Excludons: How they affect listeria, and possibly your lunch

By: Rachel Salemi

A dream house for L. monocytogenes. Source

Even if you’ve never been formally introduced to Listeria monocytogenes, you’ve likely met this bacterium before.  Enjoying the comforts of deli meats and your digestive tract, L. monocytogenes is an opportunistic pathogen, making it a formidable adversary for the immunocompromised. According to the FDA, L. monocytogenes causes 2500 cases of listeria a year, and although it is uncommon, the disease has a high mortality rate compared to other foodborne illnesses.

A clump of L. monocytogenes. Source

Interestingly, L. monocytogenes is the only pathogenic bacterium in the Listeria genus. For this reason, L. monocytogenes is often compared to its non-pathogenic relative, Listeria innocua. Since the two are so closely related, and live in similar environments, their genomes are frequently looked at side-by-side to determine which genes make L. monocytogenes pathogenic, and L. innocua harmless.

A depiction of DNA (left) compared to

folded RNA (right). Source

When researchers study organisms, they generally focus on the coding RNA. Coding RNA, also known as mRNA, is a small copy of DNA that provides instructions for building proteins. Recently, however, many types of non-coding RNA have been discovered, and although they do not act as blueprints for proteins, non-coding RNAs are able to regulate gene expression. Oftentimes, the role of mRNA in cells is overemphasized, while the importance of non-coding RNA is minimized. Taking this into consideration, Wurtzel et al. (2012) compared the non-coding RNA of  L. monocytogenes to that of L. innocua in order to figure out what makes one species pathogenic and the other benign. In doing so, they discovered a new form of gene regulation in bacteria, dubbed “excludons,” which is what I will be focusing on here.

A cartoon depiction of how asRNA functions. Source

Before we move onto the results of the study, let’s take a detour and explore the types of non-coding RNA types relevant to the paper. Small RNA, or sRNA, are strings of RNA that are much shorter than the average mRNA and involved in gene regulation, either by creating a folded, 3-D form or by binding to mRNA. Anti-sense RNA (asRNA) is able to base pair (bind) to a specific mRNA, preventing the translation of that mRNA. Wurtzle et al., however, focused mainly on long antisense RNA (lasRNA). As the name implies, these RNA function similarly to asRNA but are much longer. Although non-coding RNAs were discovered about 30 years ago, their functions are still poorly understood.

So now for the interesting stuff. In their study, Wurtzel et al. did an impressive amount of data collecting, all of which you can view using the website database they created. To make this genomic map, they identified each transcription start site (TSS). Using TSSs accounted for both non-coding and coding RNAs because regardless of function, RNA is synthesized through transcription. They created maps for both L. monocytogenes and L. innocua. Using this method, they discovered 33 new sRNAs (out of 113 total sRNAs in L. monocytogenes), and 53 novel asRNAs (out of the 70 total asRNAs in L. monocytogenes).

Although they discovered an incredible number of novel non-coding RNAs, Wurtzel et al. focused on lasRNA, and spent a hefty amount of their paper delving into the expression pattern of an lasRNA called anti0605. The "anti" refers to its role as an asRNA and 0605 refers to lmo0605, the gene this RNA regulates. Below is a figure from the paper:

Wutzel et al. 2012, Figure 4a

Admittedly, this figure looks complicated, so let’s break it down. Don’t be intimidated by the nomenclature. Lmo0605, lmo 0606, lmo0607, and lmo0608 are genes that create transmembrane protein channels, but understanding how these channels work is not necessary for understanding excludons. The important part of this figure is that anti0605, lmo0606, lmo0607, and lmo0608 are pointing in the same direction, while lmo0605 is pointed in the opposite direction. This means that the base pairs of lmo0605 are complementary to those of anti0605 because they were transcribed in opposite directions. So, anti0605 blocks the function of lmo0605 as an mRNA because they are complementary and able to bind to one another. I have included a figure below to help make this point more recognizable:

Above is a cartoon of 2 separate mRNA strands: anti0605 and lmo0605. The excludon/lasRNA anti0605 binds to lmo0605, which blocks ribosomes from binding and thus prevents translation from occurring. The section of anti0605 that is not bound to lmo0605 then acts as an mRNA for 3 genes: lmo0606, lmo0607, lmo0608.

