A Tuberculosis Enzyme Decapitates Vital Energy Molecules to Kill Cells

If you cant defeat your enemies by force, defeat them with subterfuge. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, lives by this mantra. While other disease-causing bacteria mount an all-out assault on the body, the tuberculosis bacteria lay low, hide, and slowly kill us from the inside out. M. tuberculosis is a master of stealth and deception. Like the Greeks entering Troy in a wooden horse, it hides from the immune system within our own cells – often the very same cells that guard us from bacterial infections. M. tuberculosis is a treacherous enemy.

In order to infect us, many bacteria use protein toxins to kill or manipulate our cells. The cholera and diphtheria pathogens are famous for producing toxins that attack our tissues. These toxins are fired as cannon blasts that break into our cells, and chemically change our own molecules to drive a toxic effect. Until recently, we thought that M. tuberculosis didn’t have any of these toxins. We thought that it accomplished its stealthy invasion through other means. It turns out, we were wrong: M. tuberculosis has a toxin, but it’s not a cannon, it’s an assassin’s blade.

TNT, a deadly enzyme produced by M. tuberculosis

Last year, researchers at the University of Alabama at Birmingham identified a toxin from M. tuberculosis that kills immune cells. They named the enzyme tuberculosis necrotizing toxin, or TNT, because it induces necrosis, or cell death, in the target immune cells. In a recent follow-up, they have now demonstrated exactly why the toxin is so deadly.

The TNT toxin is particularly nefarious. Rather than the upfront assault of the cholera and diphtheria toxins, it kills its host cells from the inside out. TNT breaks down the cell’s stores of NAD⁺, or nicotinamide adenine dinucleotide. This molecule is an important energy carrier molecule*, used by all life forms from the tiniest bacterium to the giant sequoia. Our cells use NAD⁺ to shuttle energy between biochemical processes. NAD⁺ harvests energy from the breakdown of glucose and other molecules, and passes that energy to other systems that drive the processes of life. If you remove a cell’s NAD⁺, the cell will die. This makes NAD⁺ a convenient target for M. tuberculosis. Destroy the NAD⁺, destroy the cell.

TNT Enzyme Decapitates NAD+. Artist's Interpretation. — TNT Enzyme Decapitates NAD+. Artist’s Interpretation.

The tuberculosis bacterium uses the TNT toxin to do exactly this. It acts the assassin’s blade, selectively destroying all of the NAD⁺ in the host cell. The enzyme “decapitates” NAD⁺ by breaking a critical bond, separating the head from the body of the molecule. Without their stores of NAD⁺, the immune cells that host the tuberculosis bacteria die, releasing them to spread to other cells. Triggering necrotic cell death also bypasses more orderly means of cell death that would allow the immune cell to sacrifice itself and quarantine the mycobacteria.

Stealth attack

In tuberculosis, some of the worst symptoms aren’t mediated by the bacteria themselves, but the immune system’s inappropriate response to the bacterium. This is part of why it wasn’t always clear that M. tuberculosis would need a toxin at all. For the most part, the M. tuberculosis lies low, waiting for its chance to strike. When it does strike, it appears to use TNT to do so in a selective and controlled way. Like the Greeks crossing the walls of Troy, the TNT enzyme has to be helped across a membrane to the host cells. Mycobateria live within compartments in the cells they infect, but in order to disrupt the metabolism of those cells, the toxin needs to reach the cytoplasm. TNT doesn’t contain any functions to get it into the cytoplasm by itself, so it has to be helped, by an export complex called ESX–1.

This is different than the cannonball toxins of cholera and diphtheria. Those toxins have their own means of forcing their way into a target cell. The TNT enzyme is actually a small enzyme, which means it doesn’t carry any parts to help cross into the host cell by itself. The researchers identified that the ESX–1 system is needed to get into the cytoplasm, although there is still a huge amount unknown about this process. This is a very interesting area of future study, because moving TNT into the cell probably involves an important switch in the bacteria’s strategy. M. tuberculosis switches from lying silently in wait, to mounting its sneak attack by cover of darkness.

