When people hear ‘anthrax’, it's often connected to outbreaks where mass death of livestock and serious human illnesses occur, both of which pose significant threats to public health systems. Anthrax also has a deep history within fundamental biological discoveries. Even from the mid-19th century, anthrax was already a prominent health problem. Its subsequent discovery by Robert Koch revealed that a bacterium caused anthrax. Overall, establishing the link that a distinct microorganism caused a specific disease. Consequently, it was understood that various life cycles and conditions allowed for the proliferation of these small but mighty microorganisms!
Figure 1. Drawings by Robert Koch representing the various stages of development of B. anthracis. This figure comes from Robert Koch and the ‘golden age’ of bacteriology.
While Bacillus anthracis, the bacterium responsible for anthrax, has many factors as to why it is such a lethal and virulent microorganism, an interesting mechanism for its sustained life cycle is its ability to form highly resistant spores that can survive for decades in the soil and within slow-decaying hosts. More information regarding its route of transmission from livestock to humans and related symptoms can be found here. Acute to moderate infections can be treated and cured with antibiotics and a 3-dose vaccine, but inhaled anthrax poses greater risks and can even be fatal.
So, how does B. anthracis even create spores?
The answer is based on one of the most interesting survival strategies within microbiology: sporulation. In times of extreme environmental stress, such as the host dying or the depletion of nutrients, B. anthracis is able to differentiate from a vegetative state into spores once exposed to oxygen. This process begins with the division of the mother cell, with the forespore as the product, with more stages of maturation in between before the spore is sent out into the environment (more about the stages of spore formation and maturation can be found here). These spores have complex mechanisms to become highly resistant to prolonged periods of UV radiation, sunlight, and other environmental stressors that are not sustainable to most bacteria when they are in their normal proliferative state.
The lifespan of B. anthracis within host/soil reservoirs has been measured in countries where Anthrax infections are common amongst grazing animals. One study focused on zebra carcass sites in Namibia and used factors similar to Koch’s (humidity and rainfall) to examine how spore counts differ from those under other extreme abiotic conditions. Researchers discovered that B. anthracis spores persisted within surface-level soil for up to 10 years after host death! Discoveries of spore concentrations, their associated cellular machinery, and survival within these reservoir sites can help provide a better understanding of how infected livestock create hotspots and transmit the disease to humans.
A novel protein has been discovered in B. anthracis!
Earlier this year, a paper was released about a novel (aka “new”) protein that was discovered and is thought to play a major role in the sporulation of B. anthracis. The authors Sangwan et al. aimed to investigate the role of a AAA+ domain-containing protein (known as “AAA+ ATPase”) that they found in B. anthracis (more about protein domains can be found here). AAA+ ATPases are a superfamily of proteins, in other words, a large group of proteins that are distantly related (in this case, they have one or more AAA+ domains).
Figure 2. The diagram shows hierarchical relationships in superfamilies and how groups within are related and separate. This figure comes from What are protein families?.
This superfamily contains the evolutionarily conserved AAA+ domain. This domain allows proteins to be responsible for a diverse range of cellular activities, as it allows them to use the energy released by ATP during the process of hydrolysis to convert ATP to ADP and carry out these activities; hence, they are named ATPases. Their name comes from this use of ATP hydrolysis: ATPases associated with diverse cellular activities (“AAA”). They take this energy and convert it into mechanical energy to do work, become molecular switches, or even become part of macromolecular machines. See this website for more on AAA+ ATPases.
Each family within this larger superfamily is referred to as a “clade”, grouped based on similarities from a common ancestor through evolution. AAA+ ATPases are classified into seven clades. Sangwan and collaborators place their novel B. anthracis AAA+ protein into clade 3 of the superfamily, the ‘classic’ clade. Within this clade, the proteins are the most functionally diverse, but all have a common role in protein remodeling.
Figure 3. C-H are diagrams that are representative of the AAA+ protein clades (clades 2-7 are shown here). The arrows are β-strands, and the cylinders are α-helices. The brown structures represent the insertions that are characteristic of each AAA+ clade. This figure comes from Current Biology: AAA+ Proteins.
What is the role of a AAA+ ATPase-related protein in B. anthracis?
The authors of the 2026 study found a protein with the AAA+ domain that is similar to a protein named PrkA in B. subtilis. Because of this, the B. anthracis AAA+ ATPase domain-containing protein was labeled “BA PrkA” due to its similarity, or homology, to the B. subtilis version. They found that BA PrkA did not exhibit similar activity to BS PrkA; therefore, the study moved on to examining other functions of this novel protein, namely, sporulation.
