Antibiotic Resistance as a Force of Nature

My research focuses on antibiotics – specifically antibiotic resistance. Last week I gave a seminar on my work, which was followed by some excellent questions about the origins and evolution of resistance. While I don’t personally get my hands dirty studying molecular evolution or microbial ecology, I think about these topics often, for a couple reasons. First, the origins and evolution of resistance factors have interesting implications that contextualize the structure and function of resistance factors – it helps me make sense of the molecules I study. Second and more importantly, the evolution of antibiotic resistance gets at a fundamental understanding of the environment and the world around us. We tend to focus on the problems that resistance create in medicine, but in nature, the relationship between microbes is much more complicated than we might assume. Antibiotics and resistance give us a window into the fascinating world of microorganisms and their strange and complicated existence.

In the discussion after my talk, I brought up a recent paper in Nature. In this report, a group at Harvard Medical School modelled the community dynamics of antibiotic-producing and -resistant microbes. The headline finding was that antibiotic production and resistance can stabilize microbial environments. The production and degradation of antibiotics are an intrinsic feature of mature microbial communities.

This seems counterintuitive – how would antibiotics, substances that kill bacteria, bring stability to an ecosystem?

It isn’t as ridiculous as it may sound. While a majority of antibiotics research focuses on the medical applications and repercussions, a less celebrated contingent of microbiologists look at the environmental role of antibiotics. These researchers find that antimicrobials play a more nuanced role than we have been led to believe. Rather than the chaotic battle royale we picture when we think of the microscopic struggle for survival, they find that antibiotic resistance often plays subtler roles in the microbial world. Let’s look into that world.

Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interactions. Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D'Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis. — **Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interaction.**
*Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D’Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis.*

What is Antibiotic Resistance?

We should get some definitions out of the way. An antibacterial is a chemical compound that kills or stops the growth of a bacterium. Antimicrobials are less well defined, and include antibacterials as well as antifungal and antiparasitic compounds, sometimes antivirals as well. Antibiotic was coined to specifically refer to a chemical that kills or stops bacteria but doesn’t affect animal cells – a nontoxic antibacterial. It since expanded to include some compounds that also target fungi and protists, but not antiviral compounds. In common speech, “antimicrobial” and “antibiotic” frequently mean “antibacterial”, so I’ll use them interchangeably here.

Antibiotic resistance is a catch-all term we give to many different mechanisms a bacterium can use to survive and/or grow, in the presence of an antibiotic. Every antibiotic has a specific target it interacts with, and anything that keeps the antibiotic and target from binding will result in resistance. Common mechanisms of antibiotic resistance include:

Chemical breakdown of the antibiotic
Molecular pumps that kick it out of the cell
Changes to the microbial target that block the antibiotic
Bypass the target molecule to allow the microbe to grow even when the target is productively blocked

Resistance factors are the molecules that give the bacterium resistance, by any of the above mechanisms. These resistance factors have diverse origins. In some cases, they’re still controversial. But broadly speaking, there’s two types of antibiotic resistance: new resistance and ancient resistance.

Two Origins of Antibiotic Resistance

New resistance makes sense. It is what we think of when we talk about antibiotic resistance as an example of Darwinian evolution. A spontaneous mutation emerges that confers resistance, and selective pressures drive it to succeed and take over the population. This is a common phenomenon that we have seen in the clinic and can induce in the lab, but is far from the only means by which antibiotic resistance happens.

Today I’ll focus on a second type of antibiotic resistance – the transfer of ancient antibiotic resistance factors from environmental bacteria into the strains that cause human disease. It was found in the 1970s that some antibiotic resistance factors appear to come from the microbes that produce the antibiotic. The thinking was that they act as a means of self-protection for the bacteria from their own toxin. This is one origin for resistance factors, although the original origin of many of these environmental resistance factors remain unknown.

Transfer of an environmental resistance factor to disease-causing bacteria results in resistance within the pathogen. In cases where this has happened multiple times, we get superbugs with resistance to multiple antibiotics. The collective environmenal pool of antibiotic resistance factors has been dubbed the “antibiotic resistome“. These resistance factors form a latent environmental reservoir, ready to jump into the strains that make us sick.

