Search

[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).

[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.