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Hi! I am a Dutch microbiologist and author and in I would like to regularly update you on my research
 

Rosanne Hertzberger

April 11 · Issue #1 · View online
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Hi! I am a Dutch microbiologist and author and in I would like to regularly update you on my research. I study metabolism of vaginal bacteria and the role they play on healthy pregnancies and healthy sex lives. My goal is to make my science as collaborative and transparant as possible, and this newsletter is trying to achieve that.

New students! Welcome Deborah Jekel & Ritesh Panchoe
After a great conference in South Africa, a lot of book writing and lecturing, I am back in the lab. Deborah Jekel is a bachelor student Life sciences at the VU University. Deborah will be studying regulation of the type 1 pullulanase. Previously I saw in our experiments that the enzymatic activity (starch degradation) is absent after growth on glucose. How? Why? We’ll be performing growth experiments to study the conditions of induction/repression.
Ritesh Panchoe is a MSc student in Biomedical Science at the VU University and he will be working on characterizing the various bacterial glycogen degrading enzymes to ultimately detect them in clinical specimens. Also, we will work together on developing different assays to study glycogen content and lactic acid concentration in vaginal specimens.
Results
This may be old news for some, but we’re studying glycogen metabolism of Lactobacillus crispatus. We compared several strains and stumbled upon a type 1 pullulanase gene whose N-terminal signal peptide sequence corresponds exactly with its ability to grow on this substrate.
Carbohydrate active enzymes in Lactobacillus crispatus – a possible link between the pullulanase gene and growth on glycogen – REBLAB
REBLAB: Carbohydrate active enzymes in Lactobacillus crispatus - a possible link between the pullulanase gene and growth on glycogen
Comparative genomics of human Lactobacillus crispatus isolates reveals genes for glycosylation and glycogen degradation: implications for in vivo dominance of the vaginal microbiota | Microbiome | Full Text
Plans
Although vaginal microbiota is strongly correlated with health, still women with comparable microbiota have vastly different outcomes. Some women with bacterial vaginosis have very serious symptoms and inflammation others don’t. Some women have biofilms, others don’t. I saw many attempts to explain these different health outcomes from a bacterial perspective, for instance differentiating between Gardnerella clades. I want to study the diversity from the perspective of the hostess by comparing the SNP data.
There is a lot of opportunism involved (in a good way): the HELIUS study center will soon have vaginal microbiome and SNP-data available for 346 women. Together with Streeklaboratorium GGD Amsterdam, (Public Health Service), Janneke vd Wijgert and Remco Kort we have send the following research proposal to the HELIUS people.
Research proposal: genetic factors of host glycogen metabolism and its relationship with the vaginal microbiome. – REBLAB
Keystone conference on the vaginal microbiome
Such an exciting gathering of clinicians, biochemists, public health specialists. I met a lot of people, learned a lot of new things. I gave a talk and presented a poster. The slides of the talk were also part of a lecture I gave in Amsterdam Zoo Artis.
Introduction to vaginal microbiome - lecture
Poster: Lactobacillus crispatus growth on glycogen is dependent on its type 1 pullulanase gene variant
Protocols
The aim is to distinguish between different enzymes of the various vaginal bacteria. If we find enzymatic activity in a clinical sample is this: an amylase? A pullulanase? Is it produced by the hostess? By one of the lactobacilli? Or by Gardnerella?
The starch assay we use is pretty straightforward, but it only allows us to look at total starch breakdown. We do not know if it is targeting the amylose moiety (only a1-4 linked glucose units) or amylopectin (which is more similar to glycogen)? Therefore, I am now using AZCL-amylose and AZCL-pullulan from Megazyme. Unsoluble substrates, that are still effectively broken down by enzymes. I am adding xanthan-gom to keep the particles in solution and prevent pelleting.
AZCL-Amylose Insoluble Chromogenic Substrates - Megazyme
AZCL-Pullulan Insoluble Chromogenic Substrates - Megazyme
I shared a first version of this protocol online, but will update with calibration curves and pictures soon. I also updated the starch assay online with some extra information and corrected a mistake (10 mg/ml instead of 1 mg/ml).
Protocols – REBLAB
Thanks everyone! I hope to update you in about three months with new results and plans.
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