Highlighted Proteins of Lyme Disease

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Revision as of 18:24, 3 October 2012

Highlighted Proteins of Lyme Disease

Lyme disease is caused by three species of bacteria belonging to the Borrelia genus, with Borrelia burgdorferi being the most common cause of the disease in the US. The bacteria are transmitted via hard-bodied ticks of the Ixodidae family. Borrelia spirochetes are motile, helical bacteria that have many lipoproteins exposed on the surfaces of their membranes. Two predominant groups of surface lipoproteins are classified as the outer surface proteins, which have been characterized as Osps A through F, and the variable major protein-like sequence expressed (VlsE). Both groups of proteins play important roles in pathogenesis, the the life cycle of Borrelia, and eliciting an immune response from the host (Figure 1).^[1]

In a introductory biology course at Stony Brook University, undergraduates are modeling and exploring B. burgdorferi surface proteins, as well as host produced antibodies to these proteins. This Proteopedia page is the product of their efforts, with a focus on highlighted proteins from the following categories: OspC, OspB and the antibodies to OspB, OspA and the antibodies to OspA, and VlsE.

The goal of this Proteopedia page is to describe Lyme disease from a structural biology perspective. What do B. burgdorferi outer surface proteins look like? How does the structure/function of these proteins relate to the infection cycle of B. burgdorferi? What are the structural targets of the human immune system and how have these targets evolved? What are the ideal structural targets for a vaccine to protect against Lyme disease?

Borrelia Gene Expression Over Life Cycle.

OspC and Lyme Disease

Migration of infected nymph from midgut to salivary glands

OspC, one of the outer surface proteins of B. burgdoferi, plays a pivotal role in transmission of B. burgdoferi from the tick vector to mammalian host. The protein gets upregulated when the tick feeds, allowing for the B. burgdoferi to adhere to the tick saliva and move to the tick's mouth and into the host.^[2] The upregulation of OspC is accompanied by a downregulation of OspA and OspB, which is thought to be induced by changes in environmental temperature and pH. ^[3]

OspC is a highly variable protein and strains of B. burgdoferi are classified according to the sequence of the OspC locus into 19 outer surface major groups (oMGs), denoted by type A to S, only four of which are invasive (disease causing).^[4] Polymorphism of OspC and abundance of invasive strains in a population of B. burgdoferiare driven by ecological factors, such as host mammalian community composition, and is a determinant of human Lyme disease risk^[5].

Researchers are attempting to take advantage of the upregulation of OspC on B. burgdorferi's surface, while the bacteria is in the host, to develop an OspC-based vaccine. However, development of OspC-based vaccination has presented difficulties due to the highly variable nature of OspC. ^[6]

Structure of OspC

spinqualitylabelsall models PDB ID 1ggq Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
1ggq, resolution 2.51Å ()
Ligands:
Related:	1f1m, 1osp, 1fj1
Structural annotation: Resources: CATH : 1Ggqa00, 1Ggqb00, 1Ggqc00, 1Ggqd00 InterPro : Ipr001800 Pfam : PF01441 SCOP : d1ggqa_, d1ggqb_, d1ggqc_, d1ggqd_ UniProt : Q07337
Functional annotation: Cellular component: cell outer membrane outer membrane membrane Biological process: defense response
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

The model presented to the right is the B. burgdorferi B31 strain (residues 38-201), which is also known as the oMG A strain. This is one of four invasive oMGs that are responsible for systematic Lyme disease. In crystal structure, OspC exists as a dimer with the coordination of divalent ion, which is modeled as a magnesium ion. Each subunit is predominantly helical, consisting of five parallel , two short antiparallel and six . The N and C termini at the membrane proximal end of two long alpha helices, (residues 38-76) and (residues 170-201) are in close proximity to each other. At the membrane distal end, there are three remaining alpha helices, (residues 95-112), (residues 121-145), including a short (residues 152-159). At the end of membrane surface, the connection between helices α1 and α2 forms two short anti-parallel β-strands, (residues 79-80),and (residues 88-89) are formed.

