Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina

Abstract

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Bacterial vaginosis is associated with increased amylolytic activity

“…Besides substrate degradation we also found differences between the regulation. Alpha-glucosidase activity of G. vaginalis was in this study also observed in cultures grown on glucose whereas we previously found L. crispatus amylopullulanase to be repressed in the presence of the products of glycogen degradation such as glucose, maltose or maltotriose (14).…”

“…Apart from the absence of catabolite repression, and the ability to degrade raw amylose, we identified a third characteristic that was found to be distinctive: unlike to what we previously found for L. crispatus (14), the Gardnerella amylolytic activity was mostly found in the extracellular environment and only to a minor extent cell-bound. This is not consistent with the presence of a C-terminal transmembrane domain in the candidate amylopullulanase gene.…”

“…All strains were anaerobically cultivated from -80 °C glycerol stocks (VU Amsterdam: NYCIII medium + 20% glycerol, Ghent: TSB + 15% glycerol) on agar plates under conditions indicated in Table S1. From these plates colonies were picked to inoculate liquid New York City (NYC)-III broth with 10% horse serum as described previously (14) or YEPG broth for Candida albicans . All cultures were handled under anaerobic conditions (VU Amsterdam: N 2 + 10% CO 2 , Ghent University: 10% H 2 , 10% CO 2 , 80% N 2 ) in an anaerobic chamber (VU Amsterdam: PLAS Labs 855 NB; Ghent university: Bugbox Plus).…”

“…Alpha-glucosidases of vaginal bacteria are hypothesized to take a central role in carbon and energy metabolism by extracellular degradation of host-associated glycogen followed by transport and metabolism of the resulting smaller dextrins. Previously, we have elucidated the genetic basis, regulation and prevalence of one of these alpha-glucosidases: the L. crispatus amylopullulanase (12–14). Strikingly, we found a relatively high number of L. crispatus strains to be deficient for this amylopullulanase and identified frequently occurring mutations in its gene in the genomes of these strains as well as in databases of the vaginal metagenome (14, 15).…”

“…Previously, we have elucidated the genetic basis, regulation and prevalence of one of these alpha-glucosidases: the L. crispatus amylopullulanase (12–14). Strikingly, we found a relatively high number of L. crispatus strains to be deficient for this amylopullulanase and identified frequently occurring mutations in its gene in the genomes of these strains as well as in databases of the vaginal metagenome (14, 15). Insight in the ability of other common vaginal microbes to digest alpha-glucans such as vaginal glycogen remains forthcoming.…”