New & Noteworthy

Mannan From Heaven

February 25, 2015


Once we started eating the yeast in breads and beer, our microbiota adapted to feast on these mannans raining down on them from heaven. Manna reigning from heaven on the Israelites (Exodus 16), from the Maciejowski Bible, c. 1250 C.E.

Thousands of years ago, humans encountered very little yeast in their diet. This all changed with the invention of beer and bread.

Now of course we didn’t include the yeast as a carbon source…we just liked fluffy bread and getting hammered. But it was a different story for the bacteria in our gut. They now found the mannans in yeast cell walls raining down on them like manna from heaven.

Unlike the Israelites, who could eat manna, most of our microbiota probably couldn’t use this newfound carbon source. But at some point, the polysaccharide utilization loci (PULs) of a few species changed so that they could now digest the mannans found in yeast cell walls. And, as a new study in Nature by Cuskin and coworkers shows, it was almost certainly a great boon for them.

They found that at least one gram negative bacterium, Bacteroides thetaiotamicron, can live on just the mannans found in the yeast cell walls. This isn’t just a cool story of coevolution either. It might actually help sick people get better one day.

Glycans, like the mannans found in yeast cell walls, have been implicated in autoimmune diseases like Crohn’s disease. One day doctors may be able to treat and/or prevent diseases like this by providing bacteria that can eliminate these potentially harmful mannans: a probiotic treatment for a terrible disease.

Cuskin and coworkers identified three loci in B. thetaiotamicron, MAN-PUL1, MAN-PUL2, and MAN-PUL3, that were activated in the presence of α-mannan. Deletion experiments showed that MAN-PUL2 was absolutely required for the bacteria to utilize these mannans.

The authors next wanted to determine whether the ability to use yeast cell walls as a food source provided an advantage to these bacteria. For this work they turned to a strain of mice that lacked any of their own gut bacteria.

The authors colonized these gnotobiotic mice with 50:50 mixtures of two different strains of B. thetaiotamicron—wild type and a mutant deleted for all three PULs. They found that in the presence of mannans, the wild type strain won out over the mutant strain and that in the absence of mannans, the opposite was true.

So as we might expect, if you feed mice mannans, bacteria that can break them down do best in the mouse gut. The second result, that mutating the PULs might be an advantage in the absence of glycans, wasn’t expected. This result suggests some energetic cost of having active PULs in the absence of usable mannans.

To better mimic the real world, Cuskin and coworkers also looked at the gut bacteria of these mice when they were fed a high bread diet. In this case, the mutant still won out over wild type but not by as much. Instead of being reduced to 10% as was true in the glycan-free diet, the final proportion of wild type bacteria in guts of mice on the high bread diet was 20%. Modest but potentially significant.

We do not have the space to discuss it here, but the authors next dissected the mannan degradation pathway in fine detail. If you are interested please read it over. It is fascinating.

The authors also analyzed 250 human metagenomic samples and found that 62% of them had PULs similar to the ones found in B. thetaiotamicron. So the majority of the people sampled, but not all, had gut microbiota that could deal with the mannans in yeast cell walls.

Human microbiota have adapted to use the energy from the bits of yeast left in bread and alcoholic beverages. Given how little there is, it might be better to think of it as the filth the peasants collected in Monty Python and the Holy Grail instead of manna. Even though it isn’t much, it has given these bacteria a niche that no one else has (or probably wants).

Still, it has allowed them to at least survive. And if these bacteria can one be repurposed as a treatment for disease like Crohn’s disease, thank goodness they adapted to using these mannans.  

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Reference Genome Annotation Update R64.2.1

February 23, 2015

SGD curators periodically update the chromosomal annotations of the S. cerevisiae Reference Genome, which is derived from strain S288C. Last November, the genome annotation was updated for the first time since the release of the major S288C resequencing update in February 2011. Note that the underlying sequence of 16 assembled nuclear chromosomes, plus the mitochondrial genome, remained unchanged in annotation release R64.2.1 (relative to genome sequence release R64.1.1).

