The Powerhouse of Borrelia burgdorferi

By: Grazia Essiam-Lindsay, Nazia Sadeq, and Nana Akua Sekyi-Appiah

The extremely common disease known as Lyme disease exists due to a predominant causative bacterial species known as Borrelia burgdorferi. The disease is transmitted by the bite of infected nymphal and adult deer ticks, which by the way are the size of a poppy seed! This disease is conveniently aggressive in the summer when people prefer to be outdoors most of the time. The attack tends to form a ring shaped mark on the skin, which is most commonly known as the “bull’s eye” rash of Lyme disease. Luckily, the rash is hardly itchy or painful. However, there are other unpleasant symptoms such as headache, fever, chills and/or fatigue. If the disease is left untreated, it can be extremely harmful as later symptoms may involve the heart and the central nervous system and become difficult to treat. Source

Figure 1. Source

Science places B. burgdorferi under the spirochete class. Spirochetes are spiral shaped bacteria that have a vigorous, unusual means of motility. Located in the space known as the periplasm lies the golden, majestic periplasmic flagella. According to Li et al., this golden token allows spirochetes to be able to move through highly viscous, gel-like medium. Such mediums can be found in delicate parts such as connective tissue and can even restrain the movement of most bacteria found in the this environment. This aggressive motility does not only enable spirochetes to infiltrate tissues in the organism, but it helps them swiftly escape immune responses accelerating through the body of the host organism. Li et al. reveals that there is clinical evidence to support the notion that pathogenic spirochetes are highly invasive; for example B. burgdorferi can be found in the vitreous humour of the eye, brain tissues, and many more tissues of patients infected with Lyme disease. This motility is no joke!

In previous studies, a number of motility genes have been inactivated in order to investigate the pathogenesis of B. burgdorferi. One such gene is FliG which encodes a flagellar motor switch complex protein and plays a huge role in the assembly and motility of flagella. B. burgdorferi consists of two FliG homologues known as FliG1 and FliG2. 

Figure 2. Western blot analysis of the fliG mutants

and their complemented strains. A. Flagella filament

protein FlaA and FlaB are absent in the fliG2- but still

present in the fliG1- mutant. B and C. The cognate

gene products were absent in the respective mutants

and were restored in the complemented strains.

In order to understand the effect of flagellation on the behavior of B. burgdorferi, Li et al. first investigated how FliG1 and FliG2 affect the structure and function of the periplasmic flagella. To do so, mutant genes FliG1- and FliG2- were constructed through a process called genetic recombination using host plasmids. The DNA products were then amplified using Polymerase Chain Reaction (PCR) in order to retrieve more copies of the genes. This is a very common technique used in genetics labs to make clones of genes just so you have more samples to work with.  FliG genes are essential for flagellation of B. burgdorferi because when expressed, they induce the flagella filament proteins FlaA and FlaB, which aid in the synthesis of the flagella. Results from a Western blot analysis, a techniques used to detect the presence of proteins in sample tissue, showed that FlaA and FlaB were present in both the wild type fliG gene and fliG1 mutant as illustrated in Figure 2. They were however absent in the FliG2 mutants, indicating that the fliG2 gene is crucial for flagellar synthesis. Without FliG2, the FlaA and FlaB genes are not expressed; no flagella formation can occur and motility is instantaneously hindered!

Therefore, it is safe to assume that the FliG2 gene codes for a normal motor switch protein which is used in the synthesis of periplasmic flagella. A functional FliG2 gene is necessary for flagellar assembly and motility in B. burgdorferi. Wild Type bacteria and FliG1 mutants are expected to have normal numbers of periplasmic flagella, ideally between 9 to 11. FliG2 mutants on the other hand, are expected to have none. Electron micrographs of the B. burgdorferi depicted in Figure 3 show that the wild type and FliG1 mutants both possess flagella, whereas the FliG2 mutant does not. This again confirms that FliG2 alone is responsible for flagellation and motility.

Figure 3. Electron microscopic analyses of the fliG1- and the fliG2-mutants. The

upper panels are the electron micrographs of outer membrane-disrupted

B. burgdorferi cells, and only one end of the cells was illustrated; the lower panel

is the thin-section electron micrographs of B. burgdorferi. Arrows point to the PFs.