Protection from a double-edged sword

There is an interesting consideration for any bacterium that makes a toxin, especially one that targets a ubiquitous molecule like NAD⁺. How does M. tuberculosis avoid killing itself? The bacterium synthesizes the toxin inside its own cells, but NAD⁺ is important for all life, including M. tuberculosis itself. How does the bacterium keep the TNT enzyme from destroying its own NAD⁺? Well, if this toxin is the assassin’s sword, a second protein, IFT (immunity factor for TNT), is the scabbard.

When the TNT enzyme is made in the mycobacterium, it is secreted, but if it somehow remains in the cell, it is bound by a molecule of IFT. This IFT blocks the part of the TNT enzyme that interacts with NAD⁺, inhibiting the enzyme. The researchers determined the structure of TNT and IFT together, and showed convincingly how the IFT would completely block TNT activity by obstructing the interaction of TNT and NAD⁺.

Outside of the bacterium, TNT and IFT are separated, and the toxin is active. Inside the cell, IFT acts like the sheathe that protects a swordsman from their own blade. It’s a cleverly-evolved means for M. tuberculosis to protect itself from its own weapon.

Every enzyme is a unique snowflake

The TNT enzyme is a great example of a reductionism-breaker. In a lot of molecular biology, if you want to probe an enzyme’s activity, you create site-directed mutants. In these mutants, the functional amino acids that interact with the target of the enzyme are replaced with non-functional amino acids. In this way you can test, in a straightforward way, what role the individual amino acids play. Reduce the activity of an enzyme to its constituent amino acids.

If you replace a functionally critical amino acid, you expect to get a non-functional protein. In TNT, removing the amino acid most important in related enzymes only reduced TNT’s activity by half. This isn’t much by molecular biology standards. This highlights that even though the same residue is present in this enzyme as related toxins, TNT appears to play by different rules than the rest. This actually is a fairly common story when studying enzymes, especially ones that are involved in bacterial disease**. A lot more work is needed to map out the mechanism that this enzyme uses, but this is a great reminder not to assume similar enzymes work exactly the same.

Why do I like this paper so much?

I really, really like this paper. Although full disclosure, I have a soft spot for bacterial toxins after doing an undergraduate placement in a toxin lab. There are a couple more points I’ll mention:

The researchers discovered the NAD⁺-breaking activity of the enzyme through some extremely clever detective work. They observed that when they produced the toxin in standard lab E. coli bacteria, the E. coli died. This happens occasionally, even with proteins that aren’t toxins. The next step they took was key. They sequenced the RNA of the expression bacteria, and found that the E. coli had up-regulated genes responsible for the synthesis of NAD⁺.

Some other bacterial toxins break down NAD⁺, notably another toxin produced by S. pyogenes. The researchers tested if TNT also acted on NAD⁺, and found the enzyme carried out the same reaction. I think this is a great case of critically evaluating your lab materials, and sharp thinking about the systems you work with. In this case troubleshooting lab problems appears to have turned into a huge discovery.

This research also identifies the first known bacterial toxin from M. tuberculosis. This bacterium is notable within microbiology because it tends to always play by different rules, growing slowly, using distinct chemistry and metabolism for everything it does. It would be easy to believe it fights the immune system in different ways, as well. This is partly true, as the TNT toxin is quite different from any other known toxin (it couldn’t be identified by comparisons to known toxins). But it seems M. tuberculosis uses a familiar weapon, just in an unfamiliar way, fitting its stealthy mode of infection.

Lastly, TNT and IFT are an interesting case study of the identification of unknown gene functions. The M. tuberculosis genome was sequenced in 1998. The NAD⁺-destroying function of mycobacteria was seen in the 1960s, as was the inhibitor function of IFT. However, without the appropriate understanding, no one could connect these functions to the genes until now. While modern sequencing technologies help us compile long lists of genes, we still need smart, careful experiments like this study to work out just what these genes do. It’s a great example of careful, inquiry-driven research in the post-genomic era.

M. tuberculosis uses the TNT toxin to decapitate a molecule that our immune cells need to live. A stealth murder weapon wielded by a treacherous infiltrator. This paper is a great piece of work illustrating how a nasty pathogen manages to sneak past our immune defences and make us sick. I’m very interested to see what we learn about this system in the future. I think this is an excellent piece of work, the UAB researchers should be proud.

