Resolution of glycogen and glycogen-degrading activities reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women

Abstract

Background: A healthy vaginal microbiome is dominated by Lactobacillus species that produce lactic acid, lowering vaginal pH and limiting colonization by pathogens. Lactobacillus dominance (LD) is established during puberty, but many women, especially those of Black race, lose LD during their reproductive years. Glycogen is thought to be a key host nutrient that supports vaginal lactobacilli and their fermentative lactic acid production, but mechanisms of glycogen utilization by Lactobacillus species are incompletely understood. By partitioning glycogen and glycogen-derived maltodextrin, as well as the activity of glycogen-degrading pullulanase enzymes, this work refines understanding of vaginal glycogen catabolism and identifies correlates of LD. Results: Vaginal swab samples were collected from a cohort of young women with limited sexual experience in Thika, Kenya (N=17, ages 17-20). Metagenomic profiling of the vaginal microbiome revealed that most samples exhibited LD, particularly dominant Lactobacillus crispatus. Amylopullulanase activity, cleavage of glycogen α-1,4 and α-1,6 linkages by individual/multifunctional enzymes, showed a significant positive correlation with glycogen-derived maltodextrin, but no relationship with L. crispatus dominance. Pullulanase activity, which specifically targets glycogen α-1,6 linkages, was 3-fold higher in L. crispatus-dominated samples and significantly correlated with D-lactic acid levels. Metagenomics and targeted PCR revealed that 36% of L. crispatus-dominated metagenomes from our African cohort lacked a functional L. crispatus pullulanase (pulA) gene, a 3-fold higher frequency of gene loss than that seen in metagenomes from European and North American women. Our findings suggest pulA gene loss or inactivation may correspond with reductions in L. crispatus abundance, pullulanase activity and lactic acid levels compared to samples dominated by pulA-competent L. crispatus. Conclusions: Our results indicate that although amylase activity drives the accumulation of glycogen catabolites in vaginal fluid, pullulanase appears to specifically contribute to maximal D-lactic acid production by L. crispatus. However, this is only possible when a functional pulA gene is present, which was not the case in a substantial proportion of young African women with dominant L. crispatus. Scaling this analysis to a larger cohort will address whether genomic and enzymatic indicators of L. crispatus pullulanase activity are predictive of sustained LD and vaginal health.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina

“…However, given the positive relationship between amylopullulanase activity and smaller maltodextrins in the study by Lithgow et al [31], we think further studies should include analyses of metatranscriptomes and carbohydrate concentration, to clarify whether the observed in vitro pulA regulation, depending on carbohydrate availability in L. crispatus, corresponds to its in vivo regulation. Based on the prevalence of these carbon sources in vaginal metabolomics studies, as well as human amylase, we expect that L. crispatus in the vaginal environment preferentially relies on glycogen cleavage by other bacteria or host amylase and will only express amylopullulanase when other carbon sources are depleted.…”

“…In most environments, L. crispatus maintains an intact amylopullulanase gene, even though disruption of the pulA gene occurs at relatively high frequencies. A recent metagenomics study and targeted PCR by Lithgow et al revealed that 36% of L. crispatus-dominated metagenomes from their African cohort lacked a functional L. crispatus pulA gene, and reported a threefold higher frequency of gene loss than that seen in metagenomes from European and North American women [31]. However, this is not reflected by the prevalence of disrupted pulA genes among the known ethnic groups in the metagenome data set of 1507 vaginal samples we have analyzed (Appendix A Table A1).…”

Relationship between Candida Species and use of Intrauterine Contraceptives Device in Women with Vulvovaginitis

“…Furthermore (Haddad et al, 2021) showed that the prevalence of vaginal candidiasis was higher in women who used contraceptives (84.1%) than in women who had not used contraceptives (15.9%), and there is a significant difference between the two groups. Previously, it was thought that oestrogen and progesterone hormones could manifest in contraceptives and increase vaginal glycogen, exposing it to lactobacilli activity (Lithgow et al, 2022). The pH of the vagina decreases as a result of the lactobacilli's function in the conversion of glycogen to lactic acid, which also inhibits the activities of the bacterial biota and promotes the growth of Candida species (Abdullah and Mohammed, 2020, Jasim, 2020).…”