alpha-(1,4)-Amylase, but not alpha- and beta-(1,3)-glucanases, may be responsible for the impaired growth and morphogenesis of Paracoccidioides brasiliensis induced by N-glycosylation inhibition

The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-beta-d-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on alpha- and beta-(1,3)-glucanases and on alpha-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 mu g/ml TM to the fungal cultures did not interfere with either alpha- or beta-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter alpha- and beta-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, alpha-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for alpha-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of alpha-(1,3)-glucan. Our results suggest that reduced fungal alpha-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. (c) 2013 The Authors. Yeast published by John Wiley \& Sons Ltd. (AU)