Human α-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus

Abstract

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus isolates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lactobacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support Lactobacillus colonization in the genital tract.

Keywords: Lactobacillus; female genital tract; glycogen; maltose; α-amylase.

Figure 1.

Lack of L. gasseri growth in glycogen. L. gasseri was cultured in MRS medium that contained either no added carbohydrates (No Carb), 2% glucose, 2% glycogen, or 2% starch for 72 hours. The OD at 600 was read each day. Each culture condition was run in duplicate and the duplicates averaged. Each culture experiment was performed at least 3 times with similar results. Abbreviations: MRS, de Man, Rogosa, and Sharpe; OD, optical density.

Figure 2.

Growth of lactobacilli in glucose polymers. L. gasseri (A), L. jensenii (B and D), and L. johnsonii (C) were cultured in MRS medium that contained either no added carbohydrates (No Carb) or 2% of either glucose, maltose, maltodextrin 4–7, maltodextrin 13–17, maltodextrin 16–19, maltotriose, or maltopentaose for 72 hours. The OD at 600 was read each day. Abbreviations: MD, maltodextrin; MRS, de Man, Rogosa, and Sharpe; OD, optical density.

Figure 3.

Growth of lactobacilli in salivary α-amylase-treated glycogen. L. gasseri (A), L. jensenii (B), or L. johnsonii (C) were cultured in MRS broth that contained either no added carbohydrates (No Carb), 2% glucose, 2% glycogen, 2.5% saliva alone, or glycogen plus saliva for 72 hours. The OD at 600 was read each day. Abbreviations: MRS, de Man, Rogosa, and Sharpe; OD, optical density.

Figure 4.

Degradation of glycogen by salivary α-amylase. Glycogen (2% oyster) was incubated for 10 minutes with no saliva (Oyster) or for 10, 30, or 120 minutes with 2.5% saliva. The size of the degraded glycogen was determined by HPSEC (A) and the products analyzed by HPAEC (B). Abbreviations: HPAEC, high-performance anion-exchange chromatography; HPSEC, high-pressure size exclusion chromatography.

Figure 5.

Temperature-dependent degradation of glycogen in genital fluid. A, Aliquots of genital fluid collected from 2 subjects (A and B) were incubated at either 37°C or 4°C for 90 minutes and the level of endogenous glycogen was measured with a colorimetric iodine assay. B, Glycogen was added to genital fluids collected from 4 other women (C–F) or saline, and aliquots were incubated at either 4°C or 37°C for 90 minutes. At the end of the incubation, the amount of glycogen in the samples was measured with the colorimetric iodine assay. C, Degradation in 21 genital fluid samples was measured as in B and the percent degradation was calculated by the following formula: 1 – glycogen 37/glycogen 4 × 100.

Figure 6.

Relationship of glycogen degradation with genital bacteria, vaginal pH, α-amylase and α-glucosidase. The percent of glycogen degradation (Figure 5C) in 21 samples was plotted along with either genital-tract levels of L. iners (A), L. crispatus (B), G. vaginalis (C), vaginal pH (D), pancreatic α-amylase (E), or α-glucosidase (F).

Figure 7.

Breakdown of glycogen by genital fluids. Glycogen was incubated for 0 hours (A) or 48 hours (B) with saline or 48 hours with genital fluid collected in saline from 2 different subjects (C and D). After incubation the size of small carbohydrates was determined by HPAEC. Abbreviation: HPAEC, high-performance anion-exchange chromatography.