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

Abstract

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.

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

“…Third, having demonstrated significant levels of glycogen-degrading enzyme activity in biobanked vaginal samples, our work underlines the critical importance of cold chain management to ensure measured glycogen/maltodextrin levels reflect those present at the time of sample collection. Finally, in contrast with previous work, we found glycogen-degrading enzyme activities, including that of pullulanases, were abundant in vaginal swab supernatants and minimally present in the cell pellet [97]. Since N-terminal motifs in the L. crispatus PulA sequence are predicted to tether the protein to the outer membrane’s Surface Layer Protein A [97], it is possible that most PulA protein is cell surface-associated in vivo , but was perhaps dislodged through the vigorous vortexing performed during swab preparation.…”

“…Finally, in contrast with previous work, we found glycogen-degrading enzyme activities, including that of pullulanases, were abundant in vaginal swab supernatants and minimally present in the cell pellet [97]. Since N-terminal motifs in the L. crispatus PulA sequence are predicted to tether the protein to the outer membrane’s Surface Layer Protein A [97], it is possible that most PulA protein is cell surface-associated in vivo , but was perhaps dislodged through the vigorous vortexing performed during swab preparation. Alternatively, PulA may be released from the cell wall in vivo through biological processes such as proteolytic activity.…”

“…Theoretically, L. crispatus could selfishly utilize glycogen by directly importing the maltodextrin products of its cell surface pullulanase and releasing few ‘free’ maltodextrin catabolites into the milieu. In contrast, when environmental disruption allows the pH to become more permissive (≥5.5), G. vaginalis , and perhaps other pullulanase-expressing BV-associated bacteria such as P. bivia and M. mulieris , efficiently deplete glycogen and release abundant maltodextrins [51, 53, 88, 97]. This supplies the broader community with a more accessible carbohydrate nutrient pool and forces L. crispatus into a more competitive mode of acquiring the fermentative substrates it needs to produce lactic acid and regain the upper hand.…”

Genetic elements orchestrating Lactobacillus crispatus glycogen metabolism in the vagina

“…A recent key study confirmed alpha-glucosidase activity of pullulanase genes from different vaginal bacteria by heterologous expression. It showed that the Lactobacillus crispatus pullulanase degrades glycogen, pullulan and amylose retaining activity at pH values that are common in a Lactobacillus -dominated vaginal environment [14]. L. crispatus strains isolated from low-glycogen environments, such as the human or poultry gut, more often lacked the pullulanase gene indicating niche-specificity [15].…”

“…Vaginal samples show alpha-glucosidase activity, which was postulated to be mostly of human origin on the basis of antibody [16,17] and proteomic analysis [18]. At the same time results from two studies have indicated that alpha-glucosidases in a subset of vaginal samples may be of bacterial origin on the basis of optimal pH and substrate and product profiling [14,19]. However, a metaproteomic study of vaginal lavages from women with a crispatus -dominated microbiome, failed to detect any L. crispatus pullulanase protein, which poses questions about host-related or genetic factors influencing its presence [18].…”