Experiment wishlist

Back in the lab! Hope to spend a lot of time here in the coming weeks. My program is getting busier in October so I hope I can get a lot done before that. On my experimental wishlist:

-Study Gardnerella vaginalis and Lactobacillus iners growth on glycogen and starch breakdown. For this I need to be able to create more strict anaerobic conditions then the growth protocols I am using now since these species seem to be more sensitive to oxygen. Let’s see if we can arrange that in the lab.

-Further compare glycogen consumers and “non-consuming” strains of L. crispatus. To be able to make this comparison I will need to find growth conditions in which I get expression of the gene, but growth of both consumers and non-consumers. Alicia told me that maltose, maltotriose or maltopentaose may do the trick. The precise mechanisms of this induction of expression (?, or whatever it may be) are interesting and may have in vivo relevance, but have low priority. I just want to compare the strains, but no growth (of the non-consuming strains on glycogen) means no pellet and no bacteria to use as a comparison! I will start with maltose, and have ordered maltotriose. These experiments will also be useful to get more replicates for the growth data in the previous blog.

-Alicia also told me more about the differences between amylose and amylopectin in starch. This may explain why we only saw partial breakdown of starch in activity assay of L. crispatus. If we are able to differentiate between amylose and amylopectin utilization, we may also learn more about the exact activity and ways to use this as a target for therapeutic approaches.

-At some point I really need to make an overview on what is actually known about glycans in the human vagina. What techniques and stains were used in the different studies sofar to characterize the glycans?

Stay tuned!

Carbohydrate active enzymes in Lactobacillus crispatus – a possible link between the pullulanase gene and growth on glycogen

Hello everyone, after moving to Rotterdam and having a baby (welcome Frank!) I am back here to show you the progress I made! The findings I present here are  work in progress, but it is time to give a first update. I believe the results I present here are promising on three different levels: 1) content-wise, I think they are a start to understanding glycogen metabolism of Lactobacillus crispatus better 2) I am showing it all: data, methods, results, questions and flaws. Exactly how I envision Open Kitchen Science, (although with a bit of delay, so less “realtime” as I would like) 3) this is the work of a collaboration with another “postacademic” scientist, who wants to contibute to science besides her regular job. Meet Alicia Brandt!

Before diving into the science, a few points of order:

Collaboration: As mentioned before, I am very interested in collaborating and receiving feedback on my blog posts. Please contact me, or leave a remark. I am also interested whether any other labs are working on the same question or have been working on this in the past.  If you are (or were ) doing experiments with Lactobacillus crispatus we could find out whether these results are true for more strains (especially using the same medium). Let’s talk! Twitter or Facebook or email rosanne dot hertzberger at gmail dot com

Manuscript: Part of the results presented here will be part of a paper that is currently in preparation by Charlotte van der Veer and Remco Kort and co-authors (including me). I will update this blog post as soon as this paper is published. When the paper is published, the full genomes of the strains will also be released online.

Internship: starting after the summer break I hope to be supervising a student working on this project, together with Jurgen Haanstra at the VU University Amsterdam. Find the description here (lab-site) or here (FigShare). BSc or MSc, please contact me if you are interested!

Warning: The results presented in this blog post are unreviewed and have not been replicated by an independent laboratory.

Open data All data and protocols used can be found in this FigShare collection.

How to cite: Hertzberger, Rosanne; Brandt, Alicia (2018): REBLAB: Carbohydrate active enzymes in Lactobacillus crispatus – a possible link between the pullulanase gene and growth on glycogen. figshare. Collection. https://doi.org/10.6084/m9.figshare.c.4133819.v2

Short overview of where we are.

I am interested in glycogen metabolism of vaginal microbes. Glycogen is an abundant vaginal source of carbohydrates and varies depending on the vaginal bacterial signature. Roughly spoken: when the vaginal microbiota are dominated by Lactobacillus glycogen is generally higher. One of the overarching questions in the field is whether vaginal lactobacilli can metabolize this glycogen to grow and produce lactic acid. I think this is a pretty big deal. I have had a Google Scholar Alert with the keywords “vaginal glycogen” since a few years and not a week goes by without a paper getting published talking about how this vaginal glycogen is supposedly turned into lactic acid by lactobacilli. In my view, there is no evidence to back up this statement.

During my postdoc at Washington University St. Louis it became clear that the BV-associated Gardnerella vaginalis and Prevotella bivia are capable of growing on glycogen, as well as Lactobacillus iners (often encountered in vaginal microbiota with and without BV). Previously on this blog I showed experiments that Lactobacillus crispatus can grow and acidify using glycogen as a source. Here we zoom in further on the variation in glycogen metabolic capacity of different Lactobacillus crispatus strains, using a group of 23 different Lactobacillus crispatus isolates.

