ABSTRACTβ-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.

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Despite the progress made by modern medicine, infectious diseases remain one of the most important threats to human health. Vaccination against pathogens is one of the primary methods used to prevent and treat infectious diseases that cause illness and death. Vaccines administered by the mucosal route are potentially a promising strategy to combat infectious diseases since mucosal surfaces are a major route of entry for most pathogens. However, this route of vaccination is not widely used in the clinic due to the lack of a safe and effective mucosal adjuvant. Therefore, the development of safe and effective mucosal adjuvants is key to preventing infectious diseases by enabling the use of mucosal vaccines in the clinic. In this study, we show that intranasal administration of a cationic liposome composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposome) has a potent mucosal adjuvant effect in mice. Intranasal vaccination with ovalbumin (OVA) in combination with DOTAP/DC-chol liposomes induced the production of OVA-specific IgA in nasal tissues and increased serum IgG1 levels, suggesting that the cationic DOTAP/DC-chol liposome leads to the induction of a Th2 immune response. Additionally, nasal-associated lymphoid tissue and splenocytes from mice treated with OVA plus DOTAP/DC-chol liposome showed high levels of IL–4 expression. DOTAP/DC-chol liposomes also enhanced OVA uptake by CD11c+ dendritic cells in nasal-associated lymphoid tissue. These data demonstrate that DOTAP/DC-chol liposomes elicit immune responses via an antigen-specific Th2 reaction. These results suggest that cationic liposomes merit further development as a mucosal adjuvant for vaccination against infectious diseases.

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A beta-D-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of beta-D-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this beta-D-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-beta-glucans like AP-FBG with such a high number of 1-6-beta-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-beta-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.

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Being external, the fungal cell wall plays a crucial role in the fungal life. By covering the underneath cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this cell wall is composed of different polysaccharides. Because of their specific composition, the fungal cell wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic fungal species. The objective of this review is to consolidate the current knowledge on the antifungal drugs targeting the cell wall and plasma membrane, mainly of Aspergillus and Candida species - the most prevalent fungal pathogens, and also to present challenges and questions conditioning the development of new antifungal drugs targeting the cell wall.