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 (CAZymes) were identified bioinformatically in Gardnerella 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 Gardnerella 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 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. 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 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.

Keywords: Gardnerella; amylase; glycogen; human microbiome; pullulanase; vagina; vaginosis.

FIG 1

Amylase activity of cell-free culture supernatants on glycogen agar. Each spot represents 1 isolate, and a total of 15 different isolates, 3 each from G. leopoldii (GL), G. piotii (GP), G. vaginalis (GV), G. swidsinskii (GS), and others (2 isolates from Gardnerella genome sp. 3 [top and middle] and 1 isolate from unknown genome species [bottom] [corresponds to the subgroup D based on the cpn60 classification system]) are shown. NC, mNYC, PC, B. licheniformis amylase (0.05 mg/mL).

FIG 2

Identification of glycogen breakdown products produced after 24 h by culture supernatants of 15 Gardnerella isolates by HPAEC-PAD (A to E) and TLC (F). GL, G. leopoldii; GV, G. vaginalis; GS, G. swidsinskii; GP, G. piotii; and others (2 isolates from Gardnerella genome sp. 3 [top and middle] and 1 isolate from unknown genome species [bottom] [corresponds to the subgroup D based on the cpn60 classification system]); MC, media control; G-M8, glucose-to-malto-octaose standards.

FIG 3

Predicted domains of G. leopoldii NR017 α-amylase-pullulanase (1,977 amino acids) (A) and α-amylase (545 amino acids) (B). Amylase and pullulanase domains belonging to GH13 subfamilies 32 and 14 are indicated in red, while three carbohydrate binding modules (CBM) are in blue. SP, signal peptide; sorting signal, LPxTG motif and a hydrophobic transmembrane helix domain (TMHelix) domain. Numbering indicates amino acid positions. The regions used for recombinant protein expression are indicated with black broken line with arrows. Predicted extracellular and intracellular regions are indicated with green lines.

FIG 4

(A) Phylogenetic tree of all α-amylase domains from 15 Gardnerella isolates and 4 reference strains (indicated with T after the strain name). The tree is rooted with Streptomyces venezuelae ATCC 15068. Trees are consensus trees of 100 bootstrap iterations and constructed using the neighbor-joining method using the Tamura-Nei distance model. The number at major branch points represents the percentage of bootstrap support. (B) Sequence alignment of GH13_32 catalytic domains of G. leopoldii NR017 α-amylase (NR017_A) and α-amylase-pullulanase (NR017_AP) with other functionally characterized GH13_32 members (α-amylases from Pseudoalteromonas haloplanktis A23 [GenPept accession number CAA41481] and S. venezuelae ATCC 15068 and amylopullulanase from Bifidobacterium breve UCC2003). Black triangles indicate conserved catalytic residues.

FIG 5

(A) Protein profiles of uninduced (UN) and induced (IN) E. coli cells containing pQE80L-AMY and pQE80L-PULL (top left) and pET41a-CO-AMY (top right) recombinant plasmids. (B) Purified 6×His-tagged amylase (Amy) (bottom left) and pullulanase (Pull) (bottom middle) and GST-6×His-tagged α-amylase (bottom right). M, molecular weight markers.

FIG 6

(A) Identification of products released by α-amylase domain (GH13_32) of α-amylase-pullulanase from maltopentose (M5) and maltodextrin (MD) and glycogen (Gly) after 24 and 48 h of incubation using TLC (top) and HPAEC-PAD (middle and bottom). (B) Products released from pullulan by the pullulanase domain (GH13_14) after 24 and 48 h of incubation were identified using TLC (top) and HPAEC-PAD (bottom). (C) Identification of products released after 24 h from pullulan or glycogen by pullulanase, α-amylase, or a mixture of α-amylase and pullulanase domains. Standards in lane M of each panel as follows: G, glucose; M2, maltose; M3, maltotriose; M4, maltotetraose; and M5, maltopentose.

FIG 7

(A) Identification of products released by the purified single-domain α-amylase enzyme from maltopentose (M5) and maltodextrin (MD) using TLC (top) and HPAEC-PAD (bottom) after 24 h of incubation. (B) Products released from glycogen by the purified single-domain α-amylase enzyme after 24 h of incubation were identified using TLC (top) and HPAEC-PAD (bottom). Standards in lane M of each panel as follows: G, glucose; M2, maltose; M3, maltotriose; M4, maltotetraose; and M5, maltopentose.

FIG 8

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 (36). Extracellular glycogen breakdown products can also be utilized by other resident microbiota.