Have you ever wondered why bacteria are so difficult to kill? Although some bacteria appear indestructible, this is due to several inherent characteristics that make them resistant to treatments and the human immune system. For example, their cell wall architecture, which includes teichoic acid, can repel detergents and toxins. One particular bacteria, Bacillus anthracis, has a specialized capsule that provides a unique example of this bacterial resistance. B. anthracis causes the well-known disease anthrax, which is an infection that affects both livestock and humans. Anthrax is a biological agent that can be used in a terrorist attack and has previously been used as a bioterror attack, including during World War I and, more recently, in the United States in 2001(Anthrax and bioterrorism).
Anthrax is caused by the B. anthracis spores entering the body via the skin, inhalation, or the GI tract, where they germinate, replicate, and begin to produce toxins (Figure 1). This toxin is produced by bacteria and is derived from the plasmid of B. anthracis. B. anthracis is a gram-positive, spore-forming, rod-shaped bacteria. Bacteria are classified as gram-positive or gram-negative based on their cell walls. Gram-positive bacteria have a thick peptidoglycan layer that turns purple when stained with gram stain (Figure 2). B. anthracis is primarily found in soil, where it can survive for long periods of time in the form of spores. Spores are highly resistant to heat, desiccation, and other environmental stresses, and they play a significant role in anthrax transmission. B. anthracis has two plasmids: pXO1, which encodes for the anthrax-causing protein toxicity, and pXO2, which encodes for a capsule that surrounds the bacterium and protects it from the immune system. The capsule is critical for the virulence factor.
Figure 1. B. anthracis transmission
Figure 2. B. anthracis gram stain at 1500x. The cells have squared ends. The spores are ellipsoidal shaped and located in the center.
A capsule is the outermost structure of bacteria and fungi that protects them from immune recognition and attack. Unlike other polysaccharide-based capsules, B. anthracis produces only one capsule structure made of poly-γ-glutamate (Figure 3). According to research, the capsule contributes to virulence by shielding the bacteria from the host's immune system. It accomplishes this by avoiding phagocytosis (engulfment of foreign objects by immune cells), which allows the bacteria to avoid being engulfed by white blood cells (Figure 4). As a result, if B. anthracis has this capsule, it can avoid being engulfed, leading to virulence. The free capsule has also been shown to inhibit dendritic cells. So, what else does B. anthracis' capsule protect it from?
Figure 3: A Schematic drawing of the structure of a generalized bacterium. Capsule in light yellow (Encyclopedia Britannica ). A heat fixed and polychrome methylene blue (McFadyean stain) B. anthracis cell. Pink/purple stained capsular material appearing as a halo around the cells (Stained Capsule)
Figure 4: Phagocytosis
That is precisely what O'Brien et al. recently investigated: the role of B. anthracis capsule in protection against human defensins and other cationic AMPs. Antimicrobial peptides (AMPs) are a type of small peptide with antimicrobial activity against gram-positive and gram-negative bacteria, fungi, and certain viruses (imagine it working like figure 5A). B. anthracis, the anthrax causative agent, encounters AMPs during all three types of infection: cutaneous (affecting the skin), inhalation (affecting the lungs), and gastrointestinal (affecting the digestive system). When B. anthracis comes into contact with AMPs, it may exhibit a variety of bactericidal activities. That is, they may disrupt bacterial membranes, form membrane pores, and damage intracellular targets (Figure 5B). Humans, microbes, arthropods, amphibians, plants, and other mammals all produce AMPs. The majority of AMPs are cationic, which means they have a positive net charge. Defensins, the cathelicidin LL-37, and histatins are the three types of cationic AMPs produced by humans.
Figure 5: AMPs Schematic presentation of bacterial killing mechanisms by antimicrobial peptide
The paper focuses on defensins, which are cationic proteins that are a major family of antimicrobial host defense peptides. The researchers present evidence that B. anthracis capsule provides resistance to many human defensins as well as some non-human AMPs. Human defensins are divided into two types: alpha and beta (Figure 6). The authors start by looking at the bactericidal activity of six different human alpha defensins. The findings show that a capsulated B. anthracis strain has significantly lower bactericidal activity(ability to kill bacteria) than an unencapsulated strain. Reduced bactericidal activity indicates that the AMP, which is normally effective in killing bacteria, is not working as well as it should. This discovery is their first proof of the importance of a capsule in resisting AMPs.
As evidence for the importance of the capsule, the researchers investigated the susceptibility of encapsulated B. anthracis to human beta defensins. With over 30 different human beta defensins (HBD), the researchers chose to focus on HBD-1-4 and compare the susceptibility of the encapsulated wild-type strain and the non-encapsulated capA mutant strain (Figure 7). The bacteria from both encapsulated and non-encapsulated strains were incubated with the selected human beta defensins, and survival percentages were calculated (Figure 8).