Summary of the findings

A dbCAN survey of the genomes of these strains shows an overview of several carbohydrate-active enzymes possibly involved in glycogen breakdown. One of the genes present in all strains is a putative S-layer linked type 1 pullulanase (GH13_13). Zooming in on this gene it appears that there is strong variation in the N-terminal sequence encoding a putative signal peptide. Only the strains that do not have a mutation in the sequence of this signal peptide show growth on glycogen. 6 out 23 strains that have a mutation in this peptide cannot grow on glycogen. Is this signal necessary for Lactobacillus crispatus  to break down the big molecules of glycogen in the vaginal environment? 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 shared their own stories about their departure from science and how this affected them. Sometimes these people find the time and energy to still contribute to science parallel to their regular jobs. We start to call ourselves “post-academic” scientists. One of these people is dr. Alicia Brandt (previously Alicia Lammerts van Bueren as she is known in the glycobiology world) who left science last year for a job in a supporting role at the Young Academy at Groningen University. 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. I went to meet her in Groningen and had a great time!

 

 

 

 

 

Alicia and me on the steps of the Groningen academy building

Lactobacillus crispatus isolates in this blog

Last year I was very lucky to join Prof. dr. Remco Kort at the VU University Amsterdam who had just isolated and sequenced about 30 Lactobacillus crispatus strains from women with and without BV ecology. These women were patients at the GGD facility in Amsterdam and the strains were isolated as part of the thesis work of Joke Dols. MSc student Jorne Swanenburg was responsible for the genomic analysis sofar and Charlotte van de Veer is currently finalizing her thesis and is writing up a paper on carbohydrate metabolism of these strains. Recently, a paper was published where the strains were used as a reference for a possible new vaginal prebiotic. The paper is open access and the isolation is described in materials&methods. At the moment we are analyzing these strains, some appear harder to culture than others. The  list of isolates that we are able to maintain in the lab is a work in progress and will be updated later. We were unable to revive RL_005 from its glycerol stock and the sequencing file of RL_022 has some problems.

Alicia offered to use her expertise to help answer some questions surrounding Lactobacillus crispatus glycogen metabolism. First thing she did was to perform an analysis using the so-called dbCAN server, detecting the presence of several glycoproteins in the genomes of a set of isolated Lactobacillus crispatus strains that can potentially be involved in glycogen metabolism.

Find the raw data here (FigShare).

Find the protocol here (FigShare).

Most important findings:

  • All L. crispatus genomes contained a putative cell-surface associated (as indicated by SLAP domain) pullulanase Type 1 enzyme (http://www.uniprot.org/uniprot/A0A135Z466) implicated in glycogen degradation. Key features of this enzyme are the presence of a CBM41 and CBM48 and a GH13_13 catalytic domain (Fig 1). More on this gene/protein later in this blog post.
  • There is a cluster (operon?) of alpha-glucan degrading enzymes in all crispatus genomes which include a GH65 (phosphorylase?), GH13_20(CBM34) and GH13_31-2 enzyme. Further analysis required to see if they are co-transcribed. (Table 1, genes located in the cluster are indicated with an (a)).
  • Other alpha-glucan metabolizing enzymes found within the L. crispatus genomes include: GH13_18, GH13_29, GH13_31, GH31 (see Table 1).
  • Only three strains of L. crispatus contained a GH13_18 (RL02, RL09, RL10) (Note: RL_022 contained this enzyme as well, but sequencing file of strain has errors).
  • RL_006 is the only strain that does not contain a GH13_29 enzyme.

Table 1: Overview of Alpha-glucan enzymes found in L. crispatus genomes (see www.cazy.org for more info on predicting enzyme activities based on amino acid sequence similarities with known enzymes within a given family)

Enzyme Family Proposed Activity Genomes
GH13_13 (CBM41, CBM48) Pullulanase type I All except RL31, RL32
GH13_18 Sucrose phosphorylase Only RL02, RL09, RL10. RL22*
GH13_20 (CBM34)(a) Pullulanase type III, cyclodextrinase All
GH13_29 Trehalose-6-phosphate hydrolase All except RL06
GH13_31-1 oligo-alpha-1,6-glucosidase All
GH13_31-2 (a) oligo-alpha-1,6-glucosidase All
GH31-1 alpha-glucosidase All
GH31-2 alpha-glucosidase All
GH65 (a) Maltose phosphorylase All

*RL22 genome needs to be resequenced (problems with sequencing file)
(a) Constitutes part of a cluster of enzymes, possibly an operon.

N-terminal signal peptide of the type 1 pullulanase gene (GH13_13) corresponds with growth on glycogen.

Glycogen is a prevalent potential carbon source in the vagina of reproductive age women. Lactobacillus crispatus and Lactobacillus iners-are the most frequently encountered species vaginally. They are generally assumed to be responsible for the low pH and high lactate concentrations- but it is unclear what sugar source they use for lactic acid production.

(To be fair: there are many assumptions and uncertainties here. To name a few: there is only circumstantial evidence that vaginal lactic acid is of bacterial origin. The same counts for the human origin of vaginal glycogen. Actually, the fact that we are dealing with glycogen and not with a different glycan is not all that well established since most studies used a PAS stain and an alpha-glucosidase. It could well be that we are dealing with a different glycan. However, I am cutting a few corners here and will assume that lactate is of bacterial origin and the glycan is in fact glycogen, produced by the hostess herself.)

