INVISIBLE INFLUENCERS

Can yeast eat mucus?

Kevin Mercurio,*† Dylan Singh,*† Kristin Baetz*†

*Department of Biochemistry, Microbiology & Immunology, University of Ottawa;

†Ottawa Institute of Systems Biology

ABSTRACT

Why:

The human gut microbiome is a vast microbial community that interacts with our cells and themselves to influence our health. To better our understanding of these interactions, researchers try and determine what organisms reside there through metagenomics techniques that search for DNA specific to certain bacteria and fungi. Dietary fungi like yeasts, found in every day foods and beverages, were found in vast quantities. Based on these methods, it is difficult to be certain whether these dietary yeasts can live in the harsh gut environment; with minimal oxygen, acidic, high temperature and a lot of mucus. Are yeasts gut microbial residents, or tourists?

How:

We investigated whether baker's yeast (Saccharomyces cerevisiae) could thrive in a mucus environment. Mucus is primarily made of proteins called mucin, which are completed surrounded with energy-rich sugars. To assess this, we introduced mucin into solid and liquid growth media and monitored cell growth. Known gut fungal residents are known to have mucin degrading proteins, so we also evaluated the importance of similar proteins called yapsins through qPCR, dot assays and fluorescence microscopy. For a more global analysis, we determined the transcriptome of yeast grown in the absence or presence of mucin by RNA sequencing, and screened for more genes that impacted growth in mucin medium using the Saccharomyces cerevisiae haploid deletion mutant array. We followed up with screen hits that had mitochondrial relevance with seahorse assays that measured differences in oxygen consumption rates.

What:

We found that yeast were able to use mucin as the main carbon source in growth media. We also found that cells reduced in size when grown in the presence of mucin. We identified an important yapsin protein, Yps7, that has a distinct role for growth in mucin conditions. We determined how mitochondria are integral for growth in mucin medium and discovered an uncharacterized protein, Ycr095w-a, for mucin-specific mitochondrial function.

 

INTRODUCTION

What we know and why we care

There are microbes living inside us

It's hard to imagine that the human body is an environment, just like homes, offices, parks, oceans, and the like. They are made up of various cell types, the smallest unit of life. The body also hosts other single-celled life as well, which include those "pesky" bacterial and fungal organisms mainly concentrated in the human gut. Surprisingly, the interactions within this gut community of single-celled life (termed microbiome) impact many different aspects of our health, and even impact our nervous system, immune response, and metabolism.

Brain Scans

Gut-Brain Axis

There is a bi-directional communication pathway between the central and enteric nervous systems, linking emotional and cognitive centres of the brain with peripheral intestinal functions.

The problem of identifying microbial residents

Researchers need to identify what species of microbes are residents that interact with the gut environment. Samples like stool and gut biopsies allow for the extraction of genetic material (DNA) which we can read and assign species, a field of research concerning metagenomics. You can imagine that a lot of this is influenced by many factors: age, gender, disease, and of course, diet. Different geographic locations vary in dietary regimes that ingest microbes in food (even washed food) and influence the composition of our individual gut microbial communities. In fact, we all have very different residential microbes, but collectively they still function to impact our health in a similar manner. The problem with this DNA approach is that it does not differentiate living or dead organisms.

To learn more, hover mouse over workflow steps.

Step 1:
Metagenomics Workflow
Collection
Step 2:
Sequencing
Metagenomics Workflow
Step 3:
Identification
Metagenomics Workflow

Dietary yeast is not known to colonize the gut

Most metagenomics research focuses on bacteria, but there has been a recent push to identify other microbes like fungi. One fungus commonly found in the human gut is Candida albicans, which is heavily studied due to its virulent ability within people with ineffective immune systems. But despite being one of the most commonly ingested fungi in western diets, there is limited information on the health impact of baker's yeast (Saccharomyces cerevisiae). We use this organism to exploit its ability to ferment sugars to create carbon dioxide (to make bread dough rise) and ethanol (for alcoholic beverages). Baker's yeast can relieve symptoms of colitis and even overturn viral infection. However, antibodies against it are an indication of inflammatory bowel disease, and it can also cause intestinal damage and increase permeability. Although baker's yeast is normally regarded as a commensal microbe of the gut, the idea that dietary yeast will always be a harmless organism within a host remains inconclusive.

Breakdown of Sugars

Sugars are taken up by yeast cells to harness energy in the form of ATP, a compound used by the cell to do any sort of work.

Learn more

Production of CO2 & Ethanol

One process of energy creation is fermentation. This process is a unique method of ATP synthesis that does not require mitochondria or oxygen. Byproducts of this process are carbon dioxide and ethanol .

