Although I have never contracted Lyme disease myself, I have seen countless friends and acquaintances affected by the horrible disease. Lyme disease is endemic to North America, central, eastern, and northern Europe, and Asia. It emerged in the 1960’s and 70’s in Lyme, Connecticut when community members there began to experience symptoms of unexplained paralysis, fatigue, and headaches. It wasn’t discovered until the 1980’s that the microorganism, Borrelia burgdorferi, carried by ticks was responsible for the sudden onset of their illness. Today, many Americans and especially New Englanders are all too familiar with the dangers that tick bites can cause and are taught to look for the dreaded erythema migrans. While this bacteria can also cause Lyme disease in other animals, this red circular rash is only seen in humans and is the prominent sign of Lyme infection. Lyme infections are also confirmed through serologic testing, although these antibody tests are subject to blurry boundaries between positive and negative test results, something that I chose to study as a part of my senior thesis!
Figure 1. Example of erythema migrans on the face of a patient infected with Lyme disease. Source
Figure 2. CDC trail sign displaying dangers and prevention of tick bites. Source
The bacteria B. burgdorferi is part of a larger phylum of bacteria, spirochetes, that also cause other infectious diseases in humans such as relapsing fever, leptospirosis, and syphilis. Although Lyme infections first emerged in the 1960’s, the first accounts of syphilis outbreaks emerged in the late 15th century. Clearly spirochete bacteria have evolved alongside animals for hundreds of years. This long standing spirochete phylum of bacteria were named as such for their long, spiral morphology that is thought to influence its virulence in hosts. While extensive research has focused on the effects of B. burgdorferi and other spirochetes on human and other animal hosts, very little is known about the basic biology of the phylum itself.
Figure 3. B. burgdorferi spiral morphology. Source
Spirochetes get their unique corkscrew structure from their peptidoglycan (PG). In gram negative bacteria like B. burgdorferi, the PG is depicted in the dark brown section in Figure 4. (below). Layered in between the inner and outer membranes, the PG is a layer of cross-linked peptides (amino acids) with a glycan (sugar) backbone. This provides the structure for the cell wall of all bacteria and depending on its synthesis, results in different cell shapes. Interestingly, bacterial daughter cells inherit old sections of PG from the mother cell and synthesize new sections of their own. As different sections of the PG grow at varying rates, the overall shape of the cell is established such as cocci, rods, or spirochetes. PG is a key topic in microbiology research particularly valued for its application in antibiotic development. One specific class of antibiotics targets the breakdown of the PG causing bacteria to rupture under osmotic pressure. Because human cells do not contain PG cell walls, this is a unique target for the treatment of pathogenic disease. A recent paper therefore decided to investigate the role of PG synthesis in B. burgdorferi to better understand its role in creating the spiral morphology (and therefore virulence) and its potential application in Lyme disease treatment.
Figure 4. Gram negative cell depiction of PG (spotted brown layer) between outer and inner membranes. Source
Figure 5. Peptidoglycan (PG) synthesis rates at different sites influence overall cell morphology. New areas of growth shown in the purple sections and lead to asymmetric growth patterns, contributing to the bent shapes. Source
In their paper, Jutras et al. first were able to track PG synthesis during B. burgdorferi replication in culture by using fluorescence microscopy. Here, they used HADA, a D-alanine analog tagged with fluorescent protein as a proxy for new PG growth and found discrete sections of PG synthesis. Remember, D-alanine is just one type of amino acid used in the peptide cross-linking of PG. The authors were able to confirm that HADA represented new PG by comparison of fluorescently tagged PG layers that represented non-forming (old) PG. In looking at the fluorescence microscopy images taken across the cell replication cycle, the authors confirmed three distinct zones of PG synthesis that correlated with relative length of the cell as shown in Figure 6., Panel A. These experiments demonstrated that PG synthesis was spatially regulated across the cell!
Figure 6. Panel A. (left) overlays phase image and the HADA fluorescence image that indicate three distinct zones of PG synthesis at the left, center, and right positions along the cell body. Panel (B) (right) shows the intensity of HADA (green) and PG sacculi (red) to confirm old PG was consistently found along the length of the cell, but that new PG synthesis was only found in three relative sites in the cell body.