Previously it was reported that at least a subset of vaginal lactobacilli (jensenii, gasseri and johnsonii) were unable to breakdown glycogen, but this study did not look at glycogen metabolism of crispatus and iners. I presented on this blog some evidence that the DSM strain of L crispatus is capable of growth and acidification (producing lactic acid) on glycogen as the carbon source.

This possible L. crispatus glycogen metabolism could be an important player in the acidification of the human vagina, and the health benefits that are associated with a vaginal community dominated by L. crispatus. Needless to say, we would like to know more about it.

Why the type 1 pullulanase had my interest – the role of serendipity 

This gene had had my attention already since I started working with a set of 4 Lactobacillus crispatus strains during my postdoc in St Louis at the Lewis lab, WUSTL School of Medicine. These strains were MV-1A-US, MV-3A-US, JV-V01 and 125-2-CHN. I found that two of these (MV-1A-US and MV-3A-US) were able to grow on glycogen as a carbon source whereas two others (JV-V01 and 125-2-CHN) were not. To find a possible genetic origin of these differences I did a blastp analysis using several enzymes as a query that were known to be involved in glycogen (or starch) breakdown. Those were: glgX of E. coli, sap of Streptococcus agalactiae (see paper and sequence ), SusB of Bacteroides theta and the amylase (amyE) of Bacillus subtilis. Three out of the four genes showed no full-length copies in the four L. crispatus genomes, but I did find genes similar to the glgX gene: a gene annotated as a type 1 pullulanase (uniprot link of the copy in strain MV-3A-US). One of the strains unable to grow on glycogen (125-2-CHN) had no copy of this gene and the other strain unable to grow on glycogen (JV-V01) had a mutation in the upstream region, that I thought might be a dealbreaker for expression. This paper on comparative genomics of Lactobacillus crispatus confirms the absence/presence of the type 1 pullulanase in these strains. (supplementary material file nr 5). All other strains in the comparison contained the gene except for 125-2-CHN and 214-1.

This was all purely speculative at that moment, but this is why the type 1 pullulanase gene caught my attention: it was the only one that showed clear variation amongst the L. crispatus strains.  There was a big chunk of serendipity that lead me to the presented finding.

All L. crispatus strains have the type 1 pullulanase gene, but the N-terminus looks different!

I was therefore disappointed that initially, from Alicia’s dbCAN analysis, and also in a screen performed by Jorne Swanenburg, it became clear that all strains had a copy of this gene. I further analysed this gene and also included the upstream region (expecting to find a mutation similar to the one in the JV-V01 strain). The sequence directly upstream this gene encodes a putative signal peptide (see Figure 1) and there is strong variation amongst the collection of genomes in this particular area (see Figure 2)

Please find the genes from all strains here. I used the EMBL Clustal Omega online Multiple Sequence Alignment tool to compare the genes and the results were striking. You could redo the analysis by using the file and just copy paste it into the Clustal Omega tool. The genes are conserved but not the starting region. In Figure 1, a schematic overview is shown of the organization of this gene in L. crispatus.  Thanks Alicia!) In Figure 2, I am showing the variation in the N-terminal sequence from the aforementioned Clustal Omega comparison.

Figure 1: Graphical Representation of GH13_13 Pullulanase Type 1. (N to C terminal): SP: signal peptide (amino acids 1-45), CBM41: carbohydrate-binding module family 41 (amino acids 110-220), CBM48: carbohydrate-binding module family 48 (amino acids 400-505), GH13_13: glycoside hydrolase family 13 subfamily 13 (amino acids 606-900), SLAP: Surface layer associated domain (1100-1259)

Figure 2: Comparison of the N-terminal sequence of the pullulanase gene in L. crispatus strains. Adjusted from Clustal Omega. Red: strains with a possible disrupted N-terminal sequence and signal peptide. Blue: strains with an N-terminal sequence indicating an intact signal peptide. Find full sequences here.

7 out of the 24 strains (RL_#-strains) have a mutation in the N-terminal locus of the pullulanase gene, more specifically, in the sequence of the signal peptide. Those strains are indicated by red in Figure 2. Further experiments should indicate whether this means that the start site of these “red” strains lies more downstream from the start site in the “blue” strains.

Remarkably, there are five different variants of this gene locus present in those seven strains. For instance, strains RL_002 and RL_009 only show a deletion of two nucleotides (a frame shift), whereas strain RL_006 and RL_007 have a completely different sequence in this region.

Growth on glycogen of the L. crispatus strains.

I performed a very straightforward cultivation experiment using glycogen as a carbon source. Initially, I only used four isolates (RL_002, RL_003, RL_007 and RL_026). When these results were promising I started to screen all 23 strains . As a benchmark I used the DSM strain, which I previously showed is able to breakdown glycogen for growth and lactic acid production. I used the same methods as described in that blog post:

I inoculated the strains in regular NYCIII glucose and after ~72 hours of growth diluted them with NYCIII glycogen (final concentration 0,5%), water (as a negative control) and glucose dissolved in water (final concentration 0,5% as a positive control). After 48 hours I measured the optical density at 600 nm to determine growth on glycogen compared to growth on NYCIII without supplemented energy source and NYCIII glucose.