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Baking Bread

Released CO2 gets caught in bread dough. As the gas continues to escape, the dough begins to fluff and bubble. This is how your bread dough rises and increases in volume.

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Creating Alcohol

After sugars have been depleted, yeast can be filtered from fermented batches and alcoholic beverages like beer and wine can be enjoyed.

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Other yeasts can eat mucus

There are various tactics that microbes use to survive in the gut. These involve evading our immune cells, and adhering to the sticky mucus coating that lines the gut cavity. Mucus helps protect our gastrointestinal tract from pathogenic invaders while easing the passage of food and waste. This layer is primarily made up of mucins, which are large proteins surrounded by sugar chains. It's clear that organisms which can break down mucins have a large advantage over other organisms, making it easier to colonize the gut. Other known fungal residents break down mucins for resources. Baker's yeast has similar proteins, called yapsins, that may have a similar function.

1/1

To learn about mucus and its components, click the picture.

 

EXPERIMENTATION

What we did and what we found

Can baker's yeast use mucin as an energy source?

We first investigated whether a laboratory strain could use mucin to grow. This strain is not the typical strain of yeast one could find at the local grocery store and varies in genetic material. However, these are still the same species, referred to as Saccharomyces cerevisiae. The growth medium is also important, and we wanted to test yeast growth on solid (a gelatinous surface, called agar) and liquid (a soup mix, called culture) media. Almost like cooking, we mixed all the ingredients together, including pig gastric mucin as our mucus supply, and incubated yeast while monitoring its growth. On solid media, this was done through a dot assay where we took pictures of surface growth on the second day. On liquid media, this was done by taking a small amount of culture and counting the number of cells in this sample volume under a microscope. But we're missing something. Every properly designed scientific experiment requires something to compare with your test, called a control. A good control allows researchers to determine whether something you modified, whether that be your growth conditions, or your yeast strain, changed the outcome of your experiment. Here, we used the exact same solid and liquid media lacking in mucin, in order to attribute any changes we see in growth to the addition of mucin.

Our results... But what would you expect? (hover mouse over to see)

Cell Count

Dot Assay

Cells

No Mucin

With Mucin

Cell colony

(multiple cells)

No Mucin

With Mucin

#

##

Individual

cell

We observed that yeast colonies grew better in medium with mucin compared to the same medium base without mucin. We also observed more individual cells in samples with mucin compared to samples without mucin. This all suggests that cells are using mucin as the main energy source in growth medium. Another interesting observation was the fact that yeast cells grown in mucin were smaller than cells grown without mucin, indicating a distinct impact of mucin on the cell like what has been shown in known gut fungal residents. Researchers already know that cell size is dependent on different energy (carbon) sources, and therefore this suggests that yeast cells reduce their size in order to grow more efficiently within mucin.

Does baker's yeast possess mucin-degrading proteins?

We next investigated whether baker's yeast has mucin-degrading proteins. A common technique for conducting any scientific experiment is to create experiments around what is already known. Other yeasts that truly colonize the gut have proteins called secreted aspartyl proteases (SAP) that have been proven to degrade mucin in the environment. Baker's yeast has similar proteins called yapsins. There are six known yapsins (YPS), with only three that have been studied and characterized. Yps1 and Yps3 are the most similar to SAPs.

To learn more, hover mouse over to click on a yapsin.

yapsins_edited.jpg

Saccharomyces

cerevisiae

Proteins are synthesized (or translated) by the cell through reading instructions within messenger RNA (mRNA) structures. These structures are created (or transcribed) from the cell's genome, containing all the genes the cell uses to make proteins that perform life functions (more on this later). We looked at these proteins in more detail. More specifically, we wondered whether the absence of these proteins would impact yeast growth on mucin medium. By transforming our laboratory yeast strain, we created six new strains that each had one of the yapsin genes deleted from the genome. We continued assessing growth on mucin medium with dot assays.

Yeasts: Microscopic Transformers

1) Creating primers for DNA insert

Polymerase chain reactions (PCRs) are performed to create DNA inserts that replace the gene in our laboratory strain. These reactions require primers, small single-stranded DNAs that help in the initiation of the reaction. Click the arrow below to learn more about PCRs.

2) Transforming cells

Cells of the working strain are collected. In a reaction tube, cells are made permeable to allow the DNA insert to be taken up by the cell. Through a process called homologous recombination, the gene of interest is swapped for the DNA insert. Click the arrow below to learn more about homologous recombination.

