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.
14Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a 15 dysbiosis in which lactobacilli dominant microbial community is replaced with mixed 16 aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of 17 multiple Gardnerella species in the vaginal environment is common, but different species 18 are dominant in different women. Competition for nutrients, particularly glycogen present 19 in the vaginal environment, could play an important role in determining the microbial 20 community structure. Digestion of glycogen into products that can be taken up by bacteria 21 requires the combined activities of several enzymes collectively known as "amylases". In 22 the present study, glycogen degrading abilities of Gardnerella spp. were assessed. We 23 found that Gardnerella spp. isolates and filtered culture supernatants had amylase activity. 24Phylogenetic analyses predicted conserved Glycoside Hydrolase family 13 (GH13) 25 members among Gardnerella spp. including a putative a-glucosidase. The gene for this 26 enzyme was cloned and expressed, and recombinant protein was purified and functionally 27 characterized. The enzyme was active on a variety of maltooligosaccharides over a broad 28 pH range (4 -8) with an optimum activity at pH 6-7. Glucose was released from maltose, 29 maltotriose and maltopentose, however, no products were detected on thin layer 30 chromatography (TLC) when the enzyme was incubated with glycogen. Our findings show 31 that Gardnerella spp. produce a secreted α-glucosidase enzyme that can contribute to the 32 complex and multistep process of glycogen breakdown by degrading smaller 33 oligosaccharides into glucose, contributing to the pool of nutrients available to the vaginal 34 microbiota. 35 36 database and G. leopoldii CG400_06090. Branch colour is based on GH13 subfamily and functionally characterized proteins are indicated with stars. The clade highlighted in grey including G. leopoldii CG400_06090 and its closest relatives is expanded in (B).
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.
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.
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. Gardnerella produces enzymes to digest glycogen, an important nutrient source for vaginal bacteria, but little is known about the mechanisms in Gardnerella for uptake of the products of this digestion, or whether Gardnerella use some or all of the products.
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