Anthrax: A Big Disease Thwarted by a Micro Problem

By: Rachel Polfer

Anthrax is an ominous word. The infamy of the disease allows it to be referenced comfortably within conversations centered on such topics as “bioterrorism” and “national threats.” Its historic use within the hands of malicious actors has added deadly connotations to the once innocuous presence of unexpected packages and letters. More recently, the federal government has begun to stockpile vaccines against the infectious disease, preparing for a day when anthrax might be weaponized against the population on a broader scale.

Bacillus anthracis is the causative agent of this notorious killer. Infection occurs through the inhalation of the bacterium’s spores (visible in the image above as the white dots within the purple B. anthracis cells). But while the bacteria may have a larger than life public profile, it has the characteristically small stature of a pathogenic bacteria making its way through the microbial world. In many ways, these small physical dimensions contribute to the success of B. anthracis as an infectious agent, but they also come with some logistical difficulties — difficulties like how a bacterium might fit a genome 1500x its own length comfortably within its cell walls, with enough space left over to allow for the day to day minutiae of life as a microbe. (The image to the right shows the genomic contents spilling from a lysed Escherichia coli cell, demonstrating how much DNA must be contained within the small dimensions of a bacterium).

The solution to this problem lies in DNA supercoiling — essentially the twisting of DNA back in on itself until it begins to compact. A good way to visualize this is to imagine what happens when you hold one end of a string while twisting the other end. At some point, the string will transform from long and straight, to short and compact.

The mechanism behind DNA supercoiling is remarkably similar, but unfortunately for the cell, DNA is not a straight length of string, but a helical biomolecule. In many ways, DNA behaves more like a slinky than a string when subjected to supercoiling. For awhile, the process proceeds unimpeded, but at some point, the continued twisting of the slinky introduces enough torsional strain to break the helical toy.

The structural integrity of supercoiled DNA is similarly threatened by torsional strain, and the mitigation of this threat involves a double stranded break within DNA that is carefully orchestrated to relieve this tension, while allowing for the break to be repaired in such a way that the supercoiling is preserved and DNA compaction is achieved (a video that uses some colorful Play-Doh to demonstrate this process can be found here).

The coordination of such a process is incredibly complex, but unsurprisingly, there is a whole host of enzymes ready and willing to tackle this particular problem — and not just on B. anthracis’ behalf. It turns out that a lot of microbes are, in fact, micro and they have developed similar enzymatic mechanisms capable of addressing this issue. As a result, the structures, roles and mechanisms of the enzymes involved are all highly conserved across various bacteria — Gram-positive and Gram-negative alike. Unfortunately for these enzymes, this means that they make a rather attractive target for researchers developing antibiotics.

Quinolones (the general structure of which is depicted in the image above) are one class of drugs designed to interact with these enzymes, particularly gyrase and topoisomerase IV. Both gyrase and topoisomerase IV are enzymes that aid in the regulation of DNA supercoiling by inducing the double stranded DNA breaks responsible for relieving torsional strain (if you want more information on this process, go ahead and rewatch the Play-Doh supercoiling video — both gyrase and topoisomerase IV are class II topoisomerases, so pay particular attention to the Type II Topo demonstration). When this break occurs, the enzymes bind to the newly exposed end of DNA and form a “cleavage complex.” Quinolones function by stabilizing this complex, preventing the cell from repairing the damage. At a high enough concentration, quinolones will increase DNA cleavage to a percentage that overwhelms the bacterium’s genome, ultimately leading to cell death.

The conservation of both gyrase and topoisomerase IV across various bacteria has made quinolones the most commonly prescribed antibiotic in the world. In fact, the breadth of quinolone success across multiple classes of bacteria has resulted in their repeated use in “cocktail” antibacterial treatments. For decades now, the conservation of the enzymes involved in DNA supercoiling has made quinolones a good bet when other, more targeted treatments are unavailable.