Update 2015.09.17 11:21: Initially, I had assumed that all TNT will interact with an IFT before it is exported. I have learned this probably isn’t true and most TNT is exported without ever seeing an IFT.

Citation:

Sun, J., Siroy, A., Lokareddy, R., Speer, A., Doornbos, K., Cingolani, G., & Niederweis, M. (2015). The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD Nature Structural & Molecular Biology, 22 (9), 672–678 DOI: 10.1038/nsmb.3064

* _{Technically, NAD⁺ is a redox shuttle, but that’s a discussion for another time.}
** _{The virulence proteins of pathogens tend to change more rapidly due to the ongoing evolutionary arms race between the pathogen and the host.}

A Blog About Biochemistry

Hi! Welcome!

If you’re reading this, you’ve found your way to my tiny corner of the web, where I will be writing on a regular basis about the things that excite and challenge me in science (with regular digressions, as mood strikes). I hope you might join me for the ride. You can also find me elsewhere @superhelical.

There’s one important thing to know about me at the start: I’m a huge nerd about molecules. While many students break into a cold sweat at the mention of the term “S_N2 reaction”, I’ve always enjoyed organic chemistry. It can be like playing with LEGO. Except the LEGO is microscopic, you assemble the LEGO by shaking it vigorously in a flask, and some types of LEGO can kill you.

benzene_lego_tabs_03 — Making a LEGO benzene that is scientifically consistent with good design is surprisingly challenging

It is fascinating to see the world through a lens of molecules and their interactions. I love thinking about the molecules that drive our lives and the environment around us. Some proteins link together when you bake a loaf of bread. Coral deposit minerals to build their skeleton. Dye molecules are linked to colour a cotton t-shirt. Even mundane things like the smell of the air after it rains or the way a hot iron straightens hair have molecular explanations. There’s a hidden richness of molecular phenomena around us. Molecules shape the world.

You can expect a heavy emphasis on protein biochemistry in this blog. Topics I find most interesting often involve biological molecules. In particular, the structure and dynamics of molecules like proteins and nucleic acids. It’s a shame that there’s a huge amount of published work on these bio-molecules that doesn’t receive much attention. I’m going to dive in and highlight some of this work, explain why I find it exciting. Hopefully I can help you to enjoy thinking about biochemistry, too.

What Exactly is Biochemistry?

So, to kick off, I’d like to start with a surprisingly difficult thing to do: define what “biochemistry” actually is.

This is a specific case of a more general question: What is any scientific discipline? Fields are labels that divide researchers and techniques into categories, even though the borders of these fields always remain fuzzy. More than techniques and objects of study, the defining features of a field is usually cultural or philosophical. I’ve learned when speaking to physicists, one of the first questions to ask is “experimental or theoretical?” because the most fundamental divide in that physics lies between theory and practice. Similarly, field biologists are a completely different breed than those who do lab work. As you get more specific, the distinctions get smaller, but you’d be very surprised how differently an immunologist and a microbiologist view the world.

When we look at the fields that study molecules of life, it doesn’t help that the names of many related disciplines are so damn confusing. Chemical Biology, Biochemistry, Molecular Biology, and Biological Chemistry all mean roughly the same thing in the dictionary. However, those of us working in these fields know that they refer to different communities that have distinct organizational cultures, and that target different types of problems using different methods and technologies.

I’ve cast around a little to try to get an idea of how some working scientists define “biochemistry” specifically, and recieved some interesting opinions from people in various fields:

It’s the necessary wet-lab work required to validate a computational model (Bioinformaticians)
It’s ELISAs and Western Blots (Immunologists)
It’s the damn prerequisite that makes you to memorize amino acids (Pre-Meds)
It’s chemistry without enough flammable solvents (Synthetic Chemists)
It stinks (Physicists)

Even people who work in closely aligned fields can’t agree on what biochemistry is:

It’s a broad umbrella, including sub-fields like chemical biology, structural biology, cell biology and systems biology
It’s an anachronism, and has been absorbed into “modern” fields like chemical biology, structural biology, cell biology and systems biology
It’s a defined set of techniques, used in fields like chemical biology, structural biology, cell biology and systems biology
It’s a historical administrative handle used to divide group chemical biologists, structural biologists, cell biologists and systems biologists into a single department

The definition I sympathised with the most is a little vague, but speaks to a simple truth:

It’s whatever you get when you look at biology with a chemical understanding

Biochemistry seeks to explain biology at the chemical scale. Start with simple building blocks like carbon, oxygen, hydrogen and nitrogen. Sprinkle in the occasional phosphorus or sulfur. How can something as complex as a tree, cat, or human emerge from these materials? Or even a single, replicating bacterial cell? There are biochemical questions to answer at every level of complexity, from shapes of sugars and amino acids, all the way to the muscle tissues of a sperm whale.