For 19 out of the 23 strains tested (we could not revive strain RL_005) I have either biological triplicates or duplicates, I am showing their results in Figure 3. I am still working on getting all data complete and aim to have at least two replicates of this experiment for each strain. In one biological replicate of strain RL_019 and one replicate of RL_006 results are very different from the others. I have no idea why and no ‘reason’ to exclude it. Not really sure what to do with this measurement at this point. All individual biological replicates are shown in the figure.

Note: I measured the cell density 10x diluted in PBS as well as undiluted. I am showing the diluted data in this blog post, since I do not have the undiluted culture measured on all dates for all strains. The difference between “growth” and “no growth” is more pronounced in the undiluted measurement since background absorption of the media is lower. I include an overview of the undiluted data in a separate tab of the excel sheet and the figure in the GraphPad Prism file. Other data that are in the file but not in the Figure shown here: the optical density in the positive (glucose) and negative control (water). Find the data here (.xls and the GraphPad Prism 7 file to generate the figure).

Figure 3: OD600 after culturing on NYCIIImedia with 0,5% glycogen. Biological replicates are shown as individual data points (some duplicates, some triplicates), vertical line indicates mean. Red or blue corresponds with red and blue in Figure 2. blue = with intact N-terminal signal peptide, red = with a disruption in this sequence. Find data here and protocol here

Although the growth data set is not complete yet, I do think we are seeing a strong connection between the N-terminal signal peptide of the type 1 pullulanase and the ability of the L. crispatus strain to grow on glycogen. All 6 strains in this experiment that have a disruption in the N-terminal signal peptide of the pullulanase gene show no growth on glycogen. The 14 strains that do have an intact signal peptide in the pullulanase gene can use glycogen as a source for growth. I see these data as strong evidence for an essential role of the pullulanase gene for glycogen consumption in Lactobacillus crispatus and, more specifically, the N-terminal signal peptide.

Thoughts, questions and new experimental plans

This finding is just that, a finding. Nothing final yet, an experiment that leads to a hypothesis: the N-terminal 29 amino acids are somehow important for Lactobacillus crispatus glycogen consumption. But how? Does this signal peptide lead to secretion of this enzyme? And if so, what is the influence of the C-terminal SLAP-domain? Are both required to localize this enzyme on the outer cell wall to be able to break down the big molecules of glycogen in its surroundings? Or does this signal peptide have a different function?

It is possible that the pullulanase without the signal peptide still functions in an intracellular metabolic pathway for glycogen breakdown, whereas the pullulanase gene that is localized on the outer cell wall can also debranch external glycans and utilize them for growth and lactic acid production.

I envision a few experiments to test these questions:

  • it is probably important at this point to establish that the pullulanase is indeed a pullulanase. I have talked with Alicia about expressing the gene in E. coli to further characterize its activity. Alternatively, we could try to capture its native activity using cell free extracts and analyse carbohydrate products with Thin Layer Chromatography. Previously, we were able to show starch breakdown, next we should take a better look at the actual breakdown products.
  • How can we study the role of the signal peptide? This is not that straightforward. Optimally we would just make L. crispatus mutants with and without it and track the enzyme’s activity and location. However, I have not seen any Lactobacillus crispatus cloning anywhere and not looking forward to try to develop my own protocols to get these isolates genetically accessible. (if someone has an idea, let me know). I hope to come up this summer with some experimental plan to test the cellular localization of this enzyme with and without the signal peptide.

What is the role of Lactobacillus crispatus glycogen metabolism in the context of the vaginal environment?

I believe that these experiments show that this activity is not something essential: the non-glycogen consumers seem to live happily in the vagina. Other lactobacilli, such as jensenii and gasseri also flourish in the vagina without being able to break down glycogen. Either, these bacteria utilize an alternative energy and carbon source. Or -what I personally expect- is that these bacteria live alongside glycogen consumers such as Lactobacillus iners or crispatus strains. Currently we don’t know whether the lactobacilli we encounter so abundantly in the vagina of reproductive age women are a collection of various strains or are a clonal population. It could well be that the non-glycogen consumers only thrive as freeloaders and can only colonize alongside a second glycogen-consuming species that does some of the glycogen breakdown. These exciting questions are definitely on my experimental wish list.

As I am still at home with baby, I will mostly do some reading and computer work to understand the genetic context and role of this pullulanase in other species. I found some interesting literature on this enzyme in Lactobacillus acidophilus and in a thermophilic bacterium called “Caldicellulosiruptor kronotskyensisencodes” (WOW! That must be one really interesting species!) Generally, pullulanases (and definitely secreted pullulanases) are of industrial relevance so there should be quite some protocols and knowledge out there. Also, I really need to read up on S-layers in lactic acid bacteria. Stay tuned!

And again: if you have suggestions how to continue, remarks or criticism, please let me know below. In general, I really appreciate any signs that this work matters to anyone because of the alternative publication route I am taking here.

