Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacilli dominant community is replaced with mixed bacteria including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400_06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. K m , k cat and k cat /K m values for the substrate 4-nitrophenyl α- d -glucopyranoside were 8.3 μM, 0.96 min −1 and 0.11 μM −1 min −1 , respectively. Glucose was released from maltose, maltotriose, maltotetraose and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

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Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

10.1101/2020.05.11.086124 ◽

2020 ◽

Cited By ~ 2

Author(s):

Pashupati Bhandari ◽

Jeffrey P. Tingley ◽

D. Wade Abbott ◽

Janet E. Hill

Keyword(s):

Microbial Community ◽

Phylogenetic Analyses ◽

Anaerobic Bacteria ◽

Glycoside Hydrolase Family ◽

Vaginal Microbiome ◽

Multistep Process ◽

Ph Range ◽

Culture Supernatants ◽

Layer Chromatography

AbstractGardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up by bacteria requires the combined activities of several enzymes collectively known as “amylases”. In the present study, glycogen degrading abilities of Gardnerella spp. were assessed. We found that Gardnerella spp. isolates and filtered culture supernatants had amylase activity. Phylogenetic analyses predicted conserved Glycoside Hydrolase family 13 (GH13) members among Gardnerella spp. including a putative α-glucosidase. The gene for this enzyme was cloned and expressed, and recombinant protein was purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4 - 8) with an optimum activity at pH 6-7. Glucose was released from maltose, maltotriose and maltopentose, however, no products were detected on thin layer chromatography (TLC) when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce a secreted α-glucosidase enzyme that can contribute to the complex and multistep process of glycogen breakdown by degrading smaller oligosaccharides into glucose, contributing to the pool of nutrients available to the vaginal microbiota.

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Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

10.1101/2021.10.27.466152 ◽

2021 ◽

Author(s):

Gregory S Bulmer ◽

Fang Wei Yuen ◽

Naimah Begum ◽

Bethan S Jones ◽

Sabine S Flitsch ◽

...

Keyword(s):

Aspergillus Niger ◽

Glycoside Hydrolase ◽

Biochemical Characterization ◽

Maximum Activity ◽

Glycoside Hydrolase Family ◽

Enzyme Specificity ◽

Fungal Enzymes ◽

Glycoside Hydrolase Family 43 ◽

Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

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Bacterial vaginosis: drivers of recurrence and challenges and opportunities in partner treatment

BMC Medicine ◽

10.1186/s12916-021-02077-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Lenka A. Vodstrcil ◽

Christina A. Muzny ◽

Erica L. Plummer ◽

Jack D. Sobel ◽

Catriona S. Bradshaw

Keyword(s):

Bacterial Vaginosis ◽

Vaginal Microbiome ◽

Epidemiological Evidence ◽

Associated Bacteria ◽

Sexually Transmitted ◽

Single Mechanism ◽

Challenges And Opportunities ◽

Key Driver ◽

Barriers And Challenges ◽

High Level

AbstractBacterial vaginosis (BV) is the most common vaginal dysbiosis to affect women globally, yet an unacceptably high proportion of women experience BV recurrence within 6 months of recommended antibiotic therapy. The low rate of sustained cure highlights our limited understanding of the pathogenesis of BV recurrence, which has been attributed to possible persistence and re-emergence of BV-associated bacteria (BVAB) or a BV-associated biofilm following antimicrobials and/or reinfection occurring from sexual partners.There is a robust body of evidence to support the exchange of bacteria between partners during sexual activity, and while the hypothesis that women treated for BV are subsequently reinfected with BVAB following sex with an untreated sexual partner is not new, failure of past partner treatment trials has eroded confidence in this concept. If reinfection is a key driver of recurrence, current antimicrobial regimens directed to women alone are unlikely to achieve a high level of sustained cure, and the approach of partner treatment to reduce reinfection is justified. In this manuscript, we present the molecular and epidemiological evidence that underlies the hypothesis that BV is sexually transmitted, and summarise why research that continues to consider sexual partnerships is necessary. We also outline the significant barriers and challenges that we have identified while undertaking partner treatment studies, and we discuss the factors that impact on our ability to determine their effectiveness.Ultimately, the pathogenesis of BV recurrence is likely to be multifaceted and not attributable to a single mechanism in all women. If we are to achieve sustained cure for women, it is likely that combined and individualised approaches to eradicate BVAB, support an optimal vaginal microbiome, and prevent reinfection from partners will be required.

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The Female Vaginal Microbiome in Health and Bacterial Vaginosis

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.631972 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xiaodi Chen ◽

Yune Lu ◽

Tao Chen ◽

Rongguo Li

Keyword(s):

Bacterial Vaginosis ◽

Therapeutic Strategies ◽

Reproductive Age ◽

Gardnerella Vaginalis ◽

Obstetric Outcomes ◽

Antimicrobial Compounds ◽

Vaginal Microbiome ◽

Sharp Decline ◽

Women Of Reproductive Age ◽

Sexually Transmitted

The vaginal microbiome is an intricate and dynamic microecosystem that constantly undergoes fluctuations during the female menstrual cycle and the woman’s entire life. A healthy vaginal microbiome is dominated by Lactobacillus which produce various antimicrobial compounds. Bacterial vaginosis (BV) is characterized by the loss or sharp decline in the total number of Lactobacillus and a corresponding marked increase in the concentration of anaerobic microbes. BV is a highly prevalent disorder of the vaginal microbiota among women of reproductive age globally. BV is confirmed to be associated with adverse gynecologic and obstetric outcomes, such as sexually transmitted infections, pelvic inflammatory disease, and preterm birth. Gardnerella vaginalis is the most common microorganism identified from BV. It is the predominant microbe in polymicrobial biofilms that could shelter G. vaginalis and other BV-associated microbes from adverse host environments. Many efforts have been made to increase our understanding of the vaginal microbiome in health and BV. Thus, improved novel and accurate diagnosis and therapeutic strategies for BV have been developed. This review covers the features of vaginal microbiome, BV, BV-associated diseases, and various strategies of diagnosis and treatment of BV, with an emphasis on recent research progresses.