After confirming these three new sites of PG synthesis the authors performed additional experiments to determine how HADA incorporates into the PG. They confirmed that HADA incorporates through a periplasmic exchange reaction in the old (existing) PG catalyzed by penicillin-binding proteins (PBPs). In essence, PBPs are periplasmic enzymes that help link together individual amino acid chains or help to disconnect them. They are named penicillin binding proteins because when bound to the antibiotic penicillin, the enzyme is unbound from the crosslinked PG, disrupting the delicate osmotic balance (of water and nutrient concentrations) between the inside and outside of the cell wall, as depicted in Figure 7.
Figure 7. Depicting the role of PBPs (in green) and their loss of function when bound to penicillin antibiotic (blue molecule) resulting in a burst bacterium. Source
Therefore, PBPs enable the newly synthesized PG to be incorporated into the existing structure and remove old sections of PG to do so. Their crosslinking mechanism in action is shown below in Figure 8.
Figure 8. Depiction of PBP in action crosslinking monomer chain units of the PG. To view, on YouTube, click here.
They used bocillin (a fluorescent analog of penicillin) as a proxy for these PBPs and found colocalization with the same HADA sites using fluorescence microscopy. They then used the bocillin and HADA proxies to examine the emergence of the new PG and its incorporation over the stages of the cell cycle by measuring fluorescence intensity. The authors found a pattern that correlated to cell length where the longer cells gew, the more HADA sites emerged (up to 3) at relative positions along the cell at the ¼, ½, and ¾ locations. This was shown in Figure 6. Panel B, where new PG growth (HADA) is marked by the green peaks at the relative positions, representing the green fluorescence, while old PG was depicted with the red fluorescence signal that remained constant. Importantly, just before cell division, the first HADA zone disappeared as shown in Figure 9-III, where the middle, original HADA zone at the ½ position is no longer visible as the cell reaches maximum length by stage III as shown in Figure 9. This indicated that new PG synthesis began after cell division but decreased over the length of the cell cycle, suggesting the first site of PG synthesis was now mature. This experiment then confirmed that not only was PG synthesis spatially regulated, but also temporally, across the length of the cell cycle!
Figure 9. Panels I, II, and III correspond to images of the growing cell with illuminated HADA sites, one in panel I, two in panel II, and the disappearance of the central site in panel III.
Finally, the authors wanted to understand the growth pattern at these sites of new PG synthesis and their incorporation into the existing PG. In this experiment they introduced a new proxy for existing (old) PG, a different D-alanine analog acronymed as NADA. They hypothesized two potential options that would result in the elongated spiral shape: PG multi-layering or elongation. In multilayering, new PG would pile on top of old PG to create multiple layers of PG, thickenicking and strengthening the PG symmetrically across the whole cell. In elongation, new PG would replace old PG asymmetrically to produce growth only laterally. If HADA zones were reflective of multilayering, they expected the signal intensity of fluorescence would appear uniformly all over the cell (colocalization). This would indicate strengthening all over existing PG reflected by the NADA sites, if new PG (HADA) layered on top of existing PG (NADA) without the old PG being removed first. Alternatively, if HADA zones were reflective of elongation of the PG, old PG would be replaced by new PG, shown through replacement of NADA (red) by HADA (green). Their results supported the latter, that new PG was replacing old PG and indicated elongation, as shown in Figure 10 (below). This explained how the B. burgdorferi attained such long, thin spiral cell shape.
Figure 10. This experiment shows the phase and fluorescence microscopy imaging identifying the replacement of old PG (NADA) in green replaced by the new PG (HADA) in red.
These exciting discoveries show that the PG of B. burgdorferi is temporally regulated across the cell cycle and spatially regulated at relative sites along the cell body. The authors were also able to extend some of these findings to other B. burgdorferi strains and found similar results of spatial regulation proportional to the length of the specific strain’s cell length. Interestingly, these spatial regulations were not seen in other spirochetes outside of the Borrelia genus. Therefore, the cumulative impact of these findings provides a wealth of new understandings that could inform newly tailored antibiotic therapies able to target only the unique PG of B. burgdorferi in treating Lyme disease. I anticipate that these results will frame new research and clinical applications, hopefully benefitting the tens of thousands diagnosed with Lyme each year.
About the author:
Helen McGunnigle F16 is a Division III student at Hampshire College studying human health, anthropology, and queer and feminist science studies with a 5-College Certificate in Culture, Health, and Science. She hopes to become a health clinician in the future, focusing on complementary/alternative medicine and nutrition. From Allentown, Pennsylvania she enjoys playing music, seeing friends, and having the kitchen to herself.
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