Speculation on the role of bacterial peroxide production in the vaginal environment.

A new commentary on the antimicrobial role of H2O2 produced by lactobacilli in the vagina was published last week.

https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0418-3

I agree with the authors, that it is not likely that H2O2 has a significant effect in keeping BV-associated bacteria at bay. Especially the experiments that dr. O’Hanlon published previously comparing the bactericidal effects of lactic acid and H2O2. They found that H2O2 is as harmful to lactobacilli as it is to several BV-associated bacteria, whereas lactobacilli can survive very high lactic acid concentrations.

I would like to add one piece of speculation here. Although the authors say that semen and cervicovaginal fluid have strong antioxidative properties that would eliminate any H2O2, I do wonder whether H2O2 production may serve a role in the onset of BV. During disruption of the vaginal environment, either through sexual arousal, intercourse,  tampon insertion etc, oxygen levels are expected to rise to atmospheric levels. Is it possible that hydrogen peroxide production and accumulation in that case can result in the demise of both lactobacilli as well as BV-bacteria? This might then open up a “window of opportunity” in which general bacterial levels are temporarily low, lactic acid levels are reduced and pH is neutral. This relatively benign environment may give BV-associated bacteria a chance to colonize and proliferate.

Just a thought about a possible alternative role of H2O2.

Podcast on The Vital Question

I have been working on two things in the past months. Soon, I will post an update here on a possibly interesting gene variant I found in L. crispatus, that might be involved in glycogen metabolism.

In the meanwhile, I am proud to present to you my first podcast! The topic is “The Vital Question”, a book by Nick Lane that provides answers to the most profound question in biology “why does life look the way it does”.  Game changing book according to the Guardian, and someone who agrees with that wholeheartedly is prof. Bas Teusink of the Free University of Amsterdam (also on Twitter). I sit down with Bas to talk about the beginning of the book where the author lays down “the problem” . That problem is that although life looks very diverse from the human eye, when you get down to the chemistry and the energetic systems we are all very similar.

I sincerely hope you enjoy it! Please let me know what you think either below or on Twitter.  I am not exactly sure where I am headed with this, whether it’s a one time thing, or a pilot to something bigger. Your feedback is much appreciated.

Work discussion september 2017 VU University

Below you can see my work discussion that I gave early September at the VU University.  I prerecorded it using PowerPoint recording function, which was new to me. Not exactly happy how it turned out, I kept dragging my own head around the slide to make sure it wasn’t in the way and it also pops up in various sizes. Also, the recording was missing from some slides and had to redo those. But now it’s done, and in my view it gives a nice overview of the background and aims of this project.

 

Update december 2017

Unfortunately, I haven’t been spending much time in the lab and was out giving talks and writing pieces for the newspaper. These weeks, I will have more time for science and I want to focus my attention on identifying any glycogen-degrading enzymes expressed by L. crispatus.

1 Screen of 20+ L. crispatus isolates for glycogen consumption.

In a project by Remco Kort together with TNO, VU University Amsterdam and GGD Amsterdam, 20+ L. crispatus strains were isolated from women with or without BV (bacterial vaginosis). The goal was to find metabolic and genetic characteristics that could predict whether a L. crispatus was more likely to be found in a BV background or in a Lactobacillus dominated background. Its genomes were sequenced and student Jorne Swanenburg, supervised by Remco Kort and Douwe Molenaar, has performed the assembly, annotation, quality control and analysis. The sequences are expected online at GenBank anytime soon, he is currently finalizing his thesis.

In addition, Charlotte van der Veer (PhD student at the GGD) has performed a preliminary API test to analyze the metabolic signatures of the strains. The API test revealed that there is quite some difference between the ability of L. crispatus strains to break down glycogen. This is relevant since glycogen is abundant in the vagina of reproductive-age women with a Lactobacillus-dominated community. Glycogen could function as a carbon source for certain L. crispatus strains to produce lactic acid and acidify the environment.

I am currently running growth experiments with these strains to verify the results of the API test and find differences amongst the strains. Previously, I have found the DSM strain (20584) to be able to grow on glycogen and I also found starch degrading activity after growth on glycogen both in supernatants as well as in the pellet. After growth on glucose, this activity was absent.

2 Genome analysis

Next, I am looking for genetic differences amongst this group of strains that correspond with their (in)ability to utilize glycogen for growth. For now, I am focusing especially on this gene, annotated for now as a “type 1 pullulanase”, expected size ~140 kDa. Previously, this gene turned up in a very small comparison between four L. crispatus strains from the Human Microbiome Project, that I carried out at the Washington University St Louis. There are some interesting differences between the genomes of the strains around this gene. Some strains lack a copy of this gene, some have one or two copies, and some have a copy that lacks the first 8 amino acids at the N-terminus. By the way, this protein has a SLAP domain, which means that it might be associated with the S-layer. Although I know very little about S-layers, it does look like the S-layer proteins are the ones that at the outer most layer of the cell wall which (warning: speculation ahead!!!) may be associated with the activity being present in both the supernatant as well as the pellet.