3) Selecting & confirming cells

Transformed cells are selected for on specific growth medium. Only cells that have incorporated the insert into its genome can grow on the selection medium. To confirm cells, another PCR is conducted and the DNA insert is confirmed by gel electrophoresis. Click the arrow below to learn more about gel electrophoresis.

Our results... But what would you expect?

(hover mouse over to see)

Mucin Medium

Cells

Normal

No YPS1

No YPS2

No YPS3

No YPS5

No YPS6

No YPS7

We observed that the yeast strain that had the YPS7 gene deleted, leading to the absence of the Yps7 yapsin protein, led to worse growth on mucin medium compared to the original strain. Yps7 is an uncharacterized yapsin. This was surprising as strains with the yapsin genes most similar to known mucin-degrading proteins (YPS1 and YPS3) were not the most deficient in growth when deleted from the genome. We investigated further and added a green fluorescent tag to Yps7 by again transforming the original laboratory strain. Through a technique called fluorescence microscopy, this tag allowed us to compare where the protein is located in the cell and its abundance in mucin medium compared to other conditions.

Our results... But what would you expect? (hover mouse over to see)

Yps7 in medium lacking a main energy (carbon) source

Cell outline

Yps7 in normal sugar medium

Yps7 in mucin medium

We observed that Yps7 fluoresced throughout the cell in mucin medium but was located in specific groups in normal sugar medium and medium lacking any major energy source. This fluorescent dispersion also indicated greater abundance of this protein in mucin medium. This change in localization suggests that this particular yapsin has a distinct role for growth in mucin conditions. Future work will look at previous research on Yps7, and whether its theorized functions coincide with a role in mucin metabolism.

What other techniques does baker's yeast have to use mucin?

We next investigated whether baker's yeast has other techniques up its sleeve (or membrane). Since we are the first group to show that baker's yeast can use mucin as an energy source, there has been no other previous research we can build upon. We needed to perform experiments that could gather a lot of data about mucin metabolism in yeast. Therefore, we took two approaches... 1) RNA sequencing: Remember those messenger RNA (mRNA) structures that contain the instructions used by the cell to build proteins for cellular functions? We can collect all the mRNA in the cell while they are growing and dividing, giving researchers an overall look at what genes are being turned on (transcribed) in a particular environment; and 2) Chemogenomics screening: Recall in the previous section how we transformed cells by deleting a single YPS gene to determine whether specific yapsins were important for growth in mucin medium. What if we could perform transformations on our laboratory strain, such that we can create a bunch of strains with a single gene deleted? Better, what if we could create these kinds of strains with every gene in the genome (excluding essential genes that would result in cell death)? Previous research groups have done just that, created a library containing thousands of strains with a single gene deleted. We can use this library to screen for genes important for growth in mucin. You can see where each approach is situated along the Central Dogma of Molecular Biology, which describes the flow of genetic information from DNA to protein.

Chemogenomics screening

focuses on these

Central Dogma of Molecular Biology

(click the picture to learn more)

FINISH

2) Genes

2) Genes

This reaction leads to a signalling cascade to the nucleus. The nucleus houses the cell's genome, the complete collection of genes for the synthesis of proteins.

3) Transcription

3) Transcription

The cell turns on (activates) genes, specific sequences of DNA that contain the instructions for protein synthesis. These activated genes are copied in the form of another structure, messenger RNA, by a protein called RNA polymerase.

6) Proteins

6) Proteins

Proteins are made up of amino acids in a specific order based on their mRNA sequences. The order is important because at such a small scale, the chemical composition of amino acids either attract or repel one another, causing the protein to twist and fold. This folding is extremely important to perform its function.

7) Modification

7) Modification

After translation, proteins usually undergo further modifications to their structures. This is done in the endoplasmic reticulum and Golgi apparatus organelles. These include adding sugar chains (like for mucin proteins) and other chemical groups to, again, allow them to perform their cellular function.

10) Adaptation

10) Adaptation

Based on the many genes activated in response to environmental cues, leading to transcription of mRNA instructions that code for the translation of specific proteins, which, along with protein modifications, finalize the 3D structure of the proteins, and transportation of these proteins to cellular locations to perform their desired functions, collectively, allow the cell to adapt to the surroundings it is in. This might be cell movement, resource uptake or compound conversion.

1) Reaction

1) Reaction

Cells sense the surroundings and react to environmental cues via protein sensors at the cell surface.

4) mRNAs

4) mRNAs

Messenger RNAs (mRNAs) are copies of the DNA sequences that correspond to genes the cell has activated. They're single-stranded, compared to their DNA counterparts which are double-stranded (humans have two copies of each gene).