Figure 6: Some of the defensins used in this research(HNP3, HBD2, and insect defensins)
Figure 7: B. anthracis with capsule on right and without capsule on left (removal by capsule depolymerase)
Figure 8: X-axis, with black bars representing the WT strain and white bars representing the capA mutant strain. The bars are separated based on the human beta defensins (HBD-1, 2, 3, 4) that were used to treat them. The y-axis displays the survival percentages, which were calculated by dividing the CFU (colony forming unit) with defensin by the CFU without defensin. The data shown is the mean of three replicates, including the standard error of the mean (SEM). The significance of differences in survival between the WT and capA strains was determined by a two-tailed Student's t-test, with ** indicating p<0.001 and **** indicating p<0.0001.
The general trend shown in figure 8 indicates that the percentage survival for non-encapsulated strains is much lower (white bars). The result shows that even encapsulated bacteria can get killed with the human beta defensins (HBD). However, this killing of encapsulated bacteria was at a lower percentage when compared to the non-encapsulated bacteria. Statistically, the percentage of encapsulated bacteria killed were 41- 95% with HBD-1-4. On the other hand, non-encapsulated bacteria had 90- 99% killing rate with HBD-1-4. These findings show that the non-encapsulated bacteria is killed more than capsulated. This trend isn’t replicated for HBD-4 (Figure 8). Overall, defensins can use different bacteria-killing mechanisms (Figure 5) to kill B. anthracis without a capsule cover more than B. anthracis with a capsule. This suggests that the B. anthracis capsule protects it from several human beta defensins.
Figure 9: A) Interference contrast (DIC) and fluorescence microscopy images of non-encapsulated capA mutant (top row) and encapsulated WT (bottom row). B & C) Fluorescence microscopy images of WT bacilli encapsulated. They used HBD-3 defensin in these experiments to see if the defensins physically bind to the capsule layer of the encapsulated WT strain. They added 5 ug of HBD-3 to both the encapsulated WT and non-encapsulated mutant strains. They observed the results using fluorescence microscopy and detected binding with rabbit anti-HBD-3 and AF594 conjugated goat anti-rabbit IgG antibodies.
They conducted further experiments with just HBD-3 to demonstrate the effect of encapsulation. When they did antibody staining and microscopy experiments (Figure 9), they found HBD-3 on the outer surfaces of the capsule of an encapsulated bacteria. However, HBD-3 was on the cell wall (past the capsule) of the non-encapsulated bacteria (Figure 9A). The antibody they used for imaging (IgG and IgM) couldn’t penetrate the capsule, so it did not reveal whether there were HBD-3 molecules on the cell wall of the WT capsulated strain. So, they took a different approach and labeled HBD-3 with the red fluorescent dye Atto-594 before incubation with the encapsulated B. anthracis. This showed the researchers that HBD-3 was in the cell wall of the encapsulated bacteria as well as within the capsule layer (Figure 9C).
They concluded that HBD-3 can gain access to and bind to the cell walls of both bacilli strains, implying that it can kill both encapsulated and non-encapsulated B. anthracis. However, because some HBD-3 is bound within the capsule layer, it has a lower chance of reaching the cell wall and thus lower killing of encapsulated bacteria. They also used flow cytometry to quantify the amount of HBD-3 bound to the cell wall and discovered that a significant amount of HBD-3 was prevented from reaching the cell wall. Encapsulation, as a result, reduces the amount of HBD-3 that can reach the cell wall. Therefore, defensins, which protect our bodies from pathogens, will be unable to effectively kill bacteria cells because the capsule acts as an effective barrier.
Scientists study the characteristics that make diseases virulent in order to find treatments. As we've seen in B. anthracis, the capsule is one of the most important virulence factors, and it's thought to be due to anti-pathogenic properties against immune cells. As a result, they conclude that this increases our understanding of how bacteria/pathogens contribute to disease and emphasizes the importance of countermeasures. In future experiments, they want to see if sequestration by capsule interferes with this defensin's function. One criticism is that they could further investigate the level of significance in killing when non-encapsulated versus capsuled B. anthracis were incubated with human alpha and beta defensins, as there were inconsistent killing results where capsulation didn't result in a difference in killing when HBD-4 was used.
Figure 10: Antibiotic resistance. AMPs the next gen therapy
Antibiotic treatments are becoming increasingly difficult as bacteria evolve and become resistant (Figure 10). This has resulted in a disease burden in healthcare, serious health issues, and a significant economic impact. Therefore, more research is being conducted to identify the specific features of bacteria that contribute to their virulence. Antibiotics, such as penicillin or ciprofloxacin are used to treat anthrax. Even if there is a cure for B. anthracis, studying its characteristics, such as the capsule described in this paper, can help us develop new treatments and improve the efficacy of existing vaccines. Understanding the capsule can also be applied to understanding and combating other diseases with similar characteristics. Furthermore, these add to our understanding of AMP, which is thought to be the next therapy that is an alternative to antibiotics (Figure 10). Overall, this research into the properties and functions of B. anthracis' capsules has the potential to uncover novel treatments to combat the evolution and resistance of this bacteria.
Ruth Fekade '23 is a Biology and Computer Science double major from Ethiopia. She works in the Camp Lab, where she studies the metabolic shutdown process of sporulating Bacillus subtilis. After graduation, she will be working as a research technician before pursuing a graduate degree. In her free time, she enjoys playing squash, trying new food, and reading fiction.
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