3 Protein purification

I hope to start my first attempts towards protein purification before the new year. I will try to fractionate all proteins in the supernatant since I hope/expect to find the glycogen-degrading activity amongst the larger proteins. I first need to find out whether I can continue using the NYCIII medium for this. It contains horse serum so has many enzymes of <100kDa (albumin, globulins). In case I do find the activity amongst the larger proteins I won’t need to try any other media. First, I will try some bench-top size exclusion chromatography using cross-linked sepharose. Wish me luck!

 

 

 

 

Update september 2017 ongoing experiments

Time for an update on last month’s progress!

LAB symposium

I attended the LAB symposium in Egmond aan Zee. Very interesting talks on a broad range of topics, from bacteriophages, to bacteriocins, to host-microbiota, to flavor and texture of lactic acid bacteria in food applications. I especially enjoyed the talk by Jens Walter who gave a broad overview of the natural history and lifestyle of the genus Lactobacillus. He mentioned that Lactobacillus iners, with the smallest genome amngst the lactobacilli, was “on its way to become an obligate symbiont”, given its loss of genes and functions. In the paper that was connected to this talk it says that the lifestyle-associated traits L. iners has “Fe-S—defense against peroxide, glycogen fermentation, adhesion”. However, I know of no published evidence that L. iners is capable of glycogen fermentation. Perhaps it is anecdotal, or just based on the genome.

Unfortunately, there were very few presenters (both posters and talks) who gave attention to vaginal LAB. I am convinced that the enormous effect of lactobacilli on reproductive health should in the future be a bigger part in the symposium and I am determined to create a lot of insight in the mechanisms underlying this colonization.

The symposium came to an abrupt and tragic end on Thursday after it became clear that one of the Irish attendees had been involved in a tragic accident after a midnight swim. Everyone was shocked about this tremendous loss in the LAB community. His name was Alan Lucid, may his memory be a blessing to his family and loved ones.

Growth of vaginal lactobacilli on glycogen

I have been growing L. iners myself, and have some preliminary indication that L. iners can grow well on glycogen, like Jens Walter wrote in his review. I also have a third replicate for L. crispatus growth on glycogen/water/glucose showing that this strain DSM 20584 can increase its cell density (grow?) on glycogen just as well as it can on glucose. (I updated the previous blog with the third replicate).

Organic acid analysis HPLC

Below you see the chromatogram of the UV detector of a L. crispatus supernatant after 24 hour of growth on NYCIII medium supplemented with either 0.5% glycogen, or water and L. iners grown on glycogen, and medium with glycogen as a control. I wanted to make sure that glycogen is indeed converted to lactic acid, and that these strains main product is lactate. At the moment I have only performed this analysis on one single experiment. This chromatogram indicates that lactate is the dominant organic acid produced by L. crispatus and L. iners. An acetate peak (expected at RT 18.9) is absent and also on the RID detector there are no big peaks detectable apart from some glucose in the medium and some lactate. I have not been able to identify all different peaks in the chromatograms. Especially the peaks at RT 21,8 and 23,9 have my interest since these might show some organic acid being produced and consumed, respectively.

Below is an overview of lactate and glucose present in each supernatant. Beware, this is a single experiment, I will have to replicate these experiments!

Starch degradation

I continued using starch to detect any possible glycogen degradation activity in the supernatants. I believe I can conclude that supernatants of L. crispatus cultures that are grown on glycogen (and not on glucose or water) have starch degradation activity, but will only breakdown ~60* of a 1% starch solution.. Unfortunately, I do  not have calibration curves for all measurements dates, so I am just showing optical density of the starch solution for now.  

Preliminary results show that this activity is sensitive to a freeze/thaw cycle. It is also present in the pellet of L. crispatus and I have not been able to detect any degradation activity in L. iners supernatants (on glycogen/water/glucose). Lastly, in answer of the question posed in the previous blog (is amylase activity product inhibited): addition of glucose to the starch assay did not result in lower activity. These are only preliminary results and I am looking into these initial observations, I do not have sufficient replicates yet to be able to say anything with certainty.

Work discussion

On September 11th I gave a work discussion to the group here at the VU. Very excited to share the data from my postdoc in St Louis, and all new ideas. I also gave this presentation at a few different meetings of clinicians. I spoke at the regular morning gynecologists meeting at the academic hospital VUMC, where I was invited by dr. Nils Lambalk. I was also invited by dr Bing Thio and dr. Marinus van Praag to speak at the “Brugge Dagen”, the yearly meeting by Dermatologists of the ErasmusMC. Dermatologists in the Netherlands treat most vulvo-vaginal disorders. These talks for clinicians were in Dutch, I will upload an English version of this talk to Figshare at a later stage.

Thoughts about product inhibition of amylase.

