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

Abstract

Gardnerella spp. are associated with bacterial vaginosis, in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake; a process that depends on the combined action of glycogen degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose and maltotetraose from glycogen was demonstrated. Carbohydrate active enzymes were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single domain -amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an -amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the -amylase-pullulanase, and the single domain -amylase were each produced in E. coli. The -amylase domain from the -amylase-pullulanase released maltose, maltotriose and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella -amylase-pullulanase is capable of hydrolyzing -1,4 and -1,6 glycosidic bonds. Similarly, the single domain -amylase protein also produced maltose, maltotriose and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as public goods that can digest glycogen into maltose, maltotriose and maltotetraose that can be used by the vaginal microbiota. ImportanceIncreased 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 food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.