The R64.2.1 annotation release included various updates and additions. The annotations of 2 existing proteins changed (GRX3/YDR098C and HOP2/YGL033W), and 1 new ORF (RDT1/YCL054W-A) and 4 RNAs (RME2, RME3, IRT1, ZOD1) were added to the genome annotation. Other additions include 8 nuclear matrix attachment sites, and 8 mitochondrial origins of replication. The coordinates of many autonomously replicating sequences (ARS) were updated, and many new ARS consensus sequences were added. Complete details can be found in the Summary of Chromosome Sequence and Annotation Updates.

Message in a Bubble

February 18, 2015


It looks like fungal and other cells may be sending out messages in tiny vesicles. We can read them using sequencing techniques, but understanding them is quite another matter! Image by Peer Kyle via Wikimedia Commons

If you’re shipwrecked on a desert island, writing a message on a scrap of paper, sealing it in a bottle, and flinging it into the ocean could be your only chance at communication. As the song goes (see below), the message in a bottle is an S.O.S. to the world.

It turns out that cells may do something very similar. But instead of using a bottle, they enclose their messages in membrane-bound bubbles.

Many different mammalian cell types have been seen to form these extracellular vesicles (EVs). In mammalian cells, it’s known that EVs are used for cell-to-cell communication. They contain signals that allow cells to influence their neighbors, both for good (for example, regulating the immune response) and for bad (transmitting viruses or toxic peptides). In most cases these signals aren’t well-characterized, but the EVs may include DNAs and proteins, and they’re rich in RNAs.

Fungi have been found to produce EVs too, but they’ve been much less studied. In a new paper in Scientific Reports, da Silva and colleagues looked at the extracellular vesicles (EVs) produced by four different fungal species, including S. cerevisiae, and found that among other things, the vesicles actually include at least parts of many RNAs, both protein-coding and non-coding.

The scientists decided to look at S. cerevisiae and three species of fungal pathogens that infect humans: Cryptococcus neoformans, Paracoccidioides brasiliensis, and Candida albicans. (S. cerevisiae can be pathogenic too, but isn’t as virulent as any of those species.) They isolated EVs from each and treated the unbroken EVs with RNase to get rid of any RNA that might be contaminating their surfaces.

Then they broke open the vesicles to see what was inside. They found that the EVs contained many small RNAs, most less than 250 nucleotides in length. The scientists used RNA-seq analysis to determine the sequences of these small RNAs, and compared them to the genomic sequences that were already known for these organisms.

Many of the sequences corresponded to noncoding RNAs. For S. cerevisiae, the RNA sequences identified included the mitochondrial small and large ribosomal RNAs, RNA components of RNase enzyme complexes, a variety of small nuclear and small nucleolar RNAs, and tRNAs.

Sequences corresponding to several dozen S. cerevisiae mRNAs were also detected. There wasn’t much rhyme or reason to the kinds of proteins they encoded.  But the set of mRNA fragments didn’t correspond simply to the set of most abundant mRNAs in the cell. So it seemed like the vesicles didn’t just contain random samples of the cytoplasm, but instead had been loaded selectively with particular mRNAs.

This study raises as many questions as it answers, and there is a lot of work to be done before fungal EVs will be understood. The intriguing discovery of RNA in EVs suggests the possibility that the RNAs could influence gene expression in cells that take up the EVs, either by regulating processes like splicing or translation, or even by encoding a protein that gets translated in the recipient cell.

Being able to influence neighboring fungal cells via EVs could be an advantage for fungi in the fierce competition for biological resources. Or perhaps EVs are used to subvert gene expression in host tissues during a fungal infection. Far from being an S.O.S., these messages could be threatening.

These speculations all need much more research. Fungi are an integral part of our world, and we need to pay careful attention to the messages that they send us!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

“Message in a Bottle,” The Police, 1979

Yeast Finds Needles in a Haystack to Combat Malaria

February 11, 2015


The awesome power of yeast genetics makes it straightforward to find the few useful drugs that are buried in a haystack of possibilities. Image by uroburos via Pixabay.com

Finding a needle in a haystack would take a long time and would be very tedious (although it’s been done!) Finding a specific drug to fight malaria by testing the effect of each drug, one at a time, on a purified protein in vitro would be at least as tedious and maybe even more so.

