Ultrastructural specialization at the host-pathogen interface in rust-infected flax

Summary Ultrastructure of the association between the rust fungus, Melampsora lini, and a compatible variety of flax, Linum usitatissimum, was studied to clarify the structural relationships and interactions at the site of host penetration and at the host-parasite interface. Results of freeze-etching as well as a special section-staining procedure consisting of periodate-chromate-phosphotungstate (PACP) are shown with a host-parasite combination for the first time. The host plasma membrane is invaginated by the fungus and forms a continuous boundary around the fungal haustoria which penetrate the host cells. No morphological continuities are observed linking the protoplasts of host and fungus. With both freeze-etching and the PACP stain, the invaginated portion of the host plasma membrane at the host-parasite interface shows distinctive features that are not characteristic of the non-invaginated portion of the membrane. This localized specialization of host plasma membrane in response to the fungus appears as a significant and consistent feature of the host-parasite interaction. The host plasma membrane is separated from the haustorial wall by an amorphous layer of sheath material which covers the body but not the neck of the haustorium. This sheath provides the environment in which the haustorium exists and functions during the course of the host-parasite association. Occasionally, a collar of wall-like material derived from the host cell forms around the haustorial neck. The collar is continuous with the host wall and is distinct and discontinuous from the haustorial sheath. In fewer than 5% of the infected cells this wall material encases entire haustoria. The fungal wall is structurally specialized around the site of host penetration, and it becomes intimately associated with the host wall where the fungus penetrates into the lumen of the host cell. During penetration, the host and fungal walls appear to be fused so that the interface between them is not clearly delineated. The haustorial wall is continuous, via the haustorial neck, with the wall of the haustorial mother cell which lies outside the host cell. Different staining properties reveal this wall continuum to consist of several well-defined regions having different structure or composition. A ring of fungal wall material midway along the haustorial neck stains densely with lead citrate, but is preferentially etched away by periodic acid. The neck ring denotes a transition in the staining reaction of the fungal wall, from that present in the region of host penetration to that of the wall surrounding the haustorium. The findings demonstrate specialization of the fungal wall in the area of host penetration as well as specialization of the host plasma membrane at the host-parasite interface to a degree not previously realized from ultrastructural information.     

Generating cell surface diversity in Candida albicans and other fungal pathogens

The fungal cell surface contributes to pathogenesis by mediating interactions with host cells and eliciting host immune responses. This review focuses on the cell wall proteome of the major fungal pathogen Candida albicans and discusses how diversity at the cell surface can be introduced by altering the expression and structure of cell wall proteins. Remodelling the cell wall architecture is critical to maintain cellular integrity in response to different environments and stresses including challenge with antifungal drugs. In addition, the dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host's immune system. Examples of the role of cell surface diversity in the pathogenesis of a number of microorganisms are described.     

Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. Here, we report that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, we find that there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host.     

Mannosylation in Candida albicans: role in cell wall function and immune recognition

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post‐translational modifications being the addition of O‐ and N‐linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O‐ and N‐linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host–fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.     

Antibacterial action of acetic acid soluble material isolated from Mucor rouxii and its application onto textile

Acetic acid soluble material (AcSM) is a chitosan-rich fraction isolated from the fungal cell wall materials. The final step in the traditional production of fungal chitosan is the separation of chitosan from the cell wall AcSM via raising the pH to 9-10 followed by centrifugation. This step results in further undesirable economic and environmental effects. The goal of this paper is to avoid that by investigating the antimicrobial effect of the whole AcSM from Mucor rouxii DSM-1191 cell wall and its application on cotton fabrics. The treated fabrics were characterized through monitoring the textile physical properties and for the antibacterial activity against Escherichia coli and Micrococcus luteus. Results showed that Mucor rouxii DSM-1191 has excellent potentials to be used for cell wall AcSM production on industrial scale with a maximum content of 40% in dry mycelia. The obtained results indicated that the physical properties of the treated fabrics, as well as the antibacterial activity, were improved after treatment with fungal AcSM.     