Unfortunately, resistance to quinolones has been on the rise, and the mutations that confer resistance on a given bacterium tend to be unique to the species of bacteria in which they develop. This is because, while the overall system of supercoiling within bacteria tends to be conserved, the specific mechanism by which gyrase and topoisomerase IV perform their roles varies ever-so-slightly across species. As a result, the way in which quinolones function to stabilize the cleavage complex within a bacterium will vary between species as well, and the specific mutations that circumvent this interaction will similarly diverge.

Therefore, in order for researchers to combat antibacterial resistance, they have to first determine the resistance mechanism particular to their bacteria of interest.

This is precisely what Ashley et. al (2017) set out to uncover with a series of mechanistic studies designed to determine the basis of B. anthracis’ emerging drug resistance, specifically in relation to the mechanism of interaction between gyrase and quinolones.

Researchers had previously investigated quinolone resistance in a variety of bacterial species, and Ashley’s team drew on the leads provided by these prior studies. In most of the bacteria studied, the efficacy of quinolone was revealed to be dependent on the presence of a metal-ion bridge. This bridge was found to consist of an Mg2+ ion bound to the C3/C4 keto acid of the antibiotic (The top image above depicts the chemical structure of typical quinolones, with the C3/C4 keto acid highlighted in green. The bottom image depicts a simplified representation of a quinolone, with the C3/C4 keto acid depicted as the green circle overlapping the blue ‘core’ structure of quinolone). The magnesium ion interacts with four water molecules that subsequently hydrogen bond with a Serine residue in gyrase. In bacteria other than B. anthracis, this water-metal ion bridge typically plays one of two roles: it either helps bind the quinolone to gyrase, or it helps position the quinolone in such a way that binding between the enzyme and the antibiotic is optimized. While such a distinction in mechanism seems subtle, it has critical implications for how specific mutations might confer drug resistance on B. anthracis.

With this background information, Ashley’s team set out to establish three aspects of the interaction between quinolone and B. anthracis gyrase: 1) to determine whether or not a metal ion bridge is present, 2) to determine whether this bridge, if present, functions mechanistically to aid in the binding between quinolones and gyrase, or whether it optimizes the position of the drug and the enzyme in order to promote binding, and 3) to determine whether this bridge, if present, is disrupted by the mutated gyrase enzymes of drug resistant B. anthracis.

Most of the experiments designed to explore these questions were based on the simple fact that, if a given quinolone antibiotic is having its intended effect on gyrase, then the percentage of DNA cleaved by gyrase will increase as the concentration of the antibiotic increases. With this fact in mind, Ashley’s team ran a variety of tests on various drug-resistant strains of B. anthracis, each with a mutated form of gyrase.

In order to establish the central role played by the proposed bridge in ensuring quinolone function, Ashley et. al (2017) examined the percent of DNA cleaved when gyrase was exposed to the antibiotic 3’-(AM)P-quinolone. This antibiotic is unusual, as it possesses a C7 functional group in addition to the typical quinolone structure (In both the top and bottom images above, this C7 functional group is colored dark green). This group allows 3’-(AM)P-quinolone to bind directly to gyrase, negating the water-metal ion bridge dependence of most quinolines.

In order to further establish this independence, the researchers tested 3’(AM)P-dione, an antibiotic that possesses the C7 functional group of 3’-(AM)P-quinolone, but lacks the C3/C4 keto acid necessary for bridge formation (shown below, with red highlighting the missing C3/C4 keto acid). These antibiotics successfully increased the percentage of DNA cleaved by gyrase, even in drug-resistant strains of B. anthracis.

These results implied that the resistance of the mutants is restricted to quinolones whose interactions with gyrase solely depend on their C3/C4 keto acid (Ashley et. al, 2017). However, to establish the role of C3/C4 in the mechanism of the quinolones, and to determine whether these drugs functioned by directly facilitating binding, or by positioning the drug within the appropriate active site of gyrase, the researchers performed a competition assay.

The previous test had established that 3’-(AM)P-dione could successfully bind with gyrase despite its lacking the C3/C4 keto acid. Ashley’s team designed dione versions of quinolones that similarly lacked the C3/C4 keto acid (In the below image, this absence of the C3/C4 keto acid is represented with a red circle). However, these diones did not possess the C7 functional group of 3’-(AM)P-dione (The absent C7 functional group is similarly depicted as a red circle in the below image). As a result, even non drug-resistant, wild-type B. anthracis gyrase was found to be immune to these dione-quinolones.

