The Microbiology Behind the FBI’s “Amerithrax” Case

By: Maya Fontanez '22

An example of a reward flyer distributed by the FBI and USPS in regard to Amerithrax. (source)

Much of the public information regarding anthrax has come from various terrorist incidents involving the bacterium in powder form. The infection can take to the human body in four different forms, on the skin, in the lungs or intestines, and in the bloodstream through injection and is often contracted from interactions with wildlife. In 2001, the United States was struck with multiple emergency cases in which envelopes laced with anthrax spores were distributed through the U.S. Postal system, endangering the lives of 22 people and killing five. The FBI and other government offices used “Amerithrax” as a codename for the attacks. Anthrax is considered both a Tier 1 biological agent as well as a bioterrorist weapon due to its ability to spread quickly and with little effort. (As a Criminal Minds fan, I recommend Season 4 Episode 24 “Amplification” as a pop culture reference to Anthrax used as a bioterrorist weapon.)

B. anthracis, Gram positive rods. (source)

In this blog post I will be diving deeper into the biology behind anthrax and its bacterium, Bacillus anthracis, through the lens of the Clark et al (2019) study “Heme catabolism in the causative agent of anthrax.” In this study, the authors investigated the process of how B. anthracis acquires and utilizes nutrients from mammalian hosts, due to the bacterium’s impressive ability to grow and spread rapidly. B. anthracis is a Gram-positive sporulating bacterium that is well known for its ability to infect mammals. Based on the findings of previous studies, we know that there are two proteins that are known to bind and degrade heme within the family of Bacillus, Heme Monooxygenase A (HmoA) and Heme Monooxygenase B (HmoB.) Because B. anthracis contains homologs of these two proteins, BAS2776 for HmoA and BAS0997 for HmoB, it is a very likely possibility that B. anthracis contains three heme-degrading enzymes rather than only one. In the Clark et al (2019) study, the authors aimed to determine how each protein contributes to heme-iron utilization and what their individual roles are.

Their experiment began by obtaining the Sterne (34F2) strain of B. anthracis and growing it on Luria-broth (LB) covered agar plates for a maximum of one week. The IsdG mutant used to develop isogenic mutants of HmoA and HmoB was received from Dr. Olaf Schneewind’s laboratory. The two mutants were then created by using pLM4, a temperature sensitive plasmid, and the restriction cloning method. To determine where HmoA and HmoB are located within the CDE superfamily, the researchers used the Sequence Similarity Network to compare both proteins to others within the family. Next, Clark et al (2019) purified B. anthracis HmoA, HmoB and IsdG, and then completed a measurement of Heme binding and degradation through separate reactions. Measurements of heme toxicity in iron deficient media (IDM), heme survival assay, heme and hemoglobin-dependent growth of bacillus in blood serum mimic, bacillus survival in macrophages , and bacillus virulence in a murine model of infection were all determined using various tests.

Focusing on the measurement of heme toxicity in IDM, the researchers completed this by inoculating IDM heme at different concentrations with overnight cultures of the bacillus strains. As shown in Figure 2C, by using LB both containing and not containing heme and inoculating it with mid-log cultures of B. anthracis strains, the strains were incubated for three hours. The survival ratio was determined after the samples had been washed with LB, serial diluted, and incubated at 37°C for one night. As shown in Figures 2B-C, the results from these procedures found that the strains of B. anthracis that did not contain either HmoA, IsdG or both were more susceptible to heme toxicity.

Figure 2: Graphs depicting B. anthracis without the presence of HmoA and IsdG, showing survival rates and susceptibility to heme toxicity. (source)

Figure 2A. depicts the decrease in heme resistance in B. anthracis strains lacking HmoA or IsdG at 4 μM, however, the Wild Type strain showed signs of significant toxicity at 8 μM. The graph shows both results at the 12 hour time point. In order to test their hypothesis further, Clark et al (2019) were able to produce combinatorial deletions within the genes in order to show levels of survival after exposure to heme at a toxic level of 10 μM. This data is depicted in both Figure 3B and 3C, and shows signs of a decreased ability to resist heme toxicity in both ΔHmoA and ΔIsdG, in comparison to the Wild Type strain. However, when deleted simultaneously, there is no sign of increased susceptibility in both strains, which suggests that there may be an alternate mechanism that is able to complete detoxification. Clark et al (2019) have considered that perhaps a transport mechanism is responsible for detoxification in the absence of both ΔHmoA and ΔIsdG. In the discussion of their findings, Clark et al (2019) suggest that HmoA and IsdG may be unessential in various strains of bacillus. Instead, it is suggested that HmoA is primarily used for detoxification of heme, while IsdG is thought to be important in the freeing of iron from heme. HmoA is vital for heme iron assimilation and growth in Bacillus, but IsdG is what is necessary for B. anthracis to acquire heme iron from mammalian hosts, the nutrient required for much of B. anthracis’ development.

The findings of Clark et al’s study mark significance within the microbiology community, especially within the realm of B. anthracis. The discovery that HmoA and IsdG work together in order to bind, degrade and protect against heme toxicity is a big step in understanding more about how the bacterium functions within a host. While I was glad that this study was able to provide such a rich understanding of HmoA and IsdG, I do wish that there could have been a larger discussion around the function of HmoB in B. anthracis. Clark et al explained the contributions that HmoB makes in B. anthracis’ ability to survive in macrophages, but I think further research on how HmoB is utilized to maintain survival in macrophages. As well as research regarding the alternate enzymes that are able to replace the role of HmoA and IsdG within B. anthracis.