几丁质合酶在人类致病真菌中的研究进展

抑制真菌细胞壁的合成常作为防治真菌感染的安全有效手段。几丁质是真菌细胞壁及隔膜的重要结构成分，几丁质合酶是催化几丁质合成的关键酶。真菌细胞中几丁质合酶家族的不同成员在调控几丁质的合成中存在着差异，因此产生不同的生物学效应。本文通过综述几丁质合酶在人体三大条件致病真菌白色念珠菌、烟曲霉、新生隐球菌中的研究进展，分析了几丁质合酶对真菌致病性影响的机制，总结了几丁质合酶调控真菌细胞增殖、形态转换、病原菌与宿主的相互作用和细胞壁损伤诱导的补偿效应，展望了抗真菌感染的新策略及关于真菌几丁质合酶的未来研究方向。     

烟曲霉细胞壁的组分、组装及其功能概述

环境中普遍存在的腐生丝状真菌烟曲霉Aspergillus fumigatus在免疫功能低下或缺陷的人群中可引起多种急慢性疾病,包括致死率很高的侵袭性曲霉病。细胞壁作为真菌的细胞外骨架结构不仅起维持细胞形状、保护细胞抵抗外界压力等作用,在病原真菌极性生长、入侵新的生态域、启动宿主免疫反应中也起重要作用。细胞壁组分还是真菌感染的分子诊断基础和开发抗真菌药物的理想靶标。近几十年来烟曲霉细胞壁的遗传、生物化学及免疫学方向的研究使其成为研究真菌细胞壁的模式真菌。本文主要概述烟曲霉细胞壁的组分、分子组装机制及其在真菌生存和感染中的作用,并对未来研究方向提出了展望。     

Formation of arthroconidia during regeneration and selection of transformed Epichloë festucae protoplasts

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species.     

The structure and synthesis of the fungal cell wall

The fungal cell wall is a dynamic structure that protects the cell from changes in osmotic pressure and other environmental stresses, while allowing the fungal cell to interact with its environment. The structure and biosynthesis of a fungal cell wall is unique to the fungi, and is therefore an excellent target for the development of anti-fungal drugs. The structure of the fungal cell wall and the drugs that target its biosynthesis are reviewed. Based on studies in a number of fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins. The biosynthesis of the various components of the fungal cell wall and the importance of the components in the formation of a functional cell wall, as revealed through mutational analyses, are discussed. There is strong evidence that the chitin, glucans and glycoproteins are covalently cross-linked together and that the cross-linking is a dynamic process that occurs extracellularly.     

Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.     

Role of the Fungal Cell Wall in Pathogenesis and Antifungal Resistance

Study of the fungal cell wall is currently an area of very active research. The relevance of the fungal cell wall for cell survival, and pathogenicity has been well established. The view of the cell wall as a tough and impenetrable structure has been left behind, and it is now conceived as a plastic shield that undergoes structural changes depending on the surrounding environmental conditions and morphological states. The fungal cell wall is also the source of most of the pathogen-associated molecular patterns that immune cells recognize, and thus facilitates establishment of a protective antifungal immunity. Paradoxically, fungi, through their cell wall, possess disguising mechanisms to avoid immune recognition. This review gathers the current knowledge about the cell wall of Candida albicans, Aspergillus fumigatus and Paracoccidioides brasiliensis, stressing the importance of the fungal cell wall for pathogenesis, immune recognition, and as a source of targets for antifungal drugs.     

Dressed to impress: impact of environmental adaptation on the Candida albicans cell wall

Candida albicans is an opportunistic fungal pathogen of humans causing superficial mucosal infections and life‐threatening systemic disease. The fungal cell wall is the first point of contact between the invading pathogen and the host innate immune system. As a result, the polysaccharides that comprise the cell wall act as pathogen associated molecular patterns, which govern the host–pathogen interaction. The cell wall is dynamic and responsive to changes in the external environment. Therefore, the host environment plays a critical role in regulating the host–pathogen interaction through modulation of the fungal cell wall. This review focuses on how environmental adaptation modulates the cell wall structure and composition, and the subsequent impact this has on the innate immune recognition of C. albicans.     