From the two preliminary growth experiments it seems that the strain of L. crispatus I am using is able to breakdown glycogen and use it for growth. If this observation holds (I am currently repeating it a third time, running the starch assay again and running the supernatants on HPLC to measure lactate production), it poses lots of questions for what this means in practice. Although the results have not been robust, it looks like starch-degrading activity is only present when L. crispatus was grown on glycogen and not when the cultures were grown on glucose. This immediately makes me think of carbon catabolite repression: the mechanism where bacteria in the presence of glucose shut down the expression of enzymes metabolizing other, less preferential, carbon sources.
My colleague Jurgen Haanstra posed an alternative hypothesis in the hallway yesterday. This doesn’t necessarily have to be regulation at the transcriptional level. This can also just be caused by product inhibition on the enzyme level! (thanks Jurgen, I really appreciate it). A few minutes later he sent me this paper from 1986 (cause that’s how he rolls):  Glucose feedback inhibition of amylase activity in Aspergillus sp. and release of this inhibition when cocultured with Saccharomyces cerevisiae.

Here, they were unable to measure amylase activity in the supernatants of this fungus, but when they dialysed the enzymes (i.e. replacing the liquid, while retaining the enzyme) amylase activity was back. If this phenomenon is also going on with the glycogen-degrading enzymes of L. crispatus this could not only explain why I am not seeing activity in glucose grown wells, but also why in the starch assay untill now only about half of the starch was degraded. If glucose accumulates, enzyme activity will seize. Pretty basic biochemistry actually. (Hundred years ago someone could have been doing the experiments I’m doing now, nonetheless it is pretty exciting. And as far as I am aware, pretty novel too.)
So, I am first going to simply add some glucose to the starch assay and also other sugars such as galactose and maltose. Maltose is also a breakdown product of glycogen/starch metabolism, so perhaps this will also provide some inhibition. Will keep you updated!

First growth experiments with L. crispatus (warning, unreviewed/unvalidated data) UPDATED

UPDATE 25/9/2017 I have a third biological replicate for L. crispatus growth that confirmed previous findings. I added a graph with average +/- standard deviations.

So, I have been growing the isolates that I ordered (see previous post) from DSM with mixed success.

-The bad news is that the Gardnerella vaginalis -80°C glycerol stock seems to be in bad shape. I inoculated twice successfully from this stock on NYCIII growth media (both liquid and agar plates), but after this it took multiple days for the culture to grow and lately they haven’t grown at all. I suspect this has to do with the aerobic condition in which they are stored. I will retry with plates and media that I will preincubate in anaerobic (N2+CO2) conditions, but there will be some influx of oxygen while inoculating, and at this point I am not sure that it is feasible to handle an study G. vaginalis outside of an anaerobic chamber. Other methods are still optional, such as using closed infusion flasks.

-The good news is that both Lactobacillus crispatus and Lactobacillus iners stocks seem to be growing well. I am using NYCIII medium for both, Lactobacillus crispatus grows within ~16 hours anaerobically, while Lactobacillus iners requires ~48 hours.

I present my first experiences with growing L. crispatus on glycogen.

Some background

The goal of this endeavor is to test whether vaginal bacteria can grow on glycogen, and are able to convert glycogen to lactic acid. Glycogen is an important carbohydrate present in the vagina, shown by old [1] and new research [2]. It is not yet understood how lactobacilli acidify the vagina of reproductive age women, and glycogen metabolism could be an important mechanism. Previously, Spear et al [3] have shown the presence of glycogen degrading enzymes in the vagina. The lactobacilli that were tested in this study showed no glycogen degrading capacity . Spear et al, asserts that the host excretes amylases (glycogen degrading enzymes) in order to assist acidification by Lactobacillus. However, the researchers did not use the most often encountered vaginal lactobacilli: Lactobacillus iners and Lactobacillus crispatus. Here I show initial growth experiments of L. crispatus on glycogen and amylase assay of the supernatants. I have gathered some evidence that Lactobacillus crispatus DSM 20584 strain can utilize glycogen as a source for growth.

Methods and results

I have used NYCIII medium supplemented with either 0.5% glycogen or nothing (water, negative control) or 0.5% glucose as a positive control. To this end I have pipetted 100 uL of a 5% glucose solution or 5% glycogen solution or water and 900 uL of a 1.1x NYCIII medium where I have left out the glucose (see protocols). I have inoculated this with 100 uL of a preculture of L. crispatus DSM 20584 grown for >24 hours at 37°C anaerobically, without shaking. . For every condition, I used three wells as technical replicates. I also have empty controls without cells. Glycogen makes the solution a bit hazy so I wanted to make sure that any increase in optical density is not due to the glycogen itself but this was not the case. I have performed this experiment on two different occasions.
I mixed the culture by pipetting with a 1 mL pipet and diluted the culture 10x with water in a flatbottom 96-well plate to measure the cell density in the plate reader at OD 600. It looked like this


The data look like this: I’ll upload this to a platform once I have a bigger data file to share.

Updated graph with mean +/- standard deviations, unpaired t-test comparing OD600 on glycogen compared to water shows p-value of below .0005.


The optical density is increased when glycogen is added to the media, comparable to the increase by glucose. This is an indication that this particular strain can use glycogen for growth. Next, I wanted to see if I could detect the enzymes that L. crispatus uses to break down glycogen for uptake and metabolism.

