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In recent years, invasive fungal infections have emerged as a common source of infections in immunosuppressed patients. All fungal cells are surrounded by a cell wall that is essential for cell integrity and survival. It prevents cell death and lysis resulting from high internal turgor pressure. Since the cell wall is not present in animal cells, it is an ideal target for selective invasive fungal infection treatments. The antifungal family known as echinocandins, which specifically inhibit the synthesis of the cell wall β(13)glucan, has been established as an alternative treatment for mycoses. To explore the mechanism of action of these antifungals, we analyzed the cell morphology and glucan synthases localization in Schizosaccharomyces pombe cells during the initial times of growth in the presence of the echinocandin drug caspofungin. S. pombe are rod-shaped cells that grow at the poles and divide by a central division septum. The cell wall and septum are formed by different glucans, which are synthesized by four essential glucan synthases: Bgs1, Bgs3, Bgs4, and Ags1. Thus, S. pombe is not only a perfect model for studying the synthesis of the fungal β(1-3)glucan, but also it is ideal for examining the mechanisms of action and resistance of cell wall antifungals. Herein, we examined the cells in a drug susceptibility test in the presence of either lethal or sublethal concentrations of caspofungin, finding that exposure to the drug for long periods at high concentrations (>10 µg/mL) induced cell growth arrest and the formation of rounded, swollen, and dead cells, whereas low concentrations (

[Background] The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non‑contaminating activities is vital., [Results] Here we examined the efficiency and reproducibility of 12 recombinant endo‑β(1,3)‑d‑glucanases for specifically degrading the cell wall β(1,3)‑d‑glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α‑glucan, β(1,3)‑d‑glucan and β(1,6)‑d‑glucan. We found that the GH16 endo‑β(1,3)‑d‑glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)‑d‑glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo‑β(1,3)‑d‑glucanase no longer commercially available. Moreover, other recombinant β(1,3)‑d‑glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides, [Conclusions] The discovery of a reliable and efficient recombinant endo‑β(1,3)‑d‑glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)‑d‑glucan. This new commercial endo‑β(1,3)‑d‑glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for othern practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis, This work was supported by grants PGC2018-098924-B-I00 (Spanish Ministry of Science and Innovation, MICINN , Spain; and the European Regional Developmental Fund, FEDER, EU), and CSI150P20 and “Escalera de Excelencia” CLU-2017-03 (Regional Government of Castile and Leon, JCYL, Spain; and the European Regional Developmental Fund, FEDER, EU).

Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins’ mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs., This research was funded by grants PGC2018-098924-B-I00 (the Spanish Ministry of Science and Innovation, MICINN, Spain; the European Regional Developmental Fund, FEDER, EU), and CSI150P20 and “Escalera de Excelencia” CLU-2017-03 (Regional Government of Castile and Leon, JCYL, Spain; and the European Regional Developmental Fund, FEDER, EU).

[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.

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/).

The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell- cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. © 2014 by the Genetics Society of America., Financial support to H.V.M. from the Comisión Interministerial de Ciencia Y Tecnología (Spain)/European Union Fondo Europeo de Desarrollo Regional program (grant BFU2010-15085) made this work possible. M.A.C. and N.d.L were supported by Formación de Personal Universitario fellowships from the Ministry of Science, and M.H. was supported by a Junta de Ampliación de Estudios predoctoral fellowship from the Consejo Superior de Investigaciones Científicas, Spain.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., In yeast, cytokinesis requires coordination between nuclear division, acto-myosin ring contraction, and septum synthesis. We studied the role of the Schizosaccharomyces pombe Bgs1p and Cfh3p proteins during cytokinesis under stress conditions. Cfh3p formed a ring in the septal area that contracted during mitosis; Cfh3p colocalized and co-immunoprecipitated with Cdc15p, showing that Cfh3p interacted with the contractile acto-myosin ring. In a wild-type strain, a significant number of contractile rings collapsed under stress conditions and this number increased dramatically in the cfh3Δ, bgs1cps1-191, and cfh3Δ bgs1/cps1-191. Our results show that after osmotic shock Cfh3p is essential for the stability of the (1,3) glucan synthase Bgs1p in the septal area, but not at the cell poles. Finally, cells adapted to stress; they repaired their contractile rings and re-localized Bgs1p to the cell surface some time after osmotic shock. A detailed analysis of the cytokinesis machinery in the presence of KCl revealed that the actomyosin ring collapsed before Bgs1p was internalized, and that it was repaired before Bgs1p re-localized to the cell surface. In the cfh3Δ, bgs1/cps1-191, and cfh3Δ bgs1/cps1-191 mutants, which have reduced glucan synthesis, the damage produced to the ring had stronger consequences, suggesting that an intact primary septum contributes to ring stability. The results show that the contractile actomyosin ring is very sensitive to stress, and that cells have efficient mechanisms to remedy the damage produced in this structure. © 2012 Sharifmoghadam et al., Financial support to the Instituto de Biología Funcional y Genómica (IBFG) from the Fundación Ramón Areces and to our group from the Comisión Interministerial de Ciencia y Tecnología (CICYT) (Spain)/European Union FEDER (Fondo Europeo de Desarrollo Regional) program (grant BFU2007-61866) made this work possible. Dr. Curto was supported by a contract for training in research and technological innovation by Junta de Castilla y León and by Formación de Personal Investigador (FPU) fellowship. Dr. de León and Dr. Hoya were supported by FPU and Junta de Ampliación de Estudios (JAE)-PREDOC fellowships from the Ministry of Science and Consejo Superior de Investigaciones Científicas (CSIC), Spain, respectively.

El pdf del artículo es la versión pre-print., Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi-including Schizosaccharomyces pombe, which has no detectable amounts of chitin-have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific. © 2010 Springer Basel AG., This work has been supported by grants BFU2007- 61866 from the CICYT and GR231 from the Junta de Castilla y León, Spain. NdL was supported by a fellowship from the Spanish Ministry of Science. RMG and PBS were supported by postgraduate I3P fellowships from the Spanish council of research (CSIC).

This is an open-access article distributed under the terms of the Creative Commons Attribution License., The regulation of cell wall synthesis by the clathrin light chain has been addressed. Schizosaccharomyces pombe clc1Δ mutant was inviable in the absence of osmotic stabilization; when grown in sorbitol-supplemented medium clc1Δ cells grew slowly, formed aggregates, and had strong defects in morphology. Additionally, clc1Δ cells exhibited an altered cell wall composition. A mutant that allowed modulating the amount of Clc1p was created to analyze in more detail the dependence of cell wall synthesis on clathrin. A 40% reduction in the amount of Clc1p did not affect acid phosphatase secretion and bulk lipid internalization. Under these conditions, β(1,3)glucan synthase activity and cell wall synthesis were reduced. Also, the delivery of glucan synthases to the cell surface, and the secretion of the Eng1p glucanase were defective. These results suggest that the defects in the cell wall observed in the conditional mutant were due to a defective secretion of enzymes involved in the synthesis/remodelling of this structure, rather than to their endocytosis. Our results show that a reduction in the amount of clathrin that has minor effects on general vesicle trafficking has a strong impact on cell wall synthesis, and suggest that this is the reason for the lethality of clc1Δ cells in the absence of osmotic stabilization., Financial support to our group from the MICINN (http://www.idi.mineco.gob.es/. Spain)/European Union FEDER program (grant BFU2010-15085) made this work possible. NdL and MAC were supported by FPU fellowships from the Ministry of Science, and MH was supported by a JAE-PREDOC fellowship from Consejo Superior de Investigaciones Cientificas, Spain.