Elucidating the mechanisms of extracellular glycogen utilization in Gardnerella spp.
Date
2023-02-10
Authors
Journal Title
Journal ISSN
Volume Title
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ORCID
0000-0002-2616-9934
Type
Thesis
Degree Level
Doctoral
Abstract
Gardnerella spp. are associated with bacterial vaginosis, in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria including phenotypically diverse Gardnerella spp. that likely differ in their role in pathogenesis. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, and different species are dominant in different women. Given the potentially different roles of Gardnerella species in the vaginal microbiome, understanding the factors contributing to their population structure is important. Competition for nutrients could play an important role in determining the microbial community structure.
Glycogen is one major nutrient available in the vagina. It is accumulated inside vaginal epithelial cells and is released into the lumen when epithelial cells are lysed. Glycogen digestion is accomplished by the coordinated action of enzymes collectively described as amylases that belong to family 13 within the glycosyl hydrolase class (GH13) of carbohydrate active enzymes. In the vagina, host secretions contain amylase enzymes; however, the contributions of Gardnerella spp. to this process and interactions of different Gardnerella spp. with glycogen are not well understood.
The first objective of this thesis was to annotate GH13 enzymes in Gardnerella spp. and to characterize the activity of an a-glucosidase enzyme conserved among Gardnerella spp. This conserved putative amylase was annotated as an a-amylase (EC 3.2.1.1), suggesting that it was an endo-acting enzyme that releases malto-oligosaccharides from glycogen, but comparison to other functionally annotated enzymes suggested that the conserved amylase sequence was an a-glucosidase (EC 3.2.1.20). Biochemical characterization of the conserved enzyme from G. leopoldii NR017 demonstrated that it had a-glucosidase activity. This intracellular enzyme was predicted to be involved in digestion of imported malto-oligosaccharides.
The second objective of this thesis was to assess the extracellular glycogen digestion ability of different Gardnerella spp., identify the extracellular glycogen hydrolyzing enzymes and characterize their activities. Culture supernatants of a total of 15 representative isolates from different Gardnerella species showed amylase activity suggesting that glycogen digestion is a conserved property in the genus Gardnerella. Glucose, maltose, maltotriose and maltotetraose were identified as glycogen breakdown products and no species-specific fingerprints of glycogen breakdown products were observed. Analysis of the predicted proteomes of the study isolates led to the identification of two predicted extracellular glycosyl hydrolases (a-amylase and a-amylase-pullulanase) (belonging to family 13 (GH13)) in all but one isolate. The amylase domains of the a-amylase-pullulanase and a-amylase enzymes released maltose, maltotriose and maltotetraose from glycogen while the pullulanase domain released maltotriose from pullulan. These results showed that a-amylase and a-amylase-pullulanase enzymes can hydrolyze glycosidic bonds in glycogen and contribute to the nutrient pool available to the vaginal microbiota.
Once glucose, maltose, maltotriose and maltotetraose are produced from glycogen digestion, how are these products transported inside the bacteria? Do all Gardnerella species utilize these substrates for growth? The third objective of this thesis was to identify the transporters associated with uptake of glycogen breakdown products and to determine if all Gardnerella spp. can utilize those sugars for growth. Five different maltose, malto-oligosaccharide and maltodextrin specific ABC transporters were identified bioinformatically in Gardnerella spp. Although some transporters are conserved across all Gardnerella species, species-specific transporters are present in G. vaginalis and G. leopoldii suggesting that these species may have access to a greater diversity of sugars or a competitive advantage in uptake. All Gardnerella isolates grew in the presence of glucose, maltose, maltotriose and maltotetraose, demonstrating their ability to utilize glycogen breakdown products. In addition, most isolates showed more growth on maltotriose and maltotetraose compared to glucose and maltose suggesting their preference for the longer chain malto-oligosaccharides.
The overall findings of the research work described in this thesis contribute to our understanding of how different Gardnerella species interact with glycogen. Understanding factors contributing to the population dynamics of more or less pathogenic Gardnerella spp. within the vaginal microbiome and identifying phenotypic characteristics that distinguish different species is important for improving diagnostics for women’s health and identifying high risk microbiomes. This in turn will help to prevent ineffective and unnecessary treatments, which can lead to increased antibiotic resistance, treatment failure, and recurrent infection.
Description
Keywords
Gardnerella, glycogen, amylase, human microbiome, pullulanase, ABC transporter
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology