Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in
            <i>Gardnerella</i>
            spp. from the Vaginal Microbiome

Bhandari, Pashupati; Tingley, Jeffrey P.; Abbott, D. Wade; Hill, Janet E.

doi:10.1128/jb.00393-22

Cited by 7 publications

(8 citation statements)

References 43 publications

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“…In a recent preprint, the catalytic domain from a homolog of G. vaginalis AmyA was characterized in vitro (77% ID full length protein, 99% ID for the catalytic domain). 53 Intriguingly, we also detected human α-amylase (AMY1) in the majority of samples ( Fig. 5c, SI Fig.…”

Section: Resultsmentioning

confidence: 65%

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Woolston

Jenkins

Hood-Pishchany

et al. 2021

Preprint

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The healthy human vaginal microbiota is generally dominated by Lactobacilli, and the transition to a more diverse community of anaerobic microbes is associated with a number of health risks. While the mechanisms underlying the stability of Lactobacillus-dominated vaginal communities are not fully understood, competition for nutrients is a likely contributing factor. Glycogen secreted by epithelial cells is widely believed to support the growth of vaginal microbes. However, the mechanism by which bacteria access sugars from this complex polymer is unclear, with evidence to support a role for both microbial and human enzymes. To shed light on the potential contribution from microbial enzymes, here we biochemically characterize six glycogen-degrading enzymes predicted to be secreted by vaginal bacteria and confirm their ability to support the growth of an amylase-deficient strain of L. crispatus on glycogen. We reveal significant differences in the pH tolerance between enzymes from different organisms, suggesting the adaptation of Lactobacilli to an acidic vaginal environment. Using a simple assay specific for the microbial enzymes, we confirm their presence in cervicovaginal lavage samples. Finally, we demonstrate the selective inhibition of glycogen-degrading enzymes from two vaginal microbes associated with dysbiosis. This work provides biochemical evidence to support the role of vaginal bacterial amylase enzymes in the breakdown of glycogen, providing insight into factors that shape the vaginal microbiota and highlighting the possibility of manipulating community structure via non-antibiotic small molecules.

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Section: Resultsmentioning

confidence: 65%

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Woolston

Jenkins

Hood-Pishchany

et al. 2021

Preprint

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“…Development of improved diagnostics and clinical interventions for BV will be facilitated by improved understanding of the growth and colonization of these bacteria in relation to utilization of available nutrients in the vagina. Glycogen is one of the most important and abundant nutrient sources in the vaginal lumen that is degraded by α-amylase and α-amylase-pullulanase into smaller malto-oligosaccharides utilized by the vaginal microbiota (6, 9). Differential expression of genes encoding glycogen degrading enzymes in response to relative abundance of relevant substrates and products has not previously been investigated for Gardnerella spp.…”

Section: Discussionmentioning

confidence: 99%

“…Glycogen digestion is achieved by the collective action of “amylase” enzymes, which cleave α-1,4 and α-1,6 glycosidic bonds either from the non-reducing end or randomly within the chain. The single domain α-amylase enzyme of G. swidsinskii hydrolyzes α-1,4 glycosidic bonds producing maltose, maltotriose, and maltotetraose from glycogen (9). The α-amylase-pullulanase enzyme contains two catalytic domains: the α-amylase and pullulanase domains (9).…”

Section: Discussionmentioning

confidence: 99%

“…Modified NYC III medium (mNYC III) was prepared, supplemented with 1% filter sterilized oyster glycogen (9005-79-2, Sigma-Aldrich), maltotriose (J66491.03, ThermoFisher), or D-glucose (D16-500, FisherChemical). mNYC III is NYC III (ATCC Medium 1685) with 10% (v/v) heat inactivated fetal bovine serum instead of horse serum and no additional glucose (9). Colonies isolated from growth on Columbia sheep blood agar plates were used to inoculate mNYC III media and cultures were incubated for 48 hours at 37°C under anaerobic conditions (GasPak™ EZ Anaerobe Container System Sachets, BD).…”

Section: Methodsmentioning

confidence: 99%

“…We have recently demonstrated that all Gardnerella spp. produce extracellular α-amylase and α-amylase-pullulanase that can release maltose and malto-oligosaccharides from glycogen (9) and that Gardnerella spp. show more growth in media supplemented with maltotriose and maltotetraose relative to glucose or maltose (10).…”

Section: Introductionmentioning

confidence: 99%

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Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Kim,

Fernando,

Kularatne

et al. 2024

Preprint

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Gardnerellaspp. express and export enzymes for the breakdown of glycogen into glucose, maltose, and malto-oligosaccharides for consumption by the vaginal microbiota but how the expression of these "public goods" is affected by substrate and product levels in the environment is not known. Accurate measurement of relative gene expression using real-time quantitative PCR relies on the identification of appropriate reference genes whose expression levels remain constant under the conditions of the study. Currently, no reference genes have been identified for gene expression analysis ofGardnerellaspp. The objectives of this study were to identify reference genes and apply them in determining the relative gene expression levels of genes encoding α-amylase and α-amylase-pullulanase in media supplemented with substrate (glycogen) or a preferred product (maltotriose). Ten candidate reference genes were evaluated and analysis of Cq values from qPCR using multiple algorithms identified uppS (encoding polyprenyl diphosphate synthase) as the top comprehensively ranked reference gene followed by gatA (encoding Asp-tRNA/Glu-tRNA amidotransferase subunit gatA). Interpretation of the Cq values for α-amylase and α-amylase-pullulanase was performed by applying these two reference genes in the calculation of relative gene expression levels. α-amylase-pullulanase gene expression was upregulated in media supplemented with 1% glycogen in comparison to media supplemented with 1% maltotriose suggesting a regulatory mechanism inG. swidsinskiithat responds to nutrient availability. No significant difference in gene expression of α-amylase was observed suggesting expression is not influenced by substrate availability. The RNA purification protocol and reference genes validated in this study will be useful in future studies of gene expression inGardnerella.

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Bacterial amylases enable glycogen degradation by the vaginal microbiome

Jenkins,

Woolston,

Hood-Pishchany

et al. 2023

Nat Microbiol

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The human vaginal microbiota is frequently dominated by lactobacilli and transition to a more diverse community of anaerobic microbes is associated with health risks. Glycogen released by lysed epithelial cells is believed to be an important nutrient source in the vagina. However, the mechanism by which vaginal bacteria metabolize glycogen is unclear, with evidence implicating both bacterial and human enzymes. Here we biochemically characterize six glycogen-degrading enzymes (GDEs), all of which are pullanases (PulA homologues), from vaginal bacteria that support the growth of amylase-deficient Lactobacillus crispatus on glycogen. We reveal variations in their pH tolerance, substrate preferences, breakdown products and susceptibility to inhibition. Analysis of vaginal microbiome datasets shows that these enzymes are expressed in all community state types. Finally, we confirm the presence and activity of bacterial and human GDEs in cervicovaginal fluid. This work establishes that bacterial GDEs can participate in the breakdown of glycogen, providing insight into metabolism that may shape the vaginal microbiota.

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

Cited by 7 publications

References 43 publications

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Identification and characterization of bacterial glycogen-degrading enzymes in the vaginal microbiome

Investigation of expression of genes encoding glycogen degrading enzymes inGardnerella swidsinskiiand identification of reference genes for quantitative real-time PCR

Bacterial amylases enable glycogen degradation by the vaginal microbiome

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