Antibiotic Resistance as a Healthcare Menace

Most of our concerns about antibiotic resistance come from the impact it has on medicine. Antibiotics are critical to our treatment of infectious disease, and are also necessary in prophylactic use for surgeries, cancer treatment, neonatal care, and many other intensive medical procedures. The spread of antibiotic resistance in pathogens could remove our ability to treat these infections, or care for many of our society’s most vulnerable members. This could lead to a transition to a “post-antibiotic era” where once again these miracle drugs are not available to us. A minor infection from a scraped knee, sore throat, or scratch off a rosebush could be fatal.

While spontaneous emergence of antibiotic resistance occurs, resistance frequently comes from environmental cross-over. A benign soil microbe meets a pathogen, shares some genetic material, and that pathogen becomes resistant. A notorious recent example of this is the emergence and worldwide spread of the New Delhi beta-Metallolactamase, a resistance factor that knocks out some of our last-resort antibiotics.

In the face of this ongoing menace, what do we do? For decades, the answer has always been “find more antibiotics”. This is important to do, but not enough. We search the world for more obscure microbes that might produce a new antibiotic, and we’re beginning to find a few, but we are still falling behind. Searching for new antibiotics is a game of whack-a-mole, finding new drugs as nature sends more sources of resistance to knock down. We’ve kept up for a while, but now we’re falling behind.

Some of the search for new ways of killing microbes involves looking for new targets to bind antibiotics to – reading the antibiotics literature, everyone and their grandmother wants to sell you a new potential antibiotic target. Other strategies include directly inhibiting antibiotic resistance factors, blocking bacterial toxins to “disarm” the bacterium, or more obscure methods like bacteriophages. But none of these strategies have yet led to sustainable long-term solutions for treating resistance. We may be doomed to fail – we’re going up against a fundamental feature of nature.

Antibiotic Resistance as a Force of Nature

Almost as long as we’ve had antibiotics, we’ve had antibiotic resistance. In his 1945 Nobel Prize Lecture for the 1928 discovery of penicillin, Alexander Fleming said:

It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them

Since Fleming’s time, any newly-developed antibiotic has had only a few years of use before a case of antibiotic resistance was identified. This observation alone suggested that antibiotic resistance existed in the environment before we began to use these compounds to cure ourselves. Recent studies of permafrost cores and isolated cave systems have also produced compelling evidence that antibiotic resistance factors have existed in the environment long before humans came around.

The idea of transfer of bacterial resistance factors took some time to catch on. Discovered in the 1950s in Japan, the greater scientific establishment received this finding with disbelief and scorn. It took many additional reports for researchers to believe that resistance could move from one bacterial species to another. By the time the greater scientific community clued in, antibiotic resistance was already running rampant in some clinical strains. Since that point we have been in a constant search for new antibiotics, often falling behind the spread of resistance.

A Microbial Cold War

Starting with the first discoveries of antibiotics, we’ve considered them to be weapons in an ongoing environmental war between microbes. Selman Waksman, Nobel laureate for the discovery of streptomycin*, developed his entire research program on this assumption – that there are microbes in the environment that produce antibiotics in order to kill those around them and gain selective advantage. This program was replicated in many labs, resulting in the 1940’s-1960’s golden age of antibiotic discovery.

Waksman envisioned the environment as an ongoing microbial war. An antibiotic is the bacterium’s sword, resistance is it’s opponent’s shield. But what if that’s the wrong way to think about it?

What if this microscopic battle between bacteria was more of a Cold War? An ongoing stand-off that only occasionally breaks out into active conflict? That’s what microbial researchers seem to keep finding. Microbes play games of rock, paper, scissors using antibiotics. They punish freeloaders, engage in brinksmanship and even cooperate in difficult times. These bacteria are in competition, but this competition generates a kind of rolling stability as they hold each other in check.