To this end, I spun down the cells for 20 minutes at 4°C and maximum speed (4754 rcf, 4499 rpm) and transferred the supernatants to plates I stored at -20°C. I wanted to test whether L. crispatus excreted any glycogen degrading enzymes in the supernatant. I added 50 uL of supernatant to 150 uL of a 1% starch solution in “amylase buffer” (100 mM Na-acetate+5mM CaCl2, pH 5.5). Starch is not the same as glycogen, the polymer has a different structure. The backbone of starch consists of the polymers amylose and amylopectin, which also consist mostly of glucose units but which are branched differently. Looking at Google images for these three polymers gives a good idea

I hope and expect that the enzymes that degrade most of the bonds between glucose units of glycogen will breakdown the bonds in amylose and amylopectin too. Starch has the advantage that it can be easily detected using iodine, an experiment that many of us already did in elementary school. I incubated this for ~24 hours at 37°C. I also checked after one and two hours but did not observe any breakdown of starch. I didn’t use any amylase control yet, only a reference calibration curve for starch. After incubation I added 10 uL, to 290 uL of iodine working solution and measured the absorption at 600 nm. I made a calibration curve using dilutions of the 1% starch concentration.

This experiment is not robust at all, since three times I measured three different things. So I will not share protocol and data yet. During the first measurement this calibration curve was not completely straight, not sure why, but the controls (starch with the supernatants of the empty control) showed an average of 7.6 gram/L starch, which is the concentration you expect. (150 uL of 10 gr/L starch + 50 uL of starchless liquid). However, in the wells with the L. crispatus supernatants something curious is going on. It seems that the amylase activity is found in the wells grown on glycogen whereas the wells grown with no carbon source (water) show the same concentration of starch as the controls (no breakdown). The glucose wells show some increase in starch. I am not sure how this happened, perhaps it has to do with pH, lactate presence, or even some growth in the wells. The wells did not look turbid, but growth can not be entirely ruled out.

The second time I did not freeze/thaw the supernatants but used fresh after spinning down the culture. I saw starch breakdown in the glycogen wells, indicating that L. crispatus excretes some soluble glycogen degrading enzyme in the supernatants (amylase?) whereas the H2O and glucose well did not show signs of starch breakdown. A third time I performed this experiment again with frozen sups I did not see any breakdown of starch.

So, I have to optimize this method, run controls with diluted amylase. I have to find out what’s going on with the increase in OD and finetune the protocol to get reproducible results. Perhaps it will turn out that this method is not robust enough, and I will have to resort to another way of measuring amylase activity.

On my long grocery wishlist with experiments:

  • Improve amylase detection.
  • Perform a HPLC on the supernatants to determine metabolite concentrations and gather more evidence of glycogen metabolism.
  • Use HPLC to measure amylase products (glucose, maltose, trisaccharides etc).
    Continue with Lactobacillus iners and Gardnerella vaginalis to study its glycogen degrading abilities.
  • I would like to partially purify the amylases to perform proteomics, we have applied for a “Hotel”-grant at ZonMW to perform this analysis together with Winclove probiotics at Radboud UMC.
  • Lastly, we want to extend the analysis to other strains of crispatus that were isolated by Remco Kort and his student Jorne Swanenburg in order to find the gene and enzyme involved in this amylase activity. Happy to announce that the biomedical/filosophy student Noa Fuks has offered her help to track down the amylase, while teaching herself some bioinformatics. Good luck Noa!

So, it’s a long grocery wishlist of experiments. If you have any comments, suggestions for alternative amylase activity assay, ideas, theories, criticism, please drop me a line.

1. Cruickshank, R., The conversion of the glycogen of the vagina into lactic acid. Journal of Pathology and Bacteriology, 1934. 29.
2. Mirmonsef, P., et al., Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH. PloS one, 2014. 9(7): p. e102467.
3. Spear, G.T., et al., Human alpha-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus. The Journal of infectious diseases, 2014. 210(7): p. 1019-1028.

Ordering and stocking strains

Getting started! I have ordered, received and stocked 3 strains at the DSM strain library. Unfortunately they do not have as many different options for vaginal bacteria, compared to all the isolates from the Human Microbiome Project for instances, and the strains I ordered were not all isolated from the vagina. I hope that they will display the same characteristics and can be used as a reference and to assist in setting up my methods. In the future I hope to with more vaginal isolates. I have

To grow the strains I used MRS medium (liquid and plates) for L. crispatus and NYCIII medium for G. vaginalis and L. iners. Unfortunately I do not have access to an anaerobic chamber so I have been using an anaerobic jar. After closing the jar I use 3x: pulling a vacuum (minimally 0.8 bar) and filling with N2+CO2 gas. I grew plates and tubes over the weekend (72 hours) at 37 degrees and stocked them using 0.5 mL 60% glycerol + 1 mL of culture and stored them at -80 degrees. I am not sure how much the oxygen will affect the bacteria during handling and storage, especially Gardnerella is sensitive to oxygen. I will check later if the stocks lead to good growth. I will upload protocols for the media, the stocking and the anaerobic jar soon.