Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Spp. from the Vaginal Microbiome

spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including spp. Co-occurrence of multiple species of is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of 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 spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-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 α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that 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. Increased abundance of 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 species that contribute to the ability of these bacteria to utilize glycogen.