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Longitudinal profiling of the macaque vaginal microbiome reveals similarities to diverse human vaginal communities: implications for use as a pre-clinical model for bacterial vaginosis

10.1101/2020.12.14.422805 ◽

2020 ◽

Author(s):

Nicholas S Rhoades ◽

Sara M Hendrickson ◽

Danielle R Gerken ◽

Kassandra Martinez ◽

Ov D Slayden ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Rhesus Macaque ◽

Rhesus Macaques ◽

Adverse Outcomes ◽

Reproductive Age ◽

Lactobacillus Species ◽

Vaginal Microbiome ◽

Clinical Model ◽

Sexually Transmitted ◽

Increased Susceptibility

The vaginal microbiota plays an important role in women's reproductive and urogenital health. Disturbances in this microbial community can lead to several adverse outcomes including pelvic inflammatory disease, bacterial vaginosis (BV) as well as increased susceptibility to sexually transmitted infections, miscarriage, and pre-term births. It is now well accepted that while the microbiome of healthy women in the developed world is dominated by Lactobacillus species, vaginal communities in asymptomatic women, especially those in the developing world, can be comprised of a diverse set of micro-organisms. The presence of a diverse vaginal microbiome has been associated with increased susceptibility to HIV infection but their implications for women's health remain poorly understood. Rhesus macaques are an excellent translational animal model due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from 16 reproductive age female rhesus macaques. Many animals showed hallmarks of BV, including Nugent scores above 7 and high vaginal pH. At both the taxonomic and functional level, the rhesus macaque vaginal microbiome was most similar to that of women who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Specifically, rhesus macaque vaginal microbiomes harbored a diverse set of anaerobic gram-negative bacteria, including; Snethia, Prevotella, Porphyromonas, and Mobilluncus. Interestingly, some animals were transiently colonized by Lactobacillus and some with Gardnerella. Our in-depth and comprehensive analysis highlights the importance of the model to test interventions for manipulating the vaginal microbiome.

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Cloning, Expression and Characterization of a Novel Cold-adapted β-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52

Marine Drugs ◽

10.3390/md16120469 ◽

2018 ◽

Vol 16 (12) ◽

pp. 469 ◽

Cited By ~ 6

Author(s):

Jingjing Sun ◽

Congyu Yao ◽

Wei Wang ◽

Zhiwei Zhuang ◽

Junzhong Liu ◽

...

Keyword(s):

Deep Sea ◽

Glycoside Hydrolase ◽

Sea Water ◽

Maximum Activity ◽

Glycoside Hydrolase Family ◽

Cold Adapted ◽

Hydrolysis Of ◽

Hydrolase Family ◽

Substrate Hydrolysis

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S−1 at 35 °C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

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Longitudinal Profiling of the Macaque Vaginal Microbiome Reveals Similarities to Diverse Human Vaginal Communities

mSystems ◽

10.1128/msystems.01322-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Nicholas S. Rhoades ◽

Sara M. Hendrickson ◽

Danielle R. Gerken ◽

Kassandra Martinez ◽

Ov D. Slayden ◽

...

Keyword(s):

Microbial Community ◽

Bacterial Vaginosis ◽

Rhesus Macaque ◽

Longitudinal Analysis ◽

Rhesus Macaques ◽

Comprehensive Analysis ◽

Reproductive Age ◽

Vaginal Microbiome ◽

Genetic Homology ◽

Microbiome Data

ABSTRACT The vaginal microbiota plays an important role in women’s reproductive and urogenital health. It is now well accepted that a “healthy” vaginal microbiome is dominated by Lactobacillus species. Disturbances in this microbial community can lead to several adverse outcomes, including pelvic inflammatory disease and bacterial vaginosis (BV), as well as increased susceptibility to sexually transmitted infections, miscarriage, and preterm births. However, vaginal communities, especially those of women in the developing world, can be comprised of a diverse set of microorganisms in the absence of overt clinical symptoms. The implications of these diverse vaginal microbiomes for women’s health remain poorly understood. Rhesus macaques are an excellent translational animal model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from 16 reproductive-age female rhesus macaques. At both the taxonomic and functional levels, the rhesus macaque vaginal microbiome was most similar to that of women who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Specifically, rhesus macaque vaginal microbiomes harbored a diverse set of anaerobic Gram-negative bacteria, including Sneathia, Prevotella, Porphyromonas, and Mobiluncus. Interestingly, some animals were transiently colonized by Lactobacillus and some with Gardnerella. Our in-depth and comprehensive analysis highlights the importance of the model to understand the health implications of a diverse vaginal microbiome and test interventions for manipulating this community. IMPORTANCE It is widely accepted that the “healthy” vaginal microbiome of women in the developed world is dominated by Lactobacillus species. However, in the developing world, many asymptomatic women harbor diverse vaginal microbial communities that are typically associated with bacterial vaginosis. Many questions remain about the drivers and health implications of a diverse vaginal microbial community. Rhesus macaques provide an excellent translational model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from a large cohort of reproductive-age rhesus macaques. At the taxonomic, genomic, and functional levels, the rhesus macaque vaginal microbiome was most similar to that of humans, who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Our in-depth and comprehensive analysis highlights the utility of macaques as a model to study diverse vaginal community state types and test interventions for manipulating the vaginal microbiome.

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