One the most significant studies that have been performed was done in 1982 when Dr. Burgdorfer and his team isolated the bacterium B. burgdorferi from the midgut of the tick species Ixodes scapularis. They were also able to isolate the bacterium in Lyme in the bodily fluids of Lyme disease patients and showed that immune responses were linked with the bacterium, proving it is the cause of disease (Burgdorfer 1982).
There has been a lot of research performed since this discovery in an attempt to better understand how B. burgdorferi infects and survives in the human host. Many landmark experiments have helped to elucidate some of the mechanisms by which the bacterium is able to infect the human host and bypass the immune system. One of the first of such experiments was the genomic sequencing of the first strain of this spirochete completed in 1997, which showed part of the genome contains a chromosome of about 1000 kilobases and at least 17 linear and circular plasmids (Fraser et al. 1997). A later sequencing study determined that the bacteria contain 12 linear and nine circular plasmids, which completed the genome-sequencing project for the organism (Casjens et al. 2000). A large number of these sequences encode for known lipoproteins, including outer-surface proteins (Osp’s) A through F, a variable surface antigen (VlsE), a fibronectin-binding protein (BBK32), and decorin-binding proteins (DbpA and DbpB) (Fraser et al. 1997). These proteins are found on the outer membrane and are crucial to the transmission and survival of the bacteria.
Transmission of Disease
B. burgdorferi stay dormant in the midgut of the tick where they express surface lipoproteins OspA and B, which function as adhesion molecules in order to colonize the midgut of the tick. This was discovered by the creation of an OspA/B-deficient mutant form of the bacteria, which showed the lack of ability to colonize and survive in the midgut of the tick (Yang 2004). It was also shown that when a tick feeds, alterations in the temperature and pH of the midgut activate the bacteria and stimulate a change in expression of surface proteins, which then allows for the bacteria to move from the midgut and into the new host (Revel et al. 2001). OspA is downregulated, allowing the bacteria to leave the midgut and OspC is upregulated and plays a crucial role in salivary gland invasion, which allows the bacteria to infect the host through tick saliva. However, the mechanism by which OspC promotes borrelial infectivity is unknown (Steere et al. 2004). Interestingly, in a mouse model, the development of an antibody to OspC induces a downregulation of OspC. Therefore, this antibody response does not completely clear the infection, which presents a possible immune evasion mechanism through OspC (Liang 2002).
Immune System Evasion
The outer surface proteins of B. burgdorferi play a crucial role in evasion of the immune system. Here we will look into two proteins that appear to be two of the most important proteins needed for this function. One protein is the OspE protein, which plays a role in evading the complement system. The other protein is VlsE, which undergoes extensive antigenic variation and is crucial to evading the adaptive immune response (Zhang et al. 1998).
In 2000, Hellwage and his collaborators showed that OspE is a specific ligand for factor H. They first used radiolabeled proteins to determine that factor H bound strongly to the B. burgdorferi strain. Then using the surface plasmon resonance technique they determined that OspE binds to factor H, which led them to believe that this specific binding may help the pathogen to evade complement attach and phagocytosis (Hellwage 2000). It was not until recently that a structure was obtained for OspE and a mechanism was determined for how this protein is capable of protecting the bacteria from the complement system. Bhattacharjee and his lab were able to solve a solution structure of OspE by NMR, which showed a protein fold that has not been see before in complement regulation. They then solved the x-ray structure of the complex between OspE and the factor H C-terminal domains 19 and 20 at 2.83Å resolution. The structure shows that OspE binds FH19-20 in a similar way to that used by endothelial cells via glycosaminoglycans. The observed interaction reveals that OspE binds to domain 20 of FH and allows domain 19 to bind to the C3b protein of the complement system on the pathogen surface, which in turn inactivates C3b function and prevents the cell from opsonization (Bhattacharjee 2013).
VlsE is one of the most studied B. burgdorferi lipoproteins because it is capable of antigenic variation and is thought to be a key protein in evading the adaptive immune response. The Lyme disease bacterium possesses an elaborate genetic system that promotes extensive antigenic variation of the surface-exposed lipoprotein, VlsE, located on the linear plasmid 28. The presence of this plasmid correlates with the high-infectivity of B. burgdorferi (Zhang et al. 1997). This linear plasmid contains a variable major protein-like sequence (vls) expression site (vlsE) and 15 vls silent cassettes upstream. The coding sequence of vlsE contains a vls cassette region in the middle of the sequence. The central vlsE cassette region is separated from the 15 silent vls cassettes by a 17-bp direct repeat sequence. The vlsE cassette region has up to 92% DNA sequence identity with the silent vls cassettes. DNA segments of the silent cassettes were shown to be able to recombine in an apparently random manner into the vlsE cassette region in immunodeficient mice throughout the course of infection (Zhang et al. 1997). The silent vls cassette locus and the vlsE noncasette regions are preserved during the course of vlsE variation. This is consistent with a gene conversion mechanism in which segments of the silent vls cassettes replace corresponding regions in the vlsE expression site leading to antigenic variability (Zhang et al. 1998). Overall, these cassettes contain six variable regions flanked by highly conserved sequences. The large numbers of distinct antigenic variants during infection promotes resistance against adaptive immunity and persistence of infection.
A crystal structure of the VlsE protein was obtained in 2002 at 2.3Å resolution. The structure showed that the six variable regions of the protein form loop structures that constitute most of the membrane distal of VlsE, covering the invariant regions of the protein, which may result in protecting the invariant regions from being identified by antibodies, and contribute to immune evasion (Eicken et al. 2002). Removal of the vls locus through telomere resolvase-mediate showed that this portion of the DNA in an absolute requirement for persistence of disease in mouse models (Bankhead 2007).
A big advancement in the VlsE field has been the identification of an immunodominant conserved region within the variable domain of VlsE. Liang et al. was able to show that there are six invariable regions (IR) interspersed within the VlsE variable region. The most conserved of these regions is IR6, which they propose acts as a decoy epitope to divert antibody activity away from other, protective regions of VlsE (Liang et al. 1999). This epitope is now used for diagnosis of Lyme disease using the C6 peptide.
A recent study investigated possible VlsE epitopes targeted by antibody response in patients with post-Lyme disease syndrome, a condition characterized by persisting symptoms despite antibiotic treatment of B. burgdorferi. A crystal structure was obtained, which showed newly described epitopes. It was determined that post-Lyme disease patients have a strong antibody response to specific sequences in the N- and C- terminal invariable domains of VlsE. However, this study only focused on examining the antibody response to a single sequence variation of the VlsE molecule and therefore likely missed other target epitopes in the protein’s variable domain (Chandra et al. 2011).