Eng1 and Exg8 Are the Major α-Glucanases Secreted by the Fungal Pathogen Histoplasma capsulatum

Abstract

Edited by Gerald W. HartFungal cell walls contain ␤-glucan polysaccharides that stimulate immune responses when recognized by host immune cells. The fungal pathogen Histoplasma capsulatum minimizes detection of ␤-glucan by host cells through at least two mechanisms: concealment of ␤-glucans beneath ␣-glucans and enzymatic removal of any exposed ␤-glucan polysaccharides by the secreted glucanase Eng1. Histoplasma yeasts also secrete the putative glucanase Exg8, which may serve a similar role as Eng1 in removing exposed ␤-glucans from the yeast cell surface. Here, we characterize the enzymatic specificity of the Eng1 and Exg8 proteins and show that Exg8 is an exo-␤1,3-glucanase and Eng1 is an endo-␤1,3-glucanase. Together, Eng1 and Exg8 account for nearly all of the total secreted glucanase activity of Histoplasma yeasts. Both Eng1 and Exg8 proteins are secreted through a conventional secretion signal and are modified posttranslationally by O-linked glycosylation. Both glucanases have near maximal activity at temperature and pH conditions experienced during infection of host cells, supporting roles in Histoplasma pathogenesis. Exg8 has a higher specific activity than Eng1 for ␤1,3-glucans; yet despite this, Exg8 does not reduce detection of yeasts by the host ␤-glucan receptor Dectin-1. Exg8 is largely dispensable for virulence in vivo, in contrast to Eng1. These results show that Histoplasma yeasts secrete two ␤1,3-glucanases and that Eng1 endoglucanase activity is the predominant factor responsible for removal of exposed cell wall ␤-glucans to minimize host detection of Histoplasma yeasts.

Metabolism of Gluconeogenic Substrates by an Intracellular Fungal Pathogen Circumvents Nutritional Limitations within Macrophages

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Differentiation of the fungus Histoplasma capsulatum into a pathogen of phagocytes

“…Loss of Eng1 attenuates Histoplasma virulence in mice but is restored in Dectin-1-deficient mice, confirming the role of Eng1 in reducing β-glucan recognition by Dectin-1 [18**]. Although yeasts also secrete a β-(1,3)-exoglucanase (Exg8), this glycosyl hydrolase has minimal effects on Histoplasma virulence [17]. Masking β-glucans and pruning away surface β-glucan fragments constitute two additive mechanisms specified by the yeast-phase regulon to minimize detection of Histoplasma yeasts by host phagocytes (Figure 2).…”

“…Loss of α-glucan due to impairment of its biosynthetic pathway increases immune cell recognition of yeasts, increases production of proinflammatory cytokines, and attenuates Histoplasma virulence in mice. Examination of the secreted proteome [16] identified Eng1, a secreted yeast phase-specific glucanase that hydrolyzes β-(1,3)-glucans [17]. Eng1 trims exposed β-glucans from the yeast cell surface, diminishing β-glucan recognition by the Dectin-1 receptor and reducing proinflammatory responses [18**].…”