Biochemistry seeks to explain how the complexity of life can emerge from simple atoms. To do this, it takes chemical principles like thermodynamics, biological principles like natural selection, and some principles from developed from scratch, like models that explain the behaviour of enzymes. Biochemistry is an amalgam of principles from various fields, pulled together and applied toward the molecules of life.

Is the Field of Biochemistry Going Extinct?

Some might say biochemistry stopped being innovative decades ago and other fields have moved on and left the biochemists behind. That all the hard problems in biochemistry have been solved, that there’s nothing new to find.

I disagree, but it is time for a pivot.

There is one thing I might complain about the the state of biochemistry in 2015. Work in the field is often descriptive, reporting on the world but not gaining a lot of insight from it. This is a real shame, because there’s a vast amount of phenomena that still remain for biochemists to look at, study, and clarify.

A descriptive focus means that while biochemistry has generated some extremely useful tools, it is not as active a field of research as it could be. I think this is a big part of why some of the scientists seem to view biochemistry as those tools, rather than an independent area of study. To be relevant going forward, biochemists need to keep in mind that there are still Grand Challenges in biochemistry, that we have to continue to work on. These questions will drive the frontier of biochemical research. I’ll describe some of the most compelling challenges.

The Grand Challenges of Biochemistry

The emergence of functional macromolecules
Starting from simple atoms, how did the first functional molecules emerge? How have these functions changed over time. Can new functions emerge?
The folding problem
A long chain molecule needs to fold back upon itself to have a function. How a molecule does this efficiently and with high fidelity is still a mystery in many cases. How does the information in a molecule convert from chemical sequence to a three-dimensional structure?
Modelling dynamics
Many important functions of molecules depend on the fact that they are flexible machines that can bend into many different shapes as part of their function. We currently do a very poor job of modelling, understanding and predicting this flexibility. How do we understand this flexibility and predict where it can have important functional roles?
Exotic chemistries in water
Living cells don’t have access to an organic chemistry lab to create complicated molecules of life. How are exotic chemical reactions carried out, all in a water-based environment?
Selectivity versus sensitivity
Some molecules need to interact with a broad range of other molecules, others a very narrow set. How is this accomplished? What features govern this switch between sensitive, broad interactions, and highly specific, tight interactions?
Non-ideal activity in real environments
The history of biochemistry has involved purifying and isolating proteins from the complex mix of things in a cell, in order to study that protein in a test tube. But their natural environment is much more complex, full of thousands of other molecules. These other molecules will greatly affect and alter function. How can we understand the natural environment of a molecule?
Ultrastructural chemistry
Many of the most interesting molecules to study in living cells are absolutely gigantic. Normal rules of molecular behaviour break down when we get to those sizes. Modelling chemical properties at large scales is an ever-pressing challenge of biochemistry. What emergent properties occur in large molecular complexes?

What is the Future of Biochemistry?

So these Grand Challenges laid out, where does the future of biochemistry lie? Every new technology helps us tackle these problems in even greater detail. I’m optimistic that there are questions that we can now start to ask and answer, that we couldn’t even 10 years ago.

Technologies that get me excited include the high-resolution structural work now accessible at free-electron laser facilities, ever stronger supercomputers to calculate the dynamic nature of the molecules, ingenious in-lab evolution experiments to probe the change of molecules over generations, single-molecule experiments that track the behaviour of individual molecules, and many others. The future of biochemistry isn’t extinction, it’s evolution. We can now probe deeper than ever before at the inner workings of our molecular selves. The future is bright indeed.

Some may tell you biochemistry isn’t worth your time. I’m going to fight to show otherwise.

I hope you’ll come along with me.