Borrelia burgdorferi: Get Outta My Knees!

By: Stella Elwood '19

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If you live in the Northeast, you probably know someone (or at least someone’s dog) who has been diagnosed with Lyme disease. This common illness, diagnosed at a rate of 30,000 new human cases per year, is caused by the bacterium Borrelia burgdorferi, which is carried and transmitted by black-legged ticks. These ticks ingest the pathogen whenever they take blood meals from infected animals, which can include deer, rodents, humans, dogs, and many others! However, if a tick already has B. burgdorferi in its system, it spreads it to whatever animal it bites (essentially by backwashing the bacteria into its prey, because ticks are messy eaters).

Once B. burgdorferi enters the bloodstream via a tick bite, it can travel to and attach to surrounding tissues, where it eventually burrows into them! This process can cause fever, joint pain, depression, and various other unpleasant symptoms in the infected person or animal. Despite effective antibiotic treatments for Lyme, if left undiagnosed for too long, chronic and currently untreatable versions of the disease can arise, which causes the symptoms to become permanent.

Figure 1. Visual representation of a spirochete drilling into tissues. Source

Although the the B. burgdorferi bacterium exhibits gram-negative traits, such as inner and outer membranes, it is technically classified as neither gram-negative or gram-positive due to its unusual staining patterns. It, unlike most bacteria, which have one circular chromosome, has a linear chromosome, and holds 21 different plasmids (extracellular genes that often code for beneficial traits, like antibiotic resistance). If detected by healthcare providers earlier on in the infection, the bacterium is sensitive to strong doses of Doxycycline and other antibiotics within the same family. However, its specialized method of tissue colonization makes total eradication of the pathogen a little tricky sometimes.

Figure 2. The structure of B. burgdorferi’s inner and outer membranes. The balloon-like and square shapes on the outer membrane are some of the outer surface proteins (Osp’s). The hair-like bundle in the middle of the picture is the internal flagellum. Source

Many features of the bacterium allow it to perform effective tissue infection. For one, B. burgdorferi is a spirochete, meaning that it has an unusual spiral structure and a thread-like appendage called a flagellum located in between its outer and inner membranes. Many researchers believe that the bacterium’s odd shape allows it to effectively twist itself into tissues, much like a corkscrew (Figure 1). However, one of the newer and more complex bodies of research pertains to B. burgdorferi’s outer membrane surface proteins (Figure 2)! The bacterium has an enormous variety of these proteins, many of which have not been fully studied yet. After years of research, microbiologists are just beginning to understand the role that each protein plays during infection, but some are known to help adhere B. burgdorferi to the extracellular matrices of animal tissue cells (Figure 3). Infectious bacteria of all kinds often use adhesion to stay put in their chosen location and evade antibiotics, which makes disease treatment much more difficult for doctors and veterinarians!

Figure 3. The process of infection via adhesion proteins. Source

A recent study, conducted by Yi-Pin Lin and associates at the Tufts School of Medicine, focuses specifically on an outer membrane surface protein called BBK32. This protein is known to bind to multiple ligands, including fibronectin, a glycoprotein, and a class of disaccharides called glycosaminoglycans (GAGs), which are both commonly found on certain types of mammalian cell surfaces. Lin et. al explore the interactions between B. burgdorferi’s outer membrane surface proteins and both fibronectin and GAGs in order to better understand the progression of Lyme disease on a microbial level.

In the first portion of their research, the scientists measured the binding affinity (or likelihood of binding to occur) of fibronectin and GAGs to the BBK32 protein, and found that both molecules had distinct binding sites on the protein. This result suggested to Lin et. al that the two ligands may carry out unique functions during B. burgdorferi tissue infection. To further investigate these different binding sites, the researchers deleted certain amino acids that make up the BBK32 protein, then tested whether or not these alterations affected the protein’s ability to attach to fibronectin or GAGs. They discovered that BBK32 that lacked amino acids 45 through 68 could not bind fibronectin, and that BBK32 without amino acids 158 through 209 could not bind GAGs. However, the loss of amino acids 45 through 68, those responsible for fibronectin binding, had no effect on GAG binding. Likewise, BBK32 proteins without amino acids 158 through 209, the ones related to GAG binding, could still bind to fibronectin. These results, which show that separate amino acids facilitate GAG and fibronectin binding, further suggests a distinct function for both molecules.

Taking the study to the next level, Lin et. al then decided to specifically explore these predicted individual roles in tissue colonization. Different types of tissues may or may not contain GAGs or fibronectin, and the B. burgdorferi may need to employ various patterns of infection depending on a cell’s type. To begin, the researchers infected human laryngeal epithelial cells, which are known to not produce fibronectin, with B. burgdorferi. They concluded that the lack of fibronectin did not hinder the adhesion of the spirochete to the tissue. However, when the GAGs were removed from the surface of the epithelial cells, the percentage of successfully binding bacteria decreased. This result suggests that the GAGs play an integral part in adhering B. burgdorferi to tissues void of fibronectin, such as those comprised of laryngeal epithelial cells. Conversely, when the research team infected Chinese hamster ovarian cells, which are incapable of making GAGs, the BBK32 protein was still able to bind to the extracellular matrix. Unlike in the laryngeal epithelial cells, fibronectin must be responsible for the adhesion of B. burgdorferi to ovarian cells. In this way, Lin et. al were able to determine that fibronectin and GAGs promote binding to different cell types, diversifying the range of tissues that the pathogen can effectively colonize.

