Characterization of a novel type of glycogen-degrading amylopullulanase from Lactobacillus crispatus

Zhang, Jie; Li, Lili; Zhang, Tong; Zhong, Jin

doi:10.1007/s00253-022-11975-2

Cited by 8 publications

(14 citation statements)

References 57 publications

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“…In contrast, the strains LMG11415 (isolated from the human saliva) and LMG18200 (isolated from the human intestine) were clearly overwhelmed (Figure 4C). Consistently, close examination of the genomes of these four L. crispatus strains revealed that these latter were lacking in any glycogen-degrading encoding gene while the proliferating vaginal-derived LB97 and LMG11440 strains carried a type II pullulanase acting on both α-1,6and α-1,4-glycosidic bonds, which therefore achieves complete glycogen degradation, as reported in recent studies (van der Veer et al, 2019;Zhang et al, 2022). Nevertheless, LB97 and LMG11440 substantially differ in their proliferation capabilities (Table S8, Bonferronicorrected p-values < 0.05; Figure 4C).…”

Section: In Vitro Evaluation Of L Crispatus Growth Performances On Se...supporting

confidence: 80%

“…To investigate how the high level of genetic heterogeneity identified within L. crispatus could influence the respective growth abilities, the four selected L. crispatus strains (LB97, LMG11440, LMG18200 and LMG11415, whose genome diversity corresponded to ANI pairwise values <98%, Table S2 ) were cultivated on six different carbohydrate sources including glycogen, which is the primary bacterial nutritional source in the vaginal lumen (Tester & Al‐Ghazzewi, 2018 ; Zhang et al, 2022 ), along with other glycogen‐like α‐glucans, which may also represent a substrate for the bacterial enzymatic arsenal involved in carbohydrates breakdown of the vaginal environment (Figure 4A , Table S5 ). The optical density (OD) was registered after 48 h of anaerobic cultivation, and the growth on MRS was used as control condition (Table S5 ).…”

Section: Resultsmentioning

confidence: 99%

“…In fact, when different L. crispatus strains were co‐cultivated in a bioreactor‐based model simulating the vaginal environment, substantial differences were noted in the colonisation and competition efficiency. Although members of the L. crispatus species were previously thought to utilise the glycogen hydrolysis products generated in the vaginal environment by the human α‐amylase (Mirmonsef et al, 2014 ; Tester & Al‐Ghazzewi, 2018 ), recent evidences showed that members of this taxon produce the enzyme to independently degrade glycogen, annotated as a type II pullulanase (van der Veer et al, 2019 ; Zhang et al, 2022 ). In this context, while the absence of this gene was noted for those L. crispatus strains unable to stably proliferate on glycogen under in vitro conditions, we identified two amino acid substitutions within the type II pullulanase carbohydrate‐binding module arising from non‐synonymous SNPs, which could explain the different proliferation and dominance abilities observed in vitro for the L. crispatus strains investigated in this study.…”

Section: Discussionmentioning

confidence: 99%

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The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract

Tarracchini

Argentini

Alessandri

et al. 2023

Microbial Biotechnology

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The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health‐promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein‐encoding genes shared across a human‐derived L. crispatus strain selection, which includes 200 currently available human‐derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra‐species micro‐diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi‐strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.

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Section: In Vitro Evaluation Of L Crispatus Growth Performances On Se...supporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract

Tarracchini

Argentini

Alessandri

et al. 2023

Microbial Biotechnology

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show abstract

“…These findings are further validated by a separate report of the glycogen-degrading activity of L. crispatus PulA (GlgU, 99% ID to PulA). 57 A critical finding of our study examining GDEs from a variety of vaginal bacteria is that, despite sharing a common annotation, the substrate preferences of the different PulAs are quite distinct. While the L. crispatus, L. iners and G. vaginalis amylopullulanases have the highest specificity for glycogen, enzymes from other organisms are most active on amylose or pullulan and have comparatively high K m values for glycogen.…”

Section: Discussionmentioning

confidence: 99%

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

Woolston

Jenkins

Hood-Pishchany

et al. 2021

Preprint

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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.

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“…Together, these studies [ 61 – 63 ] contradict the previous theory that glycogen can only be used by vaginal bacteria when it has been broken down by human α-amylase [ 29 , 58 – 60 ]. Further confirmation that vaginal bacteria carry functional glycogen-digesting enzymes has come from characterization of amylopullanases from L. crispatus, L. iners and G. vaginalis [ 64 – 66 ]. Each of the enzymes encoded by the different species we tested utilize glycogen (among others) as a substrate, cleaving it to maltose, maltotriose, malto-oligosaccharides, trehalose, limit dextrin and/or glucose, depending on the specific enzyme (BRENDA database, https://www.brenda-enzymes.org/index.php , accessed 20 Feb 2023) [ 55 , 56 , 67 , 68 ].…”

Section: Discussionmentioning

confidence: 99%

Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis

et al. 2023

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Background Glycogen metabolism by Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5–4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome. Results The medium simulating vaginal fluid (MSVF) supported growth of L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels (pH 4.0, 4.5, and 5.0), G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels – pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus. Conclusions These results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0–5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.

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Characterization of a novel type of glycogen-degrading amylopullulanase from Lactobacillus crispatus

Cited by 8 publications

References 57 publications

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract

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

Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis

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