This resistance, in contrast to the efficacy of 3’-(AM)P-dione, allowed Ashley et. al (2017) to conduct a competition assay by examining the percentage of DNA cleaved by wild type gyrase as increasing amounts of dione-quinolones were added to a solution containing wild-type gyrase as well as 3’-(AM)P-dione. If the C3/C4 keto acid aided dione-quinolones by facilitating binding between the enzyme and the antibiotic, the dione-quinolones would be unable to bind with the wild-type gyrases in solution. Therefore, regardless of the concentration of dion-quinolones in solution, there would be no molecules present competing with 3’-(AM)P-dione for the opportunity to bind with gyrase. Therefore, the bound gyrase enzymes in solution would all be interacting with 3’-(AM)P-dione, and the effectiveness of 3’-(AM)P-dione (as assessed by percentage of DNA cleaved) would remain unchanged.

Conversely, if the C3/C4 keto acid merely aided in positioning the antibiotic within the enzymes active site, binding between gyrase and the antibiotic would still occur in the absence of the C3/C4 keto acid. Therefore, competition between 3’-(AM)P-dione and the dione-quinolones would be observed as both antibiotics would attempt to bind with the gyrase enzymes present in solution. This would result in the percentage of cleaved DNA decreasing, as some gyrase enzymes would be bound to dione-quinolones, to which they have a demonstrated resistance. This bond would then prevent 3’-(AM)P-dione, the effective antibiotic, from interacting with the bound gyrase. Ashley’s team did not observe any such competition, and therefore concluded that the C3/C4 keto acid was central to the mechanism of the quinolones, in that it played a role in directly facilitating the bond between the enzyme and the antibiotic, rather than simply facilitated correct positioning of the enzyme and drug (Ashley et. al, 2017) .

However, this study only addressed half of the oringial question. While the investigation supported the hypothesis that the majority of quinolones depend mechanistically on the C3/C4 keto acid facilitating bonding interactions, it did not elucidate whether the drug-resistance of the mutant B. anthracis resulted from the disruption of this bonding activity, or through some unrelated mechanism. To examine this point of uncertainty, a similar competition assay with 3’-(AM)P-dione was conducted. However, this time, the fully functional quinolones were used, rather than their dione counterparts. While the mutant gyrases were resistant to these quinolones, the antibiotics possessed their C3/C4 keto acid. Therefore, any observed disruptions in bonding would be the result of the mutated residues of the gyrase. If the mutations did not disrupt the bonding interaction between the enzyme and the drug, competition would occur between 3’-(AM)P-dione and the quinolone, resulting in a decrease in the percentage of DNA cleaved by the mutated gyrase. If, on the other hand, the mutation disrupted this bond, there would be no competition, and 3’-(AM)P-dione would be free to bind with all of the gyrases, and there would be no drop in the percentage of DNA cleaved by gyrase. In this study, Ashley et. al (2017) did not observe competition, which supported the hypothesis that the drug resistance of the studied gyrases results from mutations that directly disrupt the ability of quinolones to bind with the enzyme gyrase.

By conducting these relatively simple comparisons and competition studies, Ashley’s team was able to elucidate the underlying mechanism behind the emerging drug-resistance of B. anthracis. This information is critical for directing the development of more effective antibiotics. Specifically, this study suggests that by changing the means by which quinolones bind to gyrase (for example, by introducing a functional group at C7 such as that seen in 3’-(AM)P-dione) emerging resistance in bacteria may be overcome, allowing for antibiotics to continue to thwart diseases — even diseases as ominous as Anthrax.


References:

Ashley RE, Lindsey RH, McPherson SA, Turnbough CL, Kerns RJ, Osheroff N. (2017). Interactions between quinolones and Bacillus anthracis gyrase and bases of drug resistance. Biochemistry, 56 (32), 4191–4200.

Todar K. (2001). University of Wisconsin.

Kavanoff R. Supercoiled chromosome of E. coli. Scitable: by Nature Education.