Hello everyone, first bigger update of the project since December. Things have been progressing slowly but surely. This will be a long blog post so I’ll start with an overview.
1 During my talk in Groningen at the Young Academy I had the pleasure to meet dr. Alicia Brandt who aspires to remain connected and contributing to science even though she left her official scientific position. Her expertise is bacterial glycobiology and she performed a “dbCan” survey to find all carbohydrate-active enzymes (CAZymes) in the genomes of the different Lactobacillus crispatus strains that were isolated from BV+ and BV- women.
2 She also presented a intriguing hypothesis on possible sugar sources in the vaginal environment. Although I am looking at glycogen as the main vaginal sugar source for acidification by lactobacilli, a different sugar called “levan” could also function as an interesting intermediate metabolite for lactic acid production. I include Alicia’s hypothesis, her analysis on the presence of levansucrases encoding genes in the human microbiome and insights into possible L. crispatus levan metabolism.
3 One of the genes that may be involved in glycogen metabolism is a putative extracellular S-layer linked type 1 pullulanase. Zooming in on this gene I several variants present in the different L. crispatus genomes. Is this a possible match with glycogen metabolic abilities? Nothing final yet, but a promising lead to look further into.
Alicia Brandt: dbCan analysis and glycogen-active enzymes
Since I started with REBLAB I encountered several people who share their own stories about their departure from science and how this affects them. Sometimes these people find the time and energy to still contribute to science besides their regular jobs. One of these people is Alicia Brandt (previously Alicia Lammerts van Bueren as she is known in the glycobiology field) who left her job as a postdoc last year for a job in a supporting role at the University of Groningen. She expressed the strong wish to keep being involved and to share her knowledge and skill. It’s a happy coincidence that her expertise is exactly what I am looking for: glycobiology and even glycogen metabolism of bacteria. She offered to help me out with several analyses. First thing she did was to perform an analysis using the so-called dbcan server, detecting the presence of several glycoproteins that potentially be involved in glycogen metabolism.
-various glycogen active enzymes
Levansucrase and the vaginal microbiota
Alicia also brought up a second possible sugar source that lactobacilli could use to produce lactic acid and acidify the vagina: levan. From her analysis it seems that especially the bacterial communities colonizing the mouth and vagina are equipped to synthesize levan. The following text is all Alicia’s. Please find the protocol she used here and the excel sheet with complete overview of the dbcan results here.
Bacterial levansucrase in the vagina
Levansucrases (EC220.127.116.11) are enzymes that catalyse the transfructosylation of sucrose into the polysaccharide levan (ref, ref). These enzymes are found within the CAZy family GH68. GH68 enzymes use sucrose as their preferential donor substrate. Many of the enzymes can create very long levan-type fructans (catalyzed by levansucrases) or inulin-type of fructans (catalyzed by inulosucrases), as well as fructooligosacharides (FOS). However, some GH68 enzymes can also use fructan as donor substrate (in the absence of sucrose or at a high fructan/sucrose ratio) (see CAZypedia).
Levans and inulins are synthesized by bacteria and some plant species (for example chickory root inulin). In lactic acid bacteria, levan is more commonly produced over inulin. In these lactic acid bacteria, levan and inulin act as exopolysaccharides that provide a protective layer around the bacteria, shielding it from the stresses of the external environment (ref). Levans can range in size from 6-kestose (DP3) up to MDa in size. It is a biologically important compound and is used in manufacturing processes as thickening agents. It was demonstrated to be a beneficial prebiotic source. Levan and inulin are degraded via the action of GH32 enzymes (CAZypedia link), thus bacteria that encode this family of enzyme are typically capable of using levan as a nutritional source. Levans can range in size from 6-kestose (DP3) up to MDa in size (for example 130MDa or 1.3 x 108 Da in Brenneria goodwinii). Levan and inulin are degraded via the action of GH32 enzymes (CAZypedia link), thus bacteria that encode this family of enzyme are typically capable of using levan or inulin as a nutritional source.
Levan and inulin are biologically important compounds and are used in manufacturing processes as thickening agents. Levan has been demonstrated to be beneficial prebiotic source.
