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.

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Who Watches the Watchmen? Blind Trust Isn’t Enough in Today’s Research Environment

This post is cross-posted from a blog post I wrote for the Science and Policy Exchange. For more insightful writing on science and how it relates to government, the media, and society at large, visit their site http://www.sp-exchange.ca

In the spring of 2012, an article appeared in the niche crystallography journal Acta Crystallographica, Section F (“Acta F” to those in the know). This article, “Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen” describes a protein structure published in a 2010 Journal of Immunology paper. Following a thorough analysis, author Bernhard Rupp (of textbook fame) concludes there is:

… no doubt that model and data of [structure] are incompatible and that the deposited [data] are not based on actual experiments, and their standard uncertainties are not based on experimental errors.

 Translated to everyday language, this reads

The data isn’t real.

 He is accusing the authors of data fabrication.

fake

 Technical language and a deferential tone mask the severity of this accusation. The author response, published in the same issue, cuts to the chase.

The University of Salzburg immediately informed and commissioned the Austrian Agency for Research Integrity (OeAWI) to carry out an investigation into possible data fraud on the part of author Robert Schwarzenbacher, the co-author solely responsible for the Bet v 1d structure and the crystallographic section of the J. Immunol. paper. The OeAWI is presently preparing a report of this investigation.

This is a great example of researchers and institutions taking the appropriate steps to address concerns of research misconduct. Then:

Author Schwarzenbacher admits to the allegations of data fabrication and deeply apologizes to the co-authors and the scientific community for all the problems this has caused.

 Oh. Well then. No need for an investigation after all, right? Well….

Note added in proof: subsequent to the acceptance of this article for publication, author Schwarzenbacher withdrew his admission of the allegations.

 Presumably he talked to a lawyer.

 So, what went wrong? We can infer that researchers working in one field (immunology) brought in a collaborator from an outside field (structural biology) to add complementary experiments and drum up the impact of the project. The steep learning curve and specialized techniques of structural biology meant that the other authors had to trust Schwarzenbacher’s work was conducted rigorously and honestly. Obviously, this trust was misplaced.

 Almost two years after the Acta F paper was published, Schwarzenbacher has lost his job, but has sued for wrongful termination. The structure in question has been mothballed in the Protein Data Bank, and his contribution to the Journal of Immunology paper has been removed. The paper still stands on its other experiments, as the authors argue that the paper’s conclusions did not depend on the fraudulent structure work. Regardless, it’s a black mark on the record of the coauthors, the journal, funding agencies, the university, and the Austrian structural biology community. The reputations of many parties have suffered from the actions of one misguided researcher.

 Most have been happy to hang the blame on Schwarzenbacher’s shoulders, and rightly so. But does he carry all of the responsibility? I’ll argue he doesn’t. 7 co-authors all approved the work for publication. The Journal of Immunology, its reviewers and editor all gave it the stamp of approval. The department and university provided the environment where this misconduct could go unnoticed. The allergy and immunology research community also missed the fraud. Everyone appears to have been content to accept the credit and conclusions from presumably legitimate work, but once fraud was brought to light immediately distanced themselves from the situation. This is not a sustainable practice.

 This isolated case could be one of many to come. We need not just consider fraud, either. Even the most scrupulous researchers are subject to the insidious influence of wishful thinking. Critical mistakes can slip through when a “ringer expert” operates on their own, without any scrutiny. As technology drives ever more complicated experiments, and granting agencies continue to reward multidisciplinary work, lone specialists will increasingly be required in collaborations, and the chances of fraud or major errors slipping through will increase.

 Schwarzenbacher’s is a particularly good example of this problem because there is no ambiguity about his fraud – a novice crystallographer would find obvious problems with the data. Passing the data past a single critical eye could have caught the fabrication before publication. This drives home how the current environment can allow ethical or methodological problems to slip through, leading to flawed or fraudulent conclusions. A mechanism is needed to improve oversight – the assumption of good faith is not sufficient. Peer review is supposed to provide this function, but here, as elsewhere, it failed.

 Besides fixing peer review, how can future incidents like the be prevented? Movements like open data will play a role. Deposition of data is already a condition for publication of structural work, and Schwarzenbacher’s fraud was discovered through curation of the database. Post-publication peer review can also help identify problematic data and correct the scientific record, but neither of these mechanisms can prevent the initial publication of bad or fraudulent data.

 What is really needed is a culture change. The institutions, journals, and researchers involved in multidisciplinary collaborations can’t escape responsibility for scientific oversight. This could mean some sort of institutional review, or a requirement to pass the data by a friendly but impartial third party. Most importantly, the community needs to accept that scientific integrity is the responsibility of everyone involved, not just the person who processed the data. Researchers need to take steps to ensure that they can stand by the integrity of all of the work to which they attach their names. Ignorance is a poor excuse.

 It’s worth considering what drove Schwarzenbacher to cross the line. Rather than some master manipulation to trick his collaborators and journal reviewers, his actions appear more like simple indifference and laziness. As highlighted by the partial (not full) retraction, the structural work wasn’t central to the study, and the faked data is obvious enough that he clearly didn’t expend much effort to cover his tracks. Perhaps he didn’t think anyone would notice or care, and if so, he was somewhat correct. His collaborators and research environment allowed him to cut a corner that he shouldn’t have cut. He chose expedience over honesty.

 The editors of Acta F make the observation that

It seems clear that the pressures on scientists early in their careers are so severe that a few are compelled to risk their careers in order to further them.

 I think it’s time everyone else assumes a little responsibility for letting it happen.

Find out more about the Schwarzenbacher debacle on RetractionWatch:
http://retractionwatch.com/2012/04/02/protein-structure-retracted-after-investigation-into-highly-improbable-features-journal-calls-it-fraud/
http://retractionwatch.com/2012/04/12/salzburg-university-fires-crystallographer-robert-schwarzenbacher-for-faking-data-in-journal-of-immunology-paper/
http://retractionwatch.com/2013/05/27/a-partial-retraction-appears-for-former-salzburg-crystallographer-who-admitted-misconduct/
And in Nature:
http://www.nature.com/news/trial-tests-austrian-integrity-body-1.10564
As well as a relevant Nature editorial from around the same time:
http://www.nature.com/nature/journal/v483/n7391/full/483509a.html

Edit 2015.09.02: I’ve updated my account since I first wrote this post. If you’d like to follow me online, find me @superhelical