While most of the OspC locus is highly variable, the sequence alignment of all oMGs reveals that towards the membrane proximal end, the surface-exposed residues on α1 and α5 are highly , and have a positively charged surface. Other than those on helices, α1 and α5, the surface-exposed residues on the remaining regions of OspC molecule are variable.^[7]

OspC Structure and Antigenicity

At the membrane distal region, the six loop regions, including two β-strands illustrates the of OspC with the presence of variable surface-exposed residues. ^[8] Among these variable regions, the outer surface-exposed residues connecting the helices α1 and α2, forming the loops, (residues 74-78), (residues 81-87), (residues 90-93), two short beta strands, β1 and β2, and (residues 146-150) are more highly variable than those present in the loops, (residues 115-119)and (residues 161-169). The surface potential of the red region that projects away from the membrane is negatively charged and primarily involved in the protein-protein or protein-ligand interactions.^[7] Only four types of oMGs (A, B, I and K), whose surface potential in the red region is highly negative relative to non-invasive strains, play a major role in pathogenesis of human Lyme disease. ^[4]The residue, , located in the red region at the membrane distal end is unique in that the replacement of this residue with other residues, except His82, Lys82, Gln82, which are present only in four invasive oMGs, enhances the possibility of turning invasive strains to non-invasive one. Thus, the stronger the electrostatic potential on red region, the higher the chance OspC will to bind with positively charged host ligands. Therefore, the altering of an amino acid residue at the 82nd position in the red region not only demonstrates OspC polymorphism, but also increases the probability of turning invasive strains to non-invasive strains.^[4]

Lyme Disease and Ecology

Life cycle of tick.[[1]]

The number of reported cases of Lyme disease is increasing annually in highly focused geographic locations of the United States (CITE). The occurrence of Lyme disease is dependent upon the abundance of ticks infected with B. burgdorferi in natural ecosystems. Ticks are born without B. burgdorferi and acquire the bacteria while feeding on the blood of natural reservoir hosts such as mice, squirrels, shrews and other small vertebrates (Figure XX). After growth and development, the infected nymphal ticks can transmit B. burgdorferi to incidental vertebrates, including humans. The ecological interaction between the competence of reservoir hosts and the ticks is an underlying measure of human Lyme disease risk.^[9]

Ecological factors responsible for human Lyme disease risk

• Vertebrate Community Composition^[10]: Two types of environment that the vertebrate hosts reside, which is also called vertebrate host density are interspecific community, which involves organisms of different species and intraspecific community, which is composed of organisms of same species. The hosts living in the community within different species or same species strongly affects the proportion of infected nymphal tick that can cause human Lyme disease.
• Distribution frequency of particular oMGs^[5]: After taking blood meal from their hosts, the proportion of host-seeking nymphs infected with each oMG differs among oMGs. As only four types of oMGs (A, B, I and K) are responsible for systemic human Lyme disease, the host-seeking nymphs that have high distribution frequency of four invasive oMGs is one of the standard measures of human Lyme disease risk.
• Transmission Probability^[5]: The transmission probability of each oMGs from individual species differs. The higher the transmission probability of a particular oMG from vertebrate host, the higher the chance of carrying that particular oMG by the ticks after receiving blood meal from their hosts is. Thus, it is one of the parameters that contributes the prevalence of human Lyme disease.

Using Ecological Models to Predict Lyme Disease Risk

Map illustrating prevalence of Lyme disease in the United States by CDC.[[2]]

Conceptual and mathematical models have been developed by researchers to characterize the ecological interaction between vertebrate host community and distribution frequency of invasive oMGs and predict the cases of human Lyme disease. In one model, the principal natural reservoir host used in the model for the epidemic of Lyme disease in northeastern and central United States is the presence of the white-footed mice (Peromyscus leucopus) population, which has both high frequency distribution in all four human infectious oMGs and high transmission probabilities of oMGs A, B, I and K.^[5] Ticks are least likely to parasitize inefficient reservoir hosts, thereby increasing high infection prevalence in the tick population, which enhances the risk of exposure of Lyme disease in humans. Many studies have found support for this "dilution-effect