At this point, we haven’t talked about any actual in vivo evidence that anti0605 represses lmo0605 expression while increasing the expression of the other three genes. So, as you might imagine, this is what Wurtzle et al. set out to do next.

Wutzel et al. 2012, Figure 4c

Above is another figure from the paper, though I have added color to make the graph more clear. In this experiment, Wurtzel et al. repressed the transcription of anti0605 and then recorded whether or not the genes previously mentioned were upregulated or downregulated. From this graph, we can see that compared to wild type L. monocytogenes, this mutant had 10 times more copies of lmo0605. Additionally, the amount of lmo0606, lmo0607, and lmo0608 decreased almost tenfold. This data supports the idea that anti0605 represses lmo0605 while activating lmo0606, lmo0607, and lmo0608, adding to the evidence for excludons.

Now, this is all well and good, but why should we care? I mentioned in the last experiment that anti0605 was repressed, but did not explain how. Anti0605 is activated by a protein called σB, which is a sigma factor, and Wurtzel et al. turned off anti0605 by deleting σB from the L. monocytogenes’s genome. If you don’t know what a sigma factor is, that’s ok—just think of it as a special bacterial gene activator. When σB is active in L. monocytogenes it “turns on” certain genes that would not be “on” otherwise. Not only does σB activate anti0605, but this activator has also been strongly correlated with the pathogenic state of  L. monocytogenes (source). What this means is that genes activated by σB (like anti0605) most likely have something to do with making L. monocytogenes pathogenic.

Wurtzel et al. 2012 Figure 4b

In the next figure of the Wurtzle et al. 2012 paper, they compare anti0605 levels in L. monocytogenes, L. innocua, and L. monocytogenes with σB turned off. In the experiment, they looked at these RNA levels during stationary (stat) and exponential (exp) growth, and found that anti0605 was most active in wild type L. monocytogenes during the stationary phase. This not only shows that anti0605 is present when the pathogenic sigma factor, σB, is active, but also that this gene is much more active in L. monocytogenes than L. innocua.

Let’s review what we’ve gone over. Wurtzel et al. created a genomic map of the transcription start sites. This lead them to the discovery of many non-coding RNAs, including 7 lasRNAs. One of these non-coding RNAs, anti0605, was capable of blocking lmo0605 expression while increasing the expression of lmo0606, lmo0607, and lmo0608. From this data, they came to the conclusion that anti0605 is an excludon, a type of RNA that acts as both asRNA and mRNA. Furthermore, anti0605 was implicated as a gene involved in pathogenicity due to its activation by σB.

In the future I would love to see more studies confirming the existence of excludons, possibly by expressing these genes in a different bacterium (perhaps E. coli) and confirming that the expression pattern is the same, even out of context. Another interesting route would be to specifically block the translation of anti0605, rather than knocking out sigB, and see if L. monocytogenes is still capable of infecting a host. Additionally, could the expression anti0605 and other lasRNAs in L. innocua make it capable of infecting a host?

The discovery of anti0605 and other excludons could be a huge breakthrough in understand the pathogenicity of L. monocytogenes. Hopefully this article has convinced you of the importance of non-coding RNAs and that they are crucial to understanding the full picture of gene expression; The quality of your ham sandwich may depend on it.


References

Kazmierczak, M. (2003). Listeria monocytogenes σB Regulates Stress Response and Virulence Functions. J Bacteriol, 185(19), 5722-5734.

Wurtzel, O., Sesto, N., Mellin, J., Karunker, I., Edelheit, S., Bécavin, C., ... Sorek, R. (2012). Comparative transcriptomics of pathogenic and non-pathogenic Listeria species. Molecular Systems Biology.