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Department of Chemical Engineering and Biotechnology

 
Bacterial cells beginning to produce spores

Researchers from our Molecular Microbiology group have identified two new enzymes responsible for breaking open bacterial spores when they germinate.

Certain kinds of bacteria are able to form spores when they encounter life-threatening conditions, such as lack of food. Rather than simply dying out, these bacteria produce very tough survival structures – spores – that can remain dormant in hostile conditions for thousands of years.

The spores are constantly sensing their environment and as soon as conditions are conducive to growth again, they germinate, turning back into the bacteria that produced them. Examples of common spore-forming bacteria are Bacillus anthracis, the causative agent of anthrax, and Clostridium difficile, which causes the highly contagious infection that plagues many hospitals.

The spores are incredibly strong structures, resistant to heat and chemicals that would kill normal bacteria cells. Dr Graham Christie, who leads our Molecular Microbiology group, wants to gain a greater understanding of the structures of spores and their germination, with a view to developing methods to inactivate them.

 

“Just after the 9/11 terrorist attacks a bunch of letters were posted – the anthrax letters in the States – and my old boss in biotechnology got a grant to develop sensors to detect anthrax spores,” says Dr Christie. “They were looking for rapid tests to establish whether the white powder being posted or left around government buildings was just flour or was anthrax spores.

“I realised when trying to make the sensors that they would work but only if you germinated the spores. It's very difficult to make a sensor for an inert particle, but if it's doing something, you can detect the by-products and so that got me into looking at how the spores germinate.

“To understand that you need to understand the structural biology of the spores: looking at which proteins are important, can we crystallise them and can we solve their molecular structures.”

New enzymes challenge current understanding

The bacterial DNA present in spores is protected by a coat, which is made up of many layers of different proteins. Christie describes the structure as similar to that of a gobstopper, with discrete shells of different proteins layered around each other.

When a spore germinates, it has to break down this very tough protective coating. Spores typically use a specific type of enzyme, known as a cortex lytic enzyme, to break down certain components of these structures. This process was thought to be well understood, but the latest paper from Christie’s group has identified two new enzymes that can break down the spore shells. 

“It was always thought there were only two enzymes involved,” says Christie, “and that if you knocked those enzymes out, the spore could trigger germination but then be trapped because it can't degrade the spore structures. We show in this paper, with a bit of molecular trickery, that other enzymes can compensate.

“People have been developing compounds to block the known enzymes thinking if we can inhibit those, we can inactivate spores. And what this work shows is that may not be the case.”

Christie’s group use genetic engineering to create spores with or without various enzymes and then try to germinate them. Much of this work was carried out by Dr Bahja Al-Riyami, former PhD student in Christie’s lab and now Assistant Professor at Sultan Qaboos University, Oman, in the Department of Biology.

 

By creating these so-called mutant spores with different combinations of enzymes, Dr Al-Riyami and the team were able to identify two new lytic enzymes that enabled the spores to break down their coating, even when the known lytic enzymes weren’t present.

Using a technique called ellipsoidal localisation microscopy, the researchers were also able to identify the location of these new enzymes in the spore structure, sitting in a region of the coat with other cortex lytic enzymes.

Collaboration the key

The discovery is the result of a longstanding collaboration between Christie’s group and Dr Eric Rees, an applied mathematician in the department who works on applying computational inference methods to imaging of biological and material systems.

After a fortuitous meeting between the two researchers several years ago, Dr Rees developed the technique of ellipsoidal localisation microscopy to help Christie’s group in their initial mission to try to resolve the exact protein structure of spore coats.

“I met Eric at an infectious diseases seminar,” says Christie. “I was talking about the layers of proteins [around the spore] and how we'd like to understand the layer order, but this was beyond our capability because they're so close together that you can't discern the difference on a standard microscope – they all look the same.

“Eric said that he thought he could develop a method that would allow us to elucidate the different layers. I still think the best thing that's come out of my group is via Eric.”

“I was quite optimistic about this technique,” says Rees. “I'm interested in inference or essentially, how do you learn from data? One way is that you can take the information that's in your image data and combine that with some other information, for example with spores you know they have a spheroidal geometry. If you can find a mathematical way of adding up those two bits of information, you can end up with a more precise measurement than you get from just looking directly at the data.

“So that's how we can take intrinsically limited fluorescence microscopy data and get precise enough results to say where the protein layers are to within 10 nm. I have no idea what it is in microbiology that you could learn by having an exact map of the spore coat, but I have enough imagination to think it can only be helpful.”

“This is the beauty of this department,” adds Christie, “We would never have established this in our own right and I doubt Eric would have been working on spore coats. It’s this fortuitous coming together in an environment that's unique in this university. This is a genuinely great example of cross-disciplinary collaboration in this department.”

Fighting spore-formers

With a better understanding of the germination process of spores, Christie’s group are looking to develop new compounds that can counter their harmful effects.

“The way you control spores is you can either inactivate them – keep them in a dormant state by throwing inhibitors at them – or if you trigger germination then the spore loses all of its resistant properties. Things that would kill normal bacterial cells have no impact on spores, but if you can germinate them artificially, you can then follow this up with a mild heat treatment or a mild chemical which will kill the bacteria.”

The current treatment for anthrax spore exposure is a 60-day course of antibiotics. The antibiotics won’t harm the spores but if the spores germinate, the antibiotics already in your blood will kill the germinated spores before they can harm you. However, not all spores will germinate at the same time, and if you finish your 60-day course before all the spores have germinated, the ones that germinate later can be fatal.

Christie’s team are looking to develop methods to stimulate germination of spores so that all of the resulting bacteria can be killed.

This method could also reduce the spread of C.difficile, a highly contagious bacterial infection that commonly affects people recently treated with antibiotics. The infection can spread by the spores produced by the bacteria and is a huge problem in hospitals.

“The spores get everywhere in hospitals, they're on the bed linen, they're on the walls…” says Christie. “Hospital bed linen is washed at 80 degrees but that's not even tickling the spores. If you could spray something onto the sheets to germinate the spores, they'd quickly die in those conditions.

“We'd like to develop these non-destructive methods of inactivating spores and the way we do that is by knowing more about the spore structure.

“We've only scratched the surface in terms of mapping the coat. We'd like to use Eric's technique to map the coat of Clostridium botulinum, the causative agent of botulism. Nothing is known of the composition of its spore coat.”

Christie’s group are also investigating using non-harmful bacterial spores as drug delivery agents. You can find out more about his research on our website. 

Read the full paper, published in FEMS Microbiology Letters.

Sidebar Items: 

July paper of the month

Read the full paper, which was selected as the Editor’s Choice article for Issue 366- 12 of FEMS Microbiology Letters: Riyami BA, Ghosh A, Rees EJ, Christie G. 2019. Novel cortex lytic enzymes in Bacillus megaterium QM B1551 spores. FEMS Microbiol Lett 366(12)

July paper of the month

Read the full paper, which was selected as the Editor’s Choice article for Issue 366- 12 of FEMS Microbiology Letters: Riyami BA, Ghosh A, Rees EJ, Christie G. 2019. Novel cortex lytic enzymes in Bacillus megaterium QM B1551 spores. FEMS Microbiol Lett 366(12)

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