Things get even more interesting when we decrease the concentration of an antibiotic below the range at which it kills – to “sub-therapeutic”, “sub-lethal”, or “sub-inhibitory” concentrations. In toxicology, we talk about how “the dose makes the poison” – the same thing applies for bacteria. At low concentrations of antibiotic, bacteria can trigger adaptive stress responses, go into dormant states, adjust their metabolism, trigger complex growth modes (biofilms) or change their behaviour in even more subtle ways. At the extreme of this concentration range, it’s even been suggested that antibiotics might instead be thought of as signalling molecules rather than toxins.

With an environment full of these compounds at various concentrations, microbes are in constant cross-talk with each other. Studies like the most recent paper on community stability find that even in mixtures of a few strains of bacteria, antibiotics and resistance can keep competing stains in check, stabilizing a community and keeping any particular one from growing out of control. With 10-50 000 different species in a single gram of soil, the interrelationships are almost limitless.

As metagenomics studies and other work teach us more about the diversity of resistance in the environment, we find that microbial communities are complex, with different producers and resistant strains in constant rolling flux. Add to this an understanding of things like quorum sensing, and a picture of a complex, dynamic ecosystem emerges. A network of chemical cross-talk forms, and we are only starting to scratch the surface of this environmental complexity.

The use of chemical compounds to influence each other are not an exception, but the rule. The diversity of microbial species also drive a diversity of chemical compounds used to fight, defend, and communicate with each other. These countless microbial compounds have a name: the parvome. We harvest chemicals from this source for use in medicine, but must remain aware that any environmental molecule will also have corresponding mechanisms of resistance.

Rather than the all-out war that Waksman envisioned, the microbial world works like international diplomacy or a financial market. Every interaction trickles through a network and affects everything else. Booms and busts happen, but on average, the system selects for a kind of greater stability, so the entire community gains as a whole. Outright conflict is a zero-sum game. Most microbes prefer a tense collaboration, quietly manipulating their neighbours but avoiding actual battle. A microscopic Cold War. That war only rarely comes to active conflict, when we strip away diversity and release the bacteria from their self-imposed order.

Fear in a Handful of Dust

When we realize that the microbial world works this way, it means that antibiotic resistance is everywhere. Screen any environmental sample for resistance and you’ll find it. If an antibiotic exists, so do its resistance factors. Even antibiotics we haven’t yet discovered have resistance in the environment. This is a fascinating and terrifying thought at the same time.

It’s terrifying because antibiotic resistance is an enormously urgent public health concern. We’re running out of time. And as we understand that resistance is all around us, we realize that we’ll never eliminate it. We can only beat it back, and we can only play the game of antibiotic-resistance whack-a-mole for so long. Resistance seems to be an intrinsic property of the microbial world that we’ll never escape. As a great mathematician once said: Life finds a way.

I’m cynical whenever I see headlines about new breakthrough antibiotics or antimicrobial game-changers. All these do is kick the eventual resistance down the road a little bit farther. Even though I study mechanisms of resistance in hopes of blocking them, I think the most important solutions to antibiotic resistance will come from systems approaches: policy, sanitation, rapid diagnostics/response, and surveillance programs. We can’t control what resistance is out there, but we can take steps to limit the transfer of that environmental resistance to pathogens. Agricultural antibiotic use requires urgent action. Improved sanitation and means of reducing the spread of pathogenic microbes is critical.

As we struggle to deal with our impending antibiotic crisis, we are starting to realize how inevitable it probably was. It emerges from a complicated network of microbial cross-talk. Countless microbes silently jostle against their neighbours, subtly nudging with chemical signals, and being poked back with molecular weapons. This microscopic opera happens around us at all times, silently shaping our world and occasionally making our worst diseases even harder to fight.

The complexity is beautiful, and it is terrible.

_{* It should be noted that Waksman’s student, Albert Schatz, was heavily involved in the discovery of the compound, and by many accounts was snubbed by the Nobel committee when they presented the award to Waksman alone.}

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.}