The scientists then moved on from isolated tissue samples to living mice, injecting each subject with BBK32 mutants. After examining various sections of the mice’s bodies, they found that the presence of BBK32 enhanced joint (ex. knee) infection, but had no effect on colonization of either the ears or the heart (Fig. 1). To determine whether joint adhesion was a product of fibronectin or GAGs, the team then injected B. burgdorferi containing BBK32 without binding sites for either of the two molecules. Unable to adhere to the joints, the bacteria localized in the heart and skin tissues. They then injected one strain without the fibronectin site and one strain without the GAG site into two separate mice, leading them to discover that the BBK32 lacking GAG sites bound to the joints much less frequently (Figure 4). In this way, Lin et. al discovered that GAGs are responsible for joint colonization, which leads to the persistent achiness commonly associated with Lyme disease!

Figure 4. Localization of B. burgdorferi mutants across various tissues. In this case, B31A is a non-infective strain of the bacterium, pBBK32 produces both GAGs and fibronectin normally, the GAG- strain does not contain GAG binding sites, and the Fn- strain does not contain fibronectin binding sites. The dots above the GAG- category demonstrates that the absence of GAG binding sites only affects binding to tibiotarsal (joint) tissues (ie. the dots for tibiotarsal tissue are much lower down on the graph’s y axis than those for the heart and ear tissue). The dots above the Fn- category shows that proteins without fibronectin binding sites are not affected, as they are similar to the dots above the pBBK32, or “normal”, category.

Although this finding is a step in the direction towards unraveling the mystery of curing Lyme disease, it is complicated by the fact that there are many different GAGs! This study focused primarily on the binding of BBK32 to dermatan sulfate, but further experiments should examine other GAGs, to fill in more missing puzzle pieces surrounding B. burgdorferi adhesion. In this way, we may be able to target the binding sites responsible for adhesion, un-stick the pathogen from infected tissues, increase antibiotic sensitivity, and effectively treat joint pain in cases of chronic Lyme disease. However, this paper creates a helpful foundation for scientists who want to start in on this work, and it specifically uncovers a key causative component of knee pain, one of the most infamous aspects of Lyme disease.

Figure 5. Cranial neuritis (nerve inflammation) in a patient with CNS B. burgdorferi infection. The white areas indicated with arrows show the presence of neuritis.

Meanwhile, there are still many (scarier) types of tissue colonization that are not yet linked to specific outer membrane surface proteins. For example, although most people who are familiar with Lyme disease rightfully associate it with knee pain, individuals who have been infected by B. burgdorferi for prolonged amounts of time without treatment suffer from central nervous system (CNS) colonization (Figure 5). During this phase of the infection, B. burgdorferi can use its outer membrane surface proteins to serve a function unrelated to tissue binding. Here, the surface protein OspE attaches to regulatory proteins found in the body, which protects the bacterium by inhibiting the host’s complement immune response. Most of the research on Lyme disease in the CNS focuses on this topic, but to my knowledge, no one has investigated the outer surface proteins in charge of adhering to neural tissues. This is especially troubling, as B. burgdorferi is known to colonize the dura mater, one of the three layers of protective tissue surrounding the brain and spinal cord. Once this infection occurs, the host may experience severe symptoms, such as an altered mental state and sensory issues. To better understand this stage of the disease, researchers must continue their important work on uncovering the specific adhesive properties of B. burgdorferi’s (at least) 19 outer membrane surface proteins!


Sources

Divan, A., Casselli, T., Narayanan, S. A., Mukherjee, S., Zawieja, D. C., Watt, J. A., . . Newell-Rogers, M. K. (2018). Borrelia burgdorferi adhere to blood vessels in the dura mater and are associated with increased meningeal T cells during murine disseminated borreliosis. Plos One, 13(5). doi:10.1371/journal.pone.0196893

Lin, Y., Chen, Q., Ritchie, J. A., Dufour, N. P., Fischer, J. R., Coburn, J., & Leong, J. M. (2015). Glycosaminoglycan binding by Borrelia burgdorferi adhesin BBK32 specifically and uniquely promotes joint colonization. Cellular Microbiology, 17(6), 860-875. doi:10.1111/cmi.12407

Panelius, J., Meri, T., Seppälä, I., Eholuoto, M., Alitalo, A., & Meri, S. (2008). Outer surface protein E antibody response and its effect on complement factor H binding to OspE in Lyme borreliosis. Microbes and Infection, 10(2), 135-142. doi:10.1016/j.micinf.2007.10.016


About the Author:



Stella Elwood '19 is a biology major and gender studies minor from Stoughton, Massachusetts. She recently committed to Tufts School of Veterinary Medicine, where she hopes to prepare for a career in shelter medicine. In her free time, Stella likes to play video games, cook delicious vegan food, and hang out with her beautiful pit bull son, Rollo.