BicA and Borrelia

Introduction

The featured image was adapted from a cartoon on the CreativeBioLabs website. This company is trying to develop a Lyme Disease vaccine. Two Borrelia proteins will be discussed. BmtA is the Borrelia metal transporter. We are not sure if it transports metal ions across the inner or outer membranes. We are also not entirely sure if BicA is in the periplasmic space or in the interior of the spirochete.

Metals inside Borrelia

Na⁺, K⁺, and Mg²⁺ are abundant metal cations in the environment and in all living cells. Trace elements in the environment may be cofactors in structural proteins and enzymes that catalyze reactions essential for life. A group of investigators from

• the Divisions of Geographic Medicine and Infectious Diseases at Tufts University
• Geographic Medicine at Tufts University
• the Trace Element Research Laboratory at Ohio State University

used a technique called Inductively Coupled Plasma-Sector Field Mass Spectrometry (ICP-MS) to measure the transition metal content of Borrelia burgdorferi. ICP-SFMS is simply more sensitive than the traditional ICP-MS. These authors discovered that manganese, iron, copper, and zinc are the most abundance trace minerals in Borrelia burgdorferi. [1] All of these metals are important cofactors in proteins needed to sustain life.

When essential Fe and Cu go bad

Iron and copper are particularly notorious for transferring electrons from one place to another. When these elements transfer electrons to molecular oxygen, reactive oxygen species are generated. Sometimes this superoxide generating reaction happens unintentionally in the electron transport chain. The two oxygen in O₂ are generally linked together by a double bond. When two of those electrons “switch” to a vacant “spot”, these unpaired electrons are available to form pairs with incoming electrons. Once this pairing takes place, there is no going back to the double bond O₂. A rule in chemistry prevents oxygen atoms from being surrounded by more than eight valence electrons. We are left with an unpaired electron, something Nature does not tolerate for too long.

Our friendly, neighborhood superoxide radical finds a suitable place to dump that extra electron: Cu²⁺. These are the Fenton and Haber Weiss reactions.

O₂^●- is superoxide. The “●” is the unpaired electron. ^●OH is the hydroxyl radical. Wikipedia authors have a lot to say about how damaging this radical is to macromolecules in both the pathogen and the host. This, good readers, is how copper kills. Make careful note of hydrogen peroxide, H₂O₂. We will encounter this reactive oxygen in the next section. How does this Lyme Disease spirochete keep these essential trace elements from doing it harm?

Introducing BicA

This study follows in the footsteps of a previous study showing that BicA, an iron binding protein that is a member of the DNA binding Proteins of Starved bacteria. [2] BicA was found to be required for Borrelia survival in the tick as well as transmission to the host. Because Borrelia can grow without iron, Wang and coauthors hypothesized that the purpose of BicA was to detoxify iron and other transition metals. They used two infectious strains of Borrelia: B31 and 297. Wang and coauthors genetically engineered BicA (the putative heavy metal chaperone) and bmtA out of Borrelia.

Fig 1 BicA concentrates Cu and Fe in Borrelia.

In panel A we see that the wild type B31 spirochete has more Fe and Cu atoms per cell than the spirochetes lacking the gene that codes for the BicA protein. In panel B we are looking at the role of bmtA. Not having it increases the number of Fe atoms per cell and almost totally eliminates Cu from the spirochetes. That Cu content was decreased more than Fe content in the BicA lacking bugs was most unexpected as dps family members are generally thought of as Fe binding proteins.

Spirochetes were grown at 33°C to early stationary phase in the BSK-H media. ICP-FSMS was used to measure the metal ion content of the bugs. Data represent mean (± s.e.m.) of three independent samples. P-values are derived from a two-way anova followed by Bonferroni post-test: ***P < 0.001; ns, P > 0.05. bmtA is also known for its transport of manganese.

Figure 2. Concentrating metals in Borrelia

What is the concentration of these transitions metals in BSK-H medium and what is the concentration in the bugs… assuming they are shaped liked cylinders. Panel A just shows the BSK-H medium concentration of these metals in micromolar. Panel B, the fold concentration of our two pathogenic strains.

For the non-scientist, concentrating over “10³ ” is a 1000x concentration. A 10¹ concentration is just 10x.

Figure 3. The Cu⁺ binding domain of BicA

Figure 3 is a followup on Figure 1. This post will not present all of this figure to the lay reader. To make a long story short, the cysteine concentrated region (CCR) has been shown in similar proteins to become fluorescent when it binds to Cu⁺. The authors were able to show a similar fluorescence with purified BicA protein. Some site directed mutagenesis was also performed. Fe likes to bind to acidic amino acids glutamate (E) and aspartate (D) in the ferritin like Dps core. These amino acids were mutated to similar amino acids with neutral side chains. In this case the positive charge (blue) neutralizes the negative (red) charge. The Fe binding sites are starred. Likewise the 6 cysteines were mutated to alanines. Alanines are cyteines without the thiol (SH) group.

Note that the wild type BicA protein binds seven coppers. Get rid of the six cysteines and the ratio goes down to only one Cu per BicA molecule. Get rid of the aspartate and the glutamate as well and the ratio is still one. Perhaps that one Cu is binding to the histidines. At any rate, this protein is most remarkable. Purified BicA seems to bind more Cu (7 atoms) than what is seen per spirochete cell (Figure 1A, 4-5 per cell). Is BicA doing anything to protect this Borrelia spirochete against the reactive oxygen species discussed in the introduction?

Figure 4 BicA, Borrelia growth in Fe and Cu

Wang and coauthors grew Borrelia in BSK-H media in the presence of indicated amounts of copper and iron. The Medscape reference range for free copper in our blood serum is 1.6-2.4 μmol/L. The Medscape reference range for free iron in males is 14-32 μmol/L. Note that 14-32 μmol/L is the same as 14-32 μM.

The “Log10 (Growth %)” units on the Y -axis requires a pause to understand. Log₁₀ of 100% is 2. At the lowest concentrations of Cu and Fe tested the bugs are lumbering along at close to 100% of normal growth. A -4 Log₁₀ Growth % is growth at 0.0001% of normal growth. There is a ± BicA tipping point. For Fe that point occurs at about 80μM. For Cu the tipping point is between 100-200 μM. Not having BicA ○ leads to less % normal growth. Recall the trouble that is generated when Cu mixes H₂O₂.

Figure 5, Enter H₂O₂

Panels 5A-5B compare Wild type (WT) and BicA knock out Borrelia in the presence of H₂O₂ with or without Cu and Fe. We can get the story with just these three panels.

Panel C tells us that H₂O₂ kills Borrelia. Having BicA does not make much difference as long as there is no extra Fe or Cu present. Note that “survival” is not the same thing as % normal growth in Figure 4. It is possible to be alive, just not growing. When we start adding Fe or Cu, the killing starts. Having BicA ● really drops the survival.

What does this mean?

The working hypothesis is that having this Cu⁺ around is a good thing until an immune cell starts releasing H₂O₂.

References

1. Li X, Wang P, Lutton A, Olesik J. Trace Element Analysis of Borrelia burgdorferi by Inductively Coupled Plasma-Sector Field Mass Spectrometry. Methods Mol Biol. 2018;1690:83-94.
2. Wang P, Lutton A, Olesik J, Vali H, Li X. A novel iron- and copper-binding protein in the Lyme disease spirochaete. Mol Microbiol. 2012 Dec;86(6):1441-51. PMC free article