An α‐(1,4)‐amylase is essential for α‐(1,3)‐glucan production and virulence in Histoplasma capsulatum

Abstract

SummaryHistoplasma capsulatum is a dimorphic fungus that causes respiratory and systemic disease and is capable of surviving and replicating within macrophages. The virulence of Histoplasma has been linked to cell wall a-(1,3)-glucan; however, the role of this polysaccharide during infection, its organization within the cell wall, and its synthesis and regulation remain poorly understood. To identify genes involved in the biosynthesis of a-(1,3)-glucan, we employed a forward genetics strategy to isolate physically marked mutants with reduced a-(1,3)-glucan. Insertional mutants were generated in a virulent strain of H. capsulatum by optimization of Agrobacterium tumefaciens-mediated transformation. Approximately 90% of these mutants possessed single insertions with no chromosomal rearrangements or deletions in the host genome. To confirm the role and specificity of identified candidate genes, we phenocopied the disrupted locus by either RNA interference or targeted gene deletion. Our findings indicate a-(1,3)-glucan production requires the function of the AMY1 gene product, a novel protein with homology to the a-amylase family of glycosyl hydrolases, and UGP1, a UTP-glucose-1-phosphate uridylyltransferase which synthesizes UDP-glucose monomers. Loss of AMY1 function attenuated the ability of Histoplasma to kill macrophages and to colonize murine lungs.

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes

“…On the basis of these data, the authors proposed a model which suggests that a small molecule consisting of a-(1,4)-linked glucose residues acts as a primer for the formation of longer, a-(1,3)-linked polymers by a-glucan synthases. As proposed by Marion et al (2006), the function of Amy1p, AmyD and related proteins could be to produce such a primer molecule. In this study, we have shown that AmyD indeed produces small maltooligosaccharides, with a preference for maltotriose, in vitro.…”

“…Although the enzymic activity of this protein has not been studied, a clear link was made with the production of cell wall aglucan, as a functional knockout was completely incapable of producing a-(1,3)-glucan (Marion et al, 2006). This hypothesis regarding the function of fungal GH13_5 aamylases is strengthened by the genomic organization of the encoding genes, many of which are arranged in a small cluster of genes also encoding an a-glucan synthase and a GPI-anchored a-glucanotransferase.…”

“…In this pathogenic, dimorphic fungus, a-(1,3)-glucan is critical for virulence (Rappleye et al, 2004). A functional knockout strain of Amy1p, a putative intracellular a-amylase, completely loses the ability to form cell wall a-(1,3)-glucan and has attenuated virulence (Marion et al, 2006). No biochemical characterization of Amy1p has been reported, to our knowledge.…”

“…A set of sequences encoding a-amylases was retrieved from GenBank (Benson et al, 2006) and SWISS-PROT (Wu et al, 2006) (Table 1). The set was supplemented with the 12 fungal sequences identified as described above, and three additional sequences: Amy1p from H. capsulatum (Marion et al, 2006), and AgtA (An09g03100) and AgtB (An12g02460) from A. niger. The alignment strategy was based on the approach described by Da Lage et al (2004).…”

“…A more distantly related homologous sequence from the genome of Cryptococcus neoformans (Loftus et al, 2005) was also included. This set of predicted proteins, combined with the A. niger AmyD and AmyE proteins and Amy1p from H. capsulatum (Marion et al, 2006), was used for a detailed sequence comparison.…”