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

Abstract

Gardnerellaspp. are associated with bacterial vaginosis, in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria includingGardnerellaspp. Co-occurrence of multiple species ofGardnerellais common in the vagina and competition for nutrients such as glycogen likely contributes to the differential abundances ofGardnerellaspp. Glycogen must be digested into smaller components for uptake; a process that depends on the combined action of enzymes collectively known as amylases. In this study, the ability of culture supernatants of 15 isolates ofGardnerellaspp. to produce glucose, maltose, maltotriose and maltotetraose from glycogen was demonstrated. Carbohydrate active enzymes were identified bioinformatically inGardnerellaproteomes using dbCAN2. Identified proteins included a single domain α-amylase (encoded by all 15 isolates) and an α-amylase-pullulanase 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 E. 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, demonstrating that theGardnerellaα-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 thatGardnerellaspp. produce extracellular amylase enzymes as 'public goods' that can digest glycogen into maltose, maltotriose and maltotetraose that can be used by the vaginal microbiota.

Bacterial vaginosis is associated with increased amylolytic activity

“…Vaginal lactobacilli as well as other common vaginal species, such as Fannyhessea vaginae, Prevotella bivia and Candida albicans were all unable to degrade raw labeled amylose. Furthermore, we saw profound differences in the raw amylolytic activity of various Gardnerella species, which went previously undetected using glycogen as a substrate (17).…”

“…High amylose corn starch is degraded by specific gut genera, one of which is Bifidobacterium that is closely related to the genus Gardnerella . The amylopullulanase of bifidobacteria has the same carbohydrate-binding, amylase and pullulanase domains, N-terminal signal peptide and C-terminal transmembrane domain as the Gardnerella gene that is likely responsible for the observed raw amylose degradation activity (17, 23).…”

“…Most studies characterizing the alpha-glucosidases of vaginal bacteria have used soluble starch or glycogen (16)(17)(18). Yet, for the gut microbiome it was shown that the structure of the alpha-glucan has an important role in distinguishing between enzymes of different bacterial taxa.…”

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

“…Although Gardnerella spp. can release glucose, maltose, maltotriose and maltotetraose from glycogen (11) the ability of each species to transport and utilize these sugars has not previously been investigated. Competition for glycogen breakdown products could play an important role in determining the relative abundances of Gardnerella spp.…”

“…Although all Gardnerella spp. can hydrolyze glycogen to produce glucose, maltose, maltotriose and maltotetraose (11), the distribution of transporters for these products, and the extent to which they are used for growth are unknown. Variation in the numbers and types of sugar transporters among species could affect their ability to compete for these sugars.…”