Human bodies are frequently described as temples, but it is more accurate to call them fortresses. The innate immune system forms moats and ramparts which repel microbial invasion, while the adaptive immune system creates specialized weaponry to eliminate hostile intruders. This fortress is almost constantly under siege by hostile microbes seeking to access the nutrient-rich environment of the human body, so immune responses have evolved to be very effective at guarding those resources (Figure 1). Unfortunately this is not the only example of evolution in this conflict. As clever as the immune system is, pathogenic bacteria is equally so in its quest to capture biometals.
Figure 1. The Battle of Neville’s Cross by Louis de Gruuthuse depicting a medieval siege
Biometals are metal ions that are used to accomplish biological functions. The most abundant biometal in the human body is calcium, but other common metals include iron, magnesium, and zinc. These metals play a role in almost every aspect that keeps the human body functional. Calcium is critical for stability from cell membranes to bone structure, magnesium is key for RNA polymerase and ATPase, and iron ensures that enough oxygen is available to keep the whole ship afloat. Interestingly (and conveniently for this post), zinc didn’t really get the attention it deserved. The scientific community identified that there was iron in blood in the 18th century, but it took until the 1960s to say that zinc is necessary for human life. Since then research has identified that zinc is a critical component in managing the immune system. In addition to its responsibility over DNA replication and RNA transcription, zinc is crucial for developing non-specific and acquired immunity cells. However, zinc is also essential for maintaining metabolism and replication for pathogenic organisms, which leads to a “tug-of-war” battle between pathogens and host’s metallic defenders over who gets to thrive.
Figure 2. Wright’s stain of blood from plague victim showing Yersinia pestis with bipolar staining (from Public Health Image)
Figure 3. Plague culture of Yersinia pestis on blood agar (from Science Photo Library)
A microbe that embodies this rivalry is Yersinia pestis, the causative agent of plague. It is a gram-negative coccobacillus bacterium with a distinctive bipolar staining effect at the microscopic level (Figure 2) along with irregular “fried egg” shaped colonies in culture (Figure 3). We have gotten very good at identifying our pathogenic enemy due to our shared and catastrophic history. Though Y. pestis was first identified in 1894, it is responsible for some of the most devestaric epidemics throughout history. The most famous example is the Black Death in which almost one-third of Europeans died, though there were countless others with plenty of devastation (Figure 4). Though it can seem like a disease of the past, Y. pestis is still present around the world. It can seem like a far away threat from times long past, but plague is endemic to prairie dogs in the Southwestern United States.
Figure 4. The Triumph of Death by Pieter Brugel the Elder depicting the Black Death
Thankfully modern medicine has treatments to control plague infections, but Y. pestis and its pathogenic toolkit are intimidating on their own. Let’s return to the castle under siege. While the defenses are prepared for a simple battle, Y. pestis takes a stealthier approach. When pathogenic cells enter a human host, the immune system responds and eliminates most of the invaders. A couple, however, can secretly survive inside of macrophages in order to synthesize their virulence factors for when they emerge from hiding. These factors include outer membrane proteins which help the organisms resist phagocytosis. Once inside, Y. pestis has a new challenge: find nutrients in order to survive. In an effort to protect its resources, the human body sequesters its metals, such as zinc, through a process called nutritional immunity. To counter this, Y. pestis uses a siderophore called yersiniabactin (Ybt) to bind and retrieve biometals from the host cell. This has been primarily looked at in terms of iron, but that leaves a less explored angle for another common transition metal in the human ecosystem: zinc.
In 2021, Price et al. published a paper examining this question by exploring how yersiniabactin contributes to virulence independent of iron acquisition. They hypothesized that yersiniabactin contributed to the acquisition of other biometals independently from its known relationship with iron during Y. pestis infection. This builds off of previous studies that reported yersiniabactin increasing Y. pestis growth in zinc-limited media in the absence of the zinc transporter ZnuABC. They additionally focused on if ZnuABC and yersiniabactin help overcome calprotectin-mediated zinc nutritional immunity when infecting mammalian hosts, as calprotectin is the primary barrier to zinc for Y. pestis. In order to prove their hypothesis, the researchers designed a series of experiments to test each step of their logical reasoning. Through this, they were able to gather information confirming that yersiniabactin contributes to Y. pestis virulence independently of iron acquisition and that Y. pestis induces calprotectin expression during infection (which then proceeds to restrict the pathogen’s growth).
Figure 5. IC50 of calprotectin when incubated with Y. pestis mutants. Mutant names represent what biological machinery is deactivated (Irp2 is responsible for encoding one yersiniabactin subunit, ZnuBC/ZnuA are ATP-binding cassette (ABC) transporters responsible for moving zinc inside of the cell, and YbtX is an inner membrane protein that helps replace normal transport machinery for biometals). Mutant names with plus signs indicate a reintroduction of genes for a specific acquisition system to ideally restore function via horizontal gene transfer. Statistical comparisons using one-way ANOVA between test groups: *P<0.05, ***P<0.001, ****P<0.0001 (adapted from Price et al.)
With that foundation established, they decided to explore their hypothesis by comparing the half-maximal inhibitory concentration (IC50) of each strain to compare their growth when exposed to calprotectin (Figure 5). The IC50 for this study represents the concentration of calprotectin needed to inhibit the growth of Y. pestis by 50%. This is similar to ID50 and LD50 in relation to antibiotic resistance, and can be interpreted in a similar manner. The higher the calprotectin IC50 value is, the more effective that strain is at resisting calprotectin-mediated nutritional immunity (and vice versa). These results support that both ZnuABC and yersiniabactin are redundant forms of zinc acquisition that do help Y. pestis evade calprotectin growth restriction. Strains with mutations affecting yersiniabactin and ZnuABC individually experienced less growth than the wild type, indicating that neither have a unique effect when resisting calprotectin. However there was another significant decrease of growth when compared to wild type and single mutation strains when both mechanisms were mutated, supporting the idea of a redundancy between these two mechanisms. Overall this study supports the initial hypothesis and demonstrates that Y. pestis uses yersiniabactin as a zinc acquisition mechanism to evade zinc limitation during infection.
This specific type of conflict between host and invader is ripe for exploration. There are many different biometals which accomplish many different known functions in organisms, but there are likely many unknown applications to be discovered. Remember zinc was only proven to be necessary for human cells in the last century, and there is still much work to be done to explore its impact on bacterial pathogenesis. Even simply from the experiment discussed here there are many avenues of possible lines of study. What about other bacterial strains that use yersiniabactin or other host proteins for sequestering metals? Is the effectiveness of yersiniabactin limited by its ability to acquire multiple types of biometals? Are there similar interactions with other types of siderophores? This paper provided useful insight into one of humanity’s greatest microbial threats, and hopefully this can help us plan modifications to our immune system stronghold to protect against further attacks.
About the Author:
Aoife Weischedel ‘25.5 is a Biology Major and Computer Science minor from Connecticut. They are primarily interested in using data science to improve the healthcare field, but they are more often than not reading or solving puzzles with their cat, Persephone. They plan to apply to PharmD programs post graduation with the hopes of working as a clinician-researcher in the future.
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