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

Gardnerella spp. 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 of Gardnerella spp. 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 in G. swidsinskii that 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 in Gardnerella . Importance Knowledge of the factors affecting growth of vaginal microbiota is critical to understanding how vaginal dysbiosis is initiated and maintained. Overgrowth of Gardnerella species including G. swidsinskii is a hallmark of bacterial vaginosis. These organisms break down vaginal glycogen and the products become available for uptake by Gardnerella and other microbiota. Measuring how expression of genes encoding glycogen degrading enzymes relates to relative abundance of substrate and products in the environment requires development of protocols for RNA purification and identification of reference genes for RT-qPCR.