Successful infection by fungal pathogens depends on subversion of host immune mechanisms that detect conserved cell wall components such as β-glucans. A less common polysaccharide, α-(1,3)-glucan, is a cell wall constituent of most fungal respiratory pathogens and has been correlated with pathogenicity or linked directly to virulence. However, the precise mechanism by which α-(1,3)-glucan promotes fungal virulence is unknown. Here, we show that α-(1,3)-glucan is present in the outermost layer of the Histoplasma capsulatum yeast cell wall and contributes to pathogenesis by concealing immunostimulatory β-glucans from detection by host phagocytic cells. Production of proinflammatory TNFα by phagocytes was suppressed either by the presence of the α-(1,3)-glucan layer on yeast cells or by RNA interference based depletion of the host β-glucan receptor dectin-1. Thus, we have functionally defined key molecular components influencing the initial host–pathogen interaction in histoplasmosis and have revealed an important mechanism by which H. capsulatum thwarts the host immune system. Furthermore, we propose that the degree of this evasion contributes to the difference in pathogenic potential between dimorphic fungal pathogens and opportunistic fungi.

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Histoplasma capsulatum α-(1,3)-glucan blocks innate immune recognition by the β-glucan receptor

Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.

RNA interference in Histoplasma capsulatum demonstrates a role for α‐(1,3)‐glucan in virulence

A number of substances that directly or indirectly affect the cell walls of fungi have been identified. Those that actively interfere with the synthesis or degradation of polysaccharide components share the property of being produced by soil microbes as secondary metabolites. Compounds specifically interfering with chitin or beta-glucan synthesis have proven effective in studies of preclinical models of mycoses, though they appear to have a restricted spectrum of coverage. Semisynthetic derivatives of some of the natural products have offered improvements in activity, toxicology, or pharmacokinetic behavior. Compounds which act on the cell wall indirectly or by a secondary mechanism of action, such as the azoles, act against diverse fungi but are usually fungistatic in nature. Overall, these compounds are attractive candidates for further development.

Compounds active against cell walls of medically important fungi.

The cell wall is commonly regarded as an assemblage of polymers, mainly polysaccharides, that occurs outside the plasma membrane of cells of plants, fungi, and bacteria. Because of its rigidity it maintains the shape of the cell and offers resistance to unlimited influx of water into the cell. In addition it offers protection and, being the outermost cover of cells, often contains molecules involved in interaction between cells.

Fungal Cell Walls: A Survey

Two species of crayfish, Astacus astacus and Pacifastacus leniusculus could recognize components from fungal cell walls, i.e. β-1,3-glucans, since a clotting process and subsequent protein (phenoloxidase) attachment to many foreign surfaces were induced by these β-1,3-glucans. The process of clotting and phenoloxidase attachment was dependent on Ca2+. The biochemical mechanism of β-1,3-glucan induction of phenoloxidase attachment is not fully understood, although preliminary observations indicate that the glucans activated a serine protease, which then induced phenoloxidase attachment.

Fungal Cell Wall β-1,3-Glucans Induce Clotting and Phenoloxidase Attachment to Foreign Surfaces of Crayfish Hemocyte Lysate

Chemical and ultrastructural studies of the cell walls of the yeastlike (Y) and mycelial (M) forms ofHistoplasma capsulatum G-184B revealed that the Y form contained about 46.5% ofα-glucan, 31.0% ofβ-glucan, 7.7% of galactomannan and 11.5% of chitin, whereas the M form cell wall contained about 18.8% ofβ-glucan, 24.7% of galactomannan, 25.8% of chitin, and essentially noα-glucan. Theα-glucan of the Y form contained mainly anα-(1 → 3)-linkage. Theβ-glucans of both forms may have mainly aβ-(1 → 3)-linkage. Chitin microfibrils were located mainly in the inner portion of the cell walls of the Y and M forms, whereas theα-glucan fibers were observed only in the outer portion of the Y form cell wall.

I. Azuma

Papers

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum.