#Microtwjc: The Evolution of Virulence

A long time ago, a bacterium noticed the odd behaviour of its cousins. It had noticed that they had formed a group, and were spending a lot of time together. An unsettling amount of time together. The bacterium's friends told it to not worry. It is perfectly fine for related bacteria to stick together, to live in colonies of individuals.

The bacterium told its friends how its cousins behaviour was different. The cousins had forfeited the life of independence, and had reduced themselves to mere parts of a greater whole, a multicellular organism. it was disgusting, it was socialism. It had to be stopped.

Bit by bit, it rallied other bacteria to it's cause, and together they came up with a plan. They would become virulent. They would evolve the resources to fight their multicellular competitors, invade their cells and feast on their nutrients. So they went to Professor Oak, who happened to have just the right thing for them...

Okay, maybe my "Pokemon Virus Vendetta" theory of bacterial evolution is a bit off the mark. In my defence, a microbiologist looking at bacterial evolution doesn't have any fossils to help them. And even if they did, they won't really tell them anything other than the shape of the bacteria, which has no real bearing on whether a bacterium is virulent or not.

Evolutionary bacteriology is primarily based on using the bacteria's genetic code. We can look at bacteria that inhabit different environments, and look at the similarities and differences between them and formulate ideas about bacterial evolution.

So there are questions as to how bacteria evolved to live inside animals, and more importantly, how they learned how to survive the immune system. One of the great threats to bacteria are phagocytes, which roam the body gobbling up bacteria and digesting them. Yet some bacteria have not only evolved ways to prevent themselves being eaten, but to survive, even thrive, within phagocytes, such as Mycobacterium tuberculosis.  One of the first steps of infection with this pathogen actually requires a phagocyte to consume it, and then it grows and proliferates within this phagocyte in order to eventually cause disease.

M. tuberculosis  possesses many genes which help its survival within the host, and the set which is studied in this paper is known as "Mammalian Cell Entry" genes, so called because when  E.coli were given these genes, it enabled them to invade mammalian cells*. In M. tuberculosis it is thought to in some way control its survival within phagocytes.

So when the "Mammalian Cell Entry" genes were found in a soil microbe, Streptomyces coelicolor , a question is raised. Why would S. coelicolor need these genes ? The answer may tell us something interesting about how bacteria evolved to attack us and cause disease.

Does Streptomyces actually use these genes ?

Just because it's in your genome doesn't mean you use it. If the genes have no function in S. coelicolor then we can only speculate on what function their ancestors had for these genes. So they measured the presence of mRNA within these cells at the different stages of Streptomyces growth, and checked its presence on different media.

This is shown on Figure 2 A, with the white bands indicating the presence of mRNA.

 hrdB is a house keeping gene which is always active, and mceA is one of the "mammalian cell entry" genes. mtrA controls whether mceA is switched on or not.

So from figure 2A, we can see that mceA is switched on when grown in YEME medium, but not in MS medium, and mtrA is switched on in this medium. This suggested that the medium's effect on mceA works independently of mtrA. 

In Figure 2 B, we are shown what happens if S.coelicolor grown in cholesterol compared to YEME. A mutant with no functional mtrA was also grown as well. This shows that mceA is not active when cholesterol is present within the medium, and it is not present when mtrA  is removed from the medium as well. This indicates that mtrA is needed for the expression of mceA, except when the bacterium is grown in MS medium, in which case not even mtrA will not prevent mceA being repressed.

So at least we know that the mammalian cell entry genes are doing something in Streptomyces, but what are they doing ?

How well does Streptomyces do without the Mammalian Cell Entry (mce) genes?

One way of working out what a gene does is getting rid of it, and watching what the bacterium now cannot do without those genes. 

Figure 3 looks at a number of things that change when Mammalian Cell Entry (mce) genes are removed.

They took a lawn of streptomyces, and then they added a drop of SDS into the middle of it. SDS is a surfactant, that attacks bacterial cell membranes kills off the streptomyces, which produces a dark pigment as if to protest***. As you can see from the image, the darker patches are larger when 20% SDS is applied compared to the 10%.

The mce mutation appears to give the two colonies we are shown an edge when growing with lysozyme, a molecule which acts to break down cell walls. Whilst the dark patches are still there, you can see some bits of white poking through, showing that some of the cells have survived, although they are still injured***

Electron microscopy revealed that mce mutants have more "wrinkled" cells, that were on average shorter than the normal cells from a sample of a hundred cells.

So what is the significance of these effects?

What happens if we feed our bacteria to an amoeba ?

An Amoeba is the natural predator of bacteria like S. coelicolor in its natural habitat within the soil. So what happens when we feed these bacteria to an amoeba, and what effect does the mutant have on this ?

In Figure 4, this is what they do.

It turns out that the mce mutant kills the amoeba.  If you look under a microscope in 1 A , the bacteria germinate and grow within the amoeba after they are eaten, and kill the amoeba in 24 hours.

In the next experiment, they made a "lawn" of S. coelicolor grow on the surface of an agar plate, before adding amoeba to the centre of the plate. When the bacteria are eaten, clear patches form on the surface of the plate. So we can see that the deletion of mce, and the Mtr gene that promotes its growth prevents the bacteria getting eaten. If you try to correct the mutation by adding a plasmid (pLS006) with functioning mce, then you find that the bacteria once again get eaten by the amoebae.

So mce expression enables bacteria to be eaten by amoebae, and when its gone, the amobae stop being a threat. 

So if all mce  does is allow bacteria to get eaten, then what is it's point ?

What effect does mce  mutation have on root colonisation ?

S. coelicolor lives on roots, and its possible that mce plays a part in root colonisation. And it turns out that when mce is deleted, bad things happen to the plants it colonises, which is shown in 5A. On the microscopic pictures we are shown, we can see that there are less bacteria on  the roots when the mce is knocked out.

In 5B, we can see that there are less of the mce mutant on these plants compared to S. coelicolor with functioning mce. 

So now we must consider what this data has told us, and then what we can deduce from it.

Summary

This paper characterises the function of a gene system in S. coelicolor which is somewhat related to an important gene in Mycobacterium tuberculosis. So what have we found out about this gene in this paper ?

When mce is active :

• It decreases resistance to Sodium dodecyl sulphate (SDS)***
• It does not prevent resistance to lysozyme.
• It makes nice and healthy looking cells.
• It does not prevent amoebae eating it
• It allows growth of the bacteria on roots

When mce is inactive:

• It increases resistance to SDS***
• In increases resistance to lysozyme, which is lucky because lysozyme is an important digestive enzyme used by amoebae to eat their prey.
• It also kills off amoebae when they attempt to eat S. coelicolor.
• It prevents plants growing very well if they get into the roots
• It also doesn't allow S. coelicolor to grow very well either on the surface of roots.

What does this tell us about the evolution of virulence ?

On its own, this data only really tells us about S. coelicolor,  and what the mce gene does in this bacterium. But when we compare it to the action of the mce in M. tuberculosis, we can start thinking about the different ways these genes have evolved to match the life cycles of their respective bacteria.

There are multiple mce's in M.tuberculosis which have slightly different functions, but mostly they play roles in surviving within macrophages, primarily in reducing the expression of cytokines by macrophages after they've been infected with the bacterium, and its general survival. It is very difficult to compare the functions of the mce's in Mycobacterium to Streptomyces because of this.

But this isn't the point of the paper. They say in the paper that Streptomyces split off from the ancestor of Mycobacteria at around 440 million years ago**. Based on this, we assume that the common ancestor of these two bacteria probably expressed some variant of the mce gene. So based on this, we can try to deduce what this ancient bacteria from the Silurian era used these genes for. This was around the time that plants were colonising the land. The authors suggest that these genes evolved to allow soil bacteria to colonize the surface of plants, and allow it to control when it expresses the genes that allow it to survive being eaten by amoebae.

In many ways, phagocytes behave like amoebae. Like amoebae, phagocytes use lysozymes to digest their prey. So soil microbes which have methods of resisting amoebae come ready made with methods of resisting phagocytes. 

So the idea is that a long long time ago, these soil microbes were minding their own business, when they end up in the body of a mammal. The mammals immune system immediately recognises them as foreign, and sends phagocytes to destroy them. The bacteria, assuming that they are being eaten by amoebae, shut down their mce system to resist being eaten, and end up causing a disease in the mammal.

 Do you see the irony here ? 

The presence of these macrophages actually make a host more likely to get a disease after ingesting these microbes.

Why does any of this matter ?

So let's imagine a world where we fully eradicate tuberculosis, and other diseases. The primary threat of disease comes from the fat tail of emerging pathogens which can now exploit the empty niches left by the other bacteria. What was originally just one bacterium causing a disease is now a hundred bacteria causing a disease.

Understanding the evolution of virulence allows us to get an idea of where possible threats can come from.

There are Mycobacteria, Legionella and Chlamydia like bacteria which currently reside in our environment and attack amoebae, which can cause pneumonia in humans if they end up in the lung. If we understand the evolutionary process which allows soil bacterium to cause disease in humans, perhaps we can devise strategies to prevent it from happening.

This paper was up for discussion by the microbiology twitter journal club this Tuesday (2nd April) at 8pm BST.

The transcript of that discussion can be found here 

Reference

Clark L.C., Seipke R.F., Prieto P., Willemse J., van Wezel G.P., Hutchings M.I. & Hoskisson P.A. (2013). Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence., Scientific reports, PMID: 23346366

*Now I should note here that it is unclear at this time how the "Mammalian Cell Entry" system actually works, and whether it actually serves any function in helping mammalian cell entry, or whether it simply allows bacteria to survive better after naturally being eaten by cells.

**I couldn't find the reference for this observation. Well, they do give a reference for fungal and plant evolution occurring around 400 million years ago, but streptomyces is a bacterium, and I'm not sure how this would apply.

*** In the original article, I assumed that the black patches were the colonies themselves, rather than zones of dead bacteria, when in actuality, they represent dead bacteria, and I thought that the SDS had the reverse effect to what it really does. Had I known that S. coelicolor  painted itself black upon death, I would have drawn a picture of it dressed as a goth. Hat tip to @clonemanager and for explaining this to me.