To investigate this, Sangwan and others experimented on three versions of B. anthracis: wild type (WT), a mutant strain created by the deletion of the prkA gene (ΔprkA), and a complementary strain where prkA was added back to the mutated ΔprkA strain (used as a control). The researchers grew these three strains, harvested their spores, and put the spores of each of the strains into two conditions for 30 minutes: heated at 75℃ or unheated at room temperature, and then counted the number of heat-resistant (aka “mature”) spores. Their results can be seen in Figure 4 below. In the heated experiment group, the wild-type and complementary strains have a comparable amount of mature spores, similar to all three strains in the unheated experiment group.
On the other hand, the ΔprkA strain had a significant 3-fold reduction in spore counts when put through intense heat. These results tell us that this protein must play a major role in sporulation, and without it, B. anthracis has a much lower chance of forming mature heat-resistant spores! This conclusion is backed up by the mature spore counts of the complementary strain being similar to the wild-type strain in both experimental groups. Complementary strains are important because restoring the original strain after deletion allows researchers to confirm that the results are due to the mutation rather than any modifications that may arise due to the process of mutating.
Figure 4. Bar graphs show the mean colony-forming units per milliliter (CFU/mL) of spores from the three B. anthracis strains: wild-type strain (light gray, WT), ΔprkA mutated strain lacking the prkA gene (dark gray, ΔprkA::pAMY1), and complementary strain (black, ΔprkA::pAMY1-prkA). This is Figure 3B in the Sangwan et al. 2026 paper.
Additionally, the researchers did further tests to see why exactly ΔprkA led to a deficiency in sporulation by staining the spores with hexidium iodide. This stain is used to hold onto nucleic acids, such as DNA. In the case of Sangwan et al., the researchers used spore-impermeable hexidium iodide, a version that cannot go through a mature spore coat. They stained the spores of all three strains and looked at their fluorescence under a microscope. In Figure 5, we can see faint staining at the outer layer of the spores in the wild-type (left image, yellow arrows) and complementary strains (right image, yellow arrows), while the ΔprkA strain (center image, blue arrows) shows bright red staining at its core. This tells us that when the prkA gene is deleted, most of the stain is able to make it past the surface spore coat and into the core to stain the DNA.
These results suggest that without the prkA gene in B. anthracis, we see a weak and permeable spore. The function of the surface coat is to be strong and resistant to the environment to protect the DNA. This helps us better understand Figure 4, further suggesting that this deficient phenotype is what leads to the heat sensitivity in the spores. Perhaps the cellular activities that this AAA+ ATPase protein is responsible for are unable to be carried out due to a lack of the protein in the mutant strain. Therefore, we see this weak surface coat that is heat-sensitive.
Figure 5. Representative microscopy images of hexidium iodide-stained B. anthracis wild-type strain spores, mutated strain spores, and complementary strain spores. The top row is the hexidium iodide-stained spores, and the bottom row is the same image in differential interference contrast (DIC) microscopy (similar to phase microscopy). The yellow arrows indicate spores with non-permeabilized and weak surface staining. The blue arrows indicate spores with strong, diffused, permeabilized core staining. This is Figure 4B in the Sangwan et al. 2026 paper.
The big picture…
To sum up, we must acknowledge that because this paper discusses a recent discovery of a novel protein, more research should be done on this protein. We now know that this protein is necessary for successful sporulation, so it would be interesting to investigate the mechanism by which the protein accomplishes this. The authors began to answer this by finding other proteins that interact with BA PrkA and found that BA PrkA interacts with specific proteins to coordinate certain responses for spore maturation. Future research could explore the direct mechanisms by which BA PrkA uses to achieve this coordination. Furthermore, a question left standing is: with the B. subtilis BS PrkA as the only other AAA+ ATPase that is closely related to this protein, we’re curious to know why BA PrkA does not have the same or even similar functions as BS PrkA if they have significant homology (88%!)?
Overall, while the anthrax disease is not completely eradicated and there are current human cases worldwide, it is predominantly a livestock and herbivore disease acquired through grazing contaminated soil and can easily be transmitted to humans if proper precautions are not put into place. Access to vaccination should not be limited to professionals in high-risk areas, but to communities that are most at risk, such as farmers. As mentioned earlier, the Namibia study reveals that B. anthracis can persist in soil for up to 10 years, and efforts to contain these microbes remain an ongoing focus. Anthrax is a virulent disease that must be better understood to be combated. Discovering more about its mechanism of survival through sporulation can lead to the creation of knowledge of how to eliminate this highly resilient bacterium and its disease. Future research is in need of tangible strategies to combat zoonotic transmission to prepare against outbreaks or future acts of bioterrorism.
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