Cell wall structure and biogenesis in Aspergillus species

Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Mechanisms of immune evasion in fungal pathogens

The incidence of life-threatening fungal infections has continued to increase in recent years, predominantly in patients debilitated by iatrogenic interventions or immunological dysfunctions. While the picture of the immunology of fungal infections grows increasingly complex, it is clear that the phagocyte-pathogen interaction is a critical determinant of establishing an infection. About 10 years ago, genome-scale approaches began to elucidate the intricate and extensive fungal response to phagocytosis and in the last few years it has become clear that some of this response actively modulates immune cell function. Fungal pathogens avoid detection by masking pathogen-associated molecular patterns, such as cell wall carbohydrates, and by downregulating the complement cascade. Once detected, various species interfere with phagocytosis and intracellular trafficking, and can repress production of antimicrobials like nitric oxide (NO). For the most part, the molecular mechanisms behind these behaviors are not yet known. This review discusses recent discoveries and insights into how fungi manipulate the host-pathogen interaction.     

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.     

Plasma membrane-cell wall adhesion is required for expression of plant defense responses during fungal penetration

Fungal pathogens almost invariably trigger cell wall-associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane-cell wall adhesion as shown by the loss of the thin plasma membrane-cell wall connections known as Hechtian strands, reduced the expression of cell wall-associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall-associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane-cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall-associated defenses, there was no change, or an increase, in plasma membrane-cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall-associated defenses and a localized decrease in plasma membrane-cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane-cell wall adhesion resulted in reduced penetration by the rust fungus and induction of cell wall-associated defenses. These results suggest that rust fungi may induce a decrease in plasma membrane-cell wall adhesion as a means of disrupting the expression of nonspecific defense responses during penetration of host cells.     

The cell wall structure: developments in diagnosis and treatment of candidiasis.

Candidiasis are among the fungal infections the most difficult to diagnose and treat. Research focused on specific fungal components which are absent in the host, such as the cell wall has lead to a better understanding of Candida albicans pathogenicity and clinical impact. The cell wall is responsible for antigenic expression and primary interaction with the host. It is composed mainly of beta-glucans, chitin and mannoproteins, which account for the rigidity of the wall and for the fungal morphology. Of these components, mannoproteins might carry a "morphogenetic code" which might modulate the molecular architecture of the cell wall. The features of specific cell wall proteins as part of building blocks to form this structure is revised, and the usefulness of monoclonal antibodies obtained against cell wall components to study those processes, together with their clinical applicability, is discussed.     

海洋芽孢杆菌(Bacillus marinus)B-9987菌株抑制病原真菌机理

[目的]探讨海洋芽孢杆菌(Bacillus marinus)B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理.[方法]分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化.[结果]BMME-1对茄链格孢菌的抑菌中浓度(MIC_(50))为6.2 mg/L,最小杀菌浓度(MFC)为50 mg/L,在MIC_(50)浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结构β-型糖苷键、碳-氧键(C-O)、碳-氢键(C-H)等基团的特征吸收强度降低,-OH、C=O的伸缩振动吸收强度升高;菌体细胞壁几丁质结构中酰胺I键吸收强度发生变化;与对照菌株的麦角甾醇含量(62.52±3.31%)相比,处理菌株麦角甾醇减少为(56.36±2.52)%,同时出现麦角固醇合成中间产物粪甾醇.[结论]BMME-1对病原真菌的抑制表现为:干扰细胞膜麦角甾醇的合成从而改变了细胞的通透性;对细胞壁葡聚糖结构的影响较大而几丁质次之.