Now, if FliG2 is solely responsible for flagella formation, what does FliG1 actually do? FliG1 encodes an odd motor switch protein, which is not essential for flagellation. Research has shown that the presence of FliG1 in B. burgdorferi enables it to move at very high velocities in thick, viscous mediums. It may sound contradictory that motility is rather sped up in mediums that are naturally supposed to retard movement of the bacteria, but let’s look at it this way; FliG1 acts as a propellant that provides the bacterium with some sort of mechanical energy to counteract the resistance of the viscous medium.

Figure 4. Based on WordNet 3.0,
Farlex clipart collection.
© 2003-2012 Princeton University,
Farlex Inc

To better understand the performance of FliG1, let us consider how a dynamo powered fan works. In this scenario, let’s assume the dynamo, which is an electric generator, represents FliG1 and the fan represents the flagella. The dynamo provides the fan the power/ torque to rotate and work against air resistance seen in Figure 4. Similarly, FliG1 is required to provide the flagella that same power/torque to move against the drag of viscous mediums.  A closeup look on the impact of the two mutants, FliG1- and FliG2-, confirmed that the FliG2 mutant was completely non-motile and that the FliG1- mutant had reduced motility in presence of thick viscous liquid. On B. burgdorferi cells, two bundles of periplasmic flagella are found on opposite ends of the cells. FliG2 genes are situated on both ends, which even elaborates further the importance of FliG2 in flagellation of B. burgdorferi and overall motility of the bacterium. On the other hand, FliG1 is located only on one bundle. A mutation to this gene leads to bacteria that are unable to move relatively fast in viscous mediums, therefore we can assume that FliG1 is a source of additional power to drive B. burgdorferi through dense mediums. In the dark field microscopy analysis, the FliG2- mutant cells were rod shaped and formed chains that were completely non-motile whereas the FliG1 mutant cells were still motile.

Li et al. also conducted a swarm plate analysis to study the relative movement of the mutants in viscous medium. A swarm plate assay was used where the bacteria sample was spread onto an agar plate and incubated for about 72 hrs. After this incubation period the diameters of the bacteria culture was measured and the results showed that the wild type had the biggest diameter (2.1mm), the FliG1 mutant which is known to lose its motility in thick viscous medium had a diameter of 1.2mm, which is considerably smaller than that of the wild type bacteria. The recovered FliG1 mutant in the complemented strain, FliG1-/+, had a diameter of 2.0 mm almost as high as that of the wild type bacteria. This indicates that FliG is crucial for the movement of B. burgdorferi through viscous medium. FliG2 mutant cells only had  a diameter of 0.9mm which matched the initial diameter reading. This confirmed that FliG2- mutant cells were non-motile. However, FliG1- mutants had reduced motility in BSK-II medium (soft surfaces) and a comparatively higher motility in more viscous liquids (1% methylcellulose). These results showed that inactivation of FliG1 somehow repelled the ability of the spirochete cells to be motile in highly a viscous medium.

FliG1 and FliG2, are important for the motility of B. burgdorferi. Though they are homologues, these genes are very different in the roles they play in B. burgdorferi motility. FliG2 is required for flagella formation and a mutation to this gene results in a non-motile and aflagellated bacterium. On the other hand, FliG1 is not a component of flagella formation but helps in motility of the bacterium in the presence of a thick, viscous medium. B. burgdorferi is infamous in the microbial world as a deadly pathogen largely due to the roles played by the FliG genes, especially FliG1, to make it motile in places where other bacteria are not! It will be interesting to investigate in other pathogenic bacteria, whether they possess genes similar to FliG1 responsible for the unique motility that makes spirochetes infectious. Perhaps if FliG1and FliG2 in B. burgdorferi, and hopefully their equivalents in other spirochetes are silenced in through evolution, Lyme disease and other infectious diseases as we know it could very well be eliminated from society.



Reference

Li C, Xu H, Zhang K and Liang FT. (2010) Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in highly viscous media and blocks its infectivity. Mol Microbiol 75: 1563–1576.