Characterization of an α-Glucosidase Enzyme Conserved in Gardnerella spp. Isolated from the Human Vaginal Microbiome

Abstract

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacilli dominant community is replaced with mixed bacteria including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400_06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. K m , k cat and k cat /K m values for the substrate 4-nitrophenyl α- d -glucopyranoside were 8.3 μM, 0.96 min −1 and 0.11 μM −1 min −1 , respectively. Glucose was released from maltose, maltotriose, maltotetraose and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

Transport and Utilization of Glycogen Breakdown Products by Gardnerella spp. from the Human Vaginal Microbiome

“…1A ). This intracellular α-glucosidase has been previously shown to hydrolyze α-1,4 glycosidic bonds of malto-oligosaccharides to release glucose ( 20 ) while pullulanase is a debranching enzyme that breaks down α-1,6 glycosidic bonds ( 21 ). Thus, the MalXFGK operon encodes all of the components needed to import malto-oligosaccharides and/or maltodextrins for debranching and hydrolysis by pullulanase and α-glucosidase to release glucose.…”

Glycogen degrading activities of catalytic domains of α-amylase and α-amylase-pullulanase enzymes conserved inGardnerellaspp. from the vaginal microbiome

“…We have previously shown that Gardnerella spp. have conserved intracellular α-glucosidase that can release glucose from smaller oligosaccharides (47).…”

“…Proposed mechanisms of extracellular glycogen utilization in Gardnerella spp. Glycogen is digested into malto-oligosaccharides by α-amylase-pullulanase and α-amylase and these breakdown products are transported inside and can be further digested to glucose by other glycosyl hydrolases including a previously characterized α-glucosidase enzyme (47). Extracellular glycogen breakdown products can also be utilized by other resident microbiota.…”

Transport and utilization of glycogen breakdown products byGardnerellaspp. from the human vaginal microbiome

“…In Gardnerella , the MalXFGK operon also encodes an α -glucosidase and a pullulanase (Figure 1A). This intracellular α -glucosidase has been previously shown to hydrolyze α -1,4 glycosidic bonds of malto-oligosaccharides to release glucose (28) while pullulanase is a debranching enzyme that breaks down α -1,6 glycosidic bonds (29). Thus the MalXFGK operon encodes all of the components needed to import malto-oligosaccharides and/or maltodextrins for debranching and hydrolysis by pullulanase and α -glucosidase to release glucose.…”