Luckily, we don’t have to sift through a haystack. In a new study in ACS Chemical Biology, Frame and colleagues used our friend S. cerevisiae to find nine drugs out of a collection of more than 64,000 that are promising candidates for stopping the malaria parasite in its tracks. It is as if yeast allowed them to set fire to the haystack and see nine needles gleaming in the ashes.

Malaria is a huge problem for global health. Plasmodium falciparum, the organism that causes malaria, is fast developing resistance to the few effective drugs that we have left.

But P. falciparum has an Achilles heel—it can’t make its own purine nucleotides! Since these are the building blocks of DNA and, obviously, essential for life, if we can keep P. falciparum from being able to take them up, we can kill it. 

P. falciparum imports purines via a major transporter protein, called PfENT1, located in the plasma membrane. So a drug that specifically inhibited this transporter could be a good way to attack the pathogen.

It’s possible to assay the activity of the transporter in vitro, adding different drugs one at a time and seeing which inhibits transport. But doing this for thousands of drugs might make you wish you were looking for a needle in a haystack. Frame and colleagues decided to harness the awesome power of yeast genetics to test a very large set of drugs more quickly.

The toxic nucleoside analog 5-fluorouridine (5-FUrd) is taken into yeast cells by the high-affinity uridine transporter Fui1. It kills normal yeast cells, but fui1 null mutant yeast can survive in the presence of 5-FUrd.

The researchers engineered a yeast codon-optimized version of pfENT1 and expressed it in the mutant, restoring 5-FUrd uptake. The nucleoside analog was again toxic to this strain, and the only way the yeast could survive was if the transporter activity of pfENT1 was inhibited.

This system allowed a simple and powerful screen for pfENT1 inhibitors. The yeast strain expressing pfENT1 would be able to grow in the presence of 5-FUrd only if pfENT1 transporter activity was blocked by the drug that was being tested.

Setting up the screen on a large scale, the scientists were able to test 64,560 compounds. They initially found 171 compounds that allowed the yeast to grow. They narrowed these down to 9 compounds that worked well and belonged to different structural classes of chemicals.

Because of the way the study was designed, it was likely that these compounds allowed yeast to grow because they prevented PfENT1 from pumping the toxic 5-FUrd into the cell. But what if the compounds were actually doing something different, and unexpected? To rule out this possibility, the researchers designed a secondary screen for the 9 top candidate drugs.

They used ade2 mutant yeast, which can’t make their own adenine and need to be fed it in order to survive.  These mutants can make do with the related compound adenosine, but it can’t normally get inside the cell; yeast doesn’t have a transporter that will take it up. However, PfENT1 can transport adenosine, so ade2 mutants can grow on it if they are expressing PfENT1.

With this system, if the candidate drugs are working as expected, they should prevent yeast growth. And that is exactly what the researchers found. This confirmed that the drugs are working because they specifically inhibit PfENT1 and do not allow growth by some other, indirect mechanism.

To be completely sure of the mechanism, the scientists did a direct test. They found that the nine drugs prevented PfENT1-expressing cells from taking up radiolabeled adenosine.

This was all fine, but the ultimate goal of the study was to affect growth of the malaria parasite. So Frame and colleagues tested the drugs on P. falciparum

In the presence of any of the nine drugs, the parasite couldn’t take up adenosine and also failed to grow. This even happened when the parasites were grown in medium containing  much higher purine concentrations than found in human blood.

Even though PfENT1 was targeted by the drugs, all nine of the drugs also killed Pfent1 null mutants. This suggested that the drugs have a secondary target or targets in addition to PfENT1. This could be a real advantage, because it could help prevent the parasites from developing resistance to the drugs.

All nine of these diverse drugs are promising candidates for the treatment of malaria. And the same approach could be used to find chemicals that affect the function of other transporters from various organisms. 

As usual, yeast is providing scientists with streamlined ways to find new treatments for serious human diseases. Instead of tediously rummaging about in a haystack, yeast lets us quickly and easily find the needles we need. 

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

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