Fig 2: Synthesis of levan by GH68 enzymes (image from here)
Glucansucrases (EC18.104.22.168) (family GH70, see CAZypedia) are processive enzymes similar to levansucrases. However these enzymes carry out transglucosylation of sucrose into alpha-glucan polymers, such as dextran, reuteran, alternan (ref). These enzymes are also mainly encoded by lactic acid bacteria and are used by these bacteria for the production of alpha-glucan exopolysaccharides. The enzymes and exopolysaccharide products also have great interest to industry in food and nutrition applications.
Fig 3: Levan versus alpha-glucan synthesis by fructansucrases and glucansucrases levan/inulin alpha-glucan
Levansucrases in the microbiome
Lactic acid bacteria make up a significant portion of our human, mainly gut, microbiota. The exopolysaccharides they produce using glucan and levan sucrases are biologically important molecules. The best known example are the exopolysaccharides produced by the oral bacteria Streptococcus mutans. In our mouths, S. mutans takes dietary sucrose and produces a sticky biofilm for attachment to the surface of tooth enamel which is the most significant contributor to tooth decay and dental caries. Glucan and levan exopolysaccharide have also been shown to serve as important nutritional sources for strains of bacteria that reside in our gastrointestinal tracts (exopolysaccharides as microbial products have prebiotic effects, my research in Groningen). Therefore, I was interested to find out in what scale the presence of genes encoding levansucrases and glucansucrases are in metagenomic sequences of human microbiome samples.
In order to determine the abundance of GH68 and GH70 enzymes in human microbiome metagenomic data, I performed a metagenomic search in Human Microbiome Project database from the JGI-DOE Integrated Microbial Genomes & Microbiomes(IMG/M) system (This analysis is from August 2014, results may be drastically different now with so much more metagenomic sequencing data available). Within the database I performed a “Find Function – Enzymes” search using EC numbers EC22.214.171.124 (levansucrases) and EC126.96.36.199 (glucansucrases). Within the database, I selected to search within metagenomic data from microbiome sample sites including the digestive system (oral, large intestine and small intestine) stool, and reproductive system. All results were tabulated in excel file (see folder).
The majority of genes encoding glucansucrase and levansucrase enzymes are found amongst the microbiota of the oral cavity. In the mouth and stool, glucan sucrases are far more abundant than levansucrases. However in the human vagina, where bacterial levels are much lower and less diverse than mouth and gut, levansucrases are more abundant than glucansucrases (49 versus 2). Thus the questions raised are:
1. Why are levansucrases more abundant than glucansucrases in the human vagina?
2. What is the importance of levan in the vagina?
3. Is levan metabolized by other species in the human vagina?
4. Is levan biologically important to the human vagina? (Health versus disease state?)
5. What is the source of the donor substrate (sucrose or fructan) for fructansucrases/levansucrases in the human vagina?
The main carbohydrate sources that would be found in the human vagina are glycogen, human N and O linked glycans, and microbially synthesized glycans, such as exopolysaccharides. Other metabolites include lactate, succinate, polyamines, amino acid breakdown products, dipeptides, These metabolites strongly vary depending on the microbiome. However, none of these sources would contribute to levan synthesis and provide the essential sucrose which acts as a donor and acceptor substrate. Where could alternative substrates for levan synthesis be found in the vagina?
In sexually active women, a source of for the sucrose or fructan donor substrate for levan synthesis could be found in semen. The limited analysis of the carbohydrate content of human semen carried out in the 1940’s and 1960’s has identified that fructose is abundant from 1- 5 mg/ml.
(there are only some reports from the 40’s and 60’s but, funny enough, there are no recent reports of the carbohydrate analysis of human semen. This should be done because our modern methods could really identify the carbohydrate composition of human semen.)
Levan is a biologically important polysaccharide in the human vagina.
Hypothesis 1: The source of the donor substrate for fructansucrase activity (sucrose/fructose/fructans) comes from human semen.
Hypothesis 2: lactic acid bacteria harboring fructan/levansucrase enzymes synthesize levan from the fructose/fructans found in male semen. Since the presence of semen is transient, and carbohydrate sources in the human vagina are limited, levan synthesis by lactic acid bacteria strains in the vagina serves three functions
-At times when fructose/fructans are abundant (such as after sexual intercourse), lactic acid bacteria synthesize levan in order to create an equilibrium in their environment.
-At times when carbohydrate sources are limited, levan can be used as a nutritional source and metabolized by bacteria in the human vagina (that contain GH32 enzymes).
-Metabolism of levan by bacteria in the human vagina contribute to the acidification of the vaginal environment, which contribute to vaginal health.