at the base of the hyphal extension zone. Two individual wall polymers, chitin and beta-glucan, extruded at the apex are modified within the domain of the wall. Among the modifications observed are the formation of covalent crosslinks between these two polymers and hydrogen bonds between the homologous polymer chains, leading to the formation of chitin microfibrils crosslinked to a glucan matrix. This process is thought to convert an initially plastic wall into a rigid wall as the polymers fall behind the advancing tip. We have called this the steady-state growth theory for apical wall extension because a steady-state amount of plastic wall is always maintained at the growing apex. Excretion of lytic enzymes is a vital process in filamentous fungi because, in nature, they thrive on organic polymers which must be degraded extracellularly. Such enzymes are also necessary for infection processes. Cytological data suggest that such enzymes are extruded by the vesicles that continuously fuse with the plasma membrane at the growing apex. We have shown that a large portion of the excreted enzymes indeed leaves the hypha at the growing apex but another portion may be retained by the wall and is slowly released into the medium. In relation to the steady-state growth theory we hypothesize that enzymes can pass the wall at the apex by bulk flow, that is, by being carried by the flow of plastic wall material, making pores in the wall less important than previously thought, Proteins excreted by filamentous fungi not only serve dissimilatory purposes but are also important for a variety of other activities of the whole mycelium, including morphogenesis. By cloning genes abundantly expressed during formation of aerial hyphae and fruit bodies, we have discovered a class of proteins, named hydrophobins, which are only produced when the mycelium has reached a certain stage of maturity. Whilst excreted by submerged hyphae as monomers into the medium, they self-assemble as insoluble complexes in the walls of emergent hyphae. In aerial hyphae a particular hydrophobin takes the form of rodlets which probably coat the hyphae with an impermeable layer. During fruit-body formation other hydrophobins are produced which may function in the aggregation of hyphae to form a multicellular tissue. Apart from such specific morphogenetic functions, the hydrophobins may play a general role in insulating hyphae from the environment, converting the differentiating structures into sinks for translocation of water and nutrients from the assimilating mycelium.

This study evaluated the antifungal activities of synthetic naphthoquinones against opportunistic and dermatophytic fungi and their preliminary mechanisms of action. The minimum inhibitory concentrations (MICs) of four synthetic naphthoquinones for 89 microorganisms, including opportunistic yeast agents, dermatophytes and opportunistic filamentous fungi, were determined. The compound that exhibited the best activity was assessed for its action against the cell wall (sorbitol test), for interference associated with ergosterol interaction, for osmotic balance (K+ efflux) and for membrane leakage of substances that absorb at the wavelength of 260 nm. All tested naphthoquinones exhibited antifungal activity, and compound IVS320 (3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione) demonstrated the lowest MICs across the tested species. The MIC of IVS320 was particularly low for dermatophytes (values ranging from 5-28 μg/mL) and Cryptococcus spp. (3-5 μg/mL). In preliminary mechanism-of-action tests, IVS320 did not alter the fungal cell wall but did cause problems in terms of cell membrane permeability (efflux of K+ and leakage of substances that absorb at 260 nm). This last effect was unrelated to ergosterol interactions with the membrane. © 2014 Ferreira et al.