5) Translation

5) Translation

mRNAs are transported out of the nucleus and into the cytoplasm, the inside of the cell only surrounded by the cell membrane. mRNAs head to the endoplasmic reticulum, an organelle that holds the ribosomal machinery for translation (protein synthesis). Ribosomes read mRNA sequences and signal the binding of specific amino acids, serving as the backbone of all proteins.

8) Transportation

8) Transportation

Once the protein is fully formed, it needs to be transported to a specific location inside (or outside) of the cell to perform its function. For example, proteins involved in transcription are transported to the nucleus, while proteins involved in protein modifications are transported to the endoplasmic reticulum or Golgi apparatus. A specific portion of its amino acid sequence notifies other shuttling proteins where to bring the protein.

Function

Function

Once the protein is transported to its location, it can now perform the desired function for the cell. Remember, this function is not only based on its specific amino acid composition, but also its 3D structure, and the modifications that occurred after translation.

START

RNA sequencing

focuses on these

Through both approaches, we grouped specific genes based on their cellular functions to see what genetic pathways are activated/deactivated (RNA sequencing) and beneficial/detrimental (chemogenomics screening) in the cell when grown in mucin medium. Remember that we can see which genes are turned on/off through RNA sequencing, while we can see which gene products are good/bad through chemogenomics screening.

Signalling

Pathways

RNA

Processing

Mitochondrial

Function

Activated/

Beneficial

Protein

Sorting

Mitosis

(Cell Division)

Deactivated/

Detrimental

Click the magnifying glass to learn more about these pathways

Phosphorus

Metabolism

We observed a variety of different pathways that were either activated/beneficial or deactivated/detrimental to the cell when grown in mucin medium. Here, we list the largest genetic groupings. Genes that were activated and served to benefit the cell were those involved in signalling pathways (how the cell adapts based on environmental cues), protein sorting (how the cell groups new proteins for more efficient transportation), RNA processing (for transcription and ease into the cytoplasm), and mitochondrial function (how the cell obtains energy from the environment while using oxygen). Genes that were deactivated and were detrimental to the cell were those involved in mitosis (how the cell multiplies into two identical copies) and phosphorus metabolism (how the cell deals with phosphorus uptake and incorporation into desired chemical molecules). Each "node" is sized based on how many genes appeared in RNA sequencing and chemogenomics screen data. The largest group of genes were in some way involved with the mitochondria.

Let us explain...

in song

(Click to learn more)

Focusing on the mitochondrial genes, we noticed something very interesting. There were very few genes that when removed from the genome (and therefore no protein product formation), resulted in strains that grew worse on mucin medium compared to our control medium (which was the exact same medium base lacking mucin). In fact, there was one gene, YCR095W-A, whose gene product (or protein) localizes to the mitochondria but has no attributed function. We investigated whether deleting this gene resulted in defects in mitochondrial function. We can assess this by understanding that mitochondria need oxygen to produce ATP, the compound needed by cells to perform important biological functions. We performed a seahorse assay that compares the depletion of oxygen in mucin medium of our laboratory strain and the transformed strain that has YCR095W-A deleted from its genome.

Our results... But what would you expect? (hover mouse over to see)

Normal Cells

No YCR095W-A

Medium Container

Oxygen Molecule

Cell

We observed that the strain missing YCR095W-A had consumed less oxygen compared to the original laboratory strain specifically in mucin medium. This suggests that this gene is important for proper mitochondrial function in mucin conditions. Future work will look at characterizing this gene and determining what role it plays within the mitochondria and why its impact is mucin-specific in baker's yeast.

 

CONCLUSION

What this means and why you should care

Let us briefly summarize everything. We used classical growth assays, yeast genetics, RNA sequencing and chemogenomics screening methods to show that even laboratory strains of baker's yeast can grow using mucin as the main energy source. Our work also identified the importance of an uncharacterized yapsin (Yps7) to grow on mucin. Additional remodelling events showed the significance of the mitochondria for cells to grow and metabolize mucin, and determined the specific importance of an unknown protein (Ycr095w-a) for growth in mucin.

Yapsin Presence

Required

Mitochondrial Function

Required

Our work supports baker's yeast as a gut colonizer as opposed to just an environmental organism passing through our digestive system. The more we know about which organisms are a part of our gut microbiome, the more we can determine which organisms impact our health the most. So have a piece of bread, or a pint of beer, and celebrate that dietary yeast has the potential to be a major player in the microbial dynamics within the human microbiome.

Special thanks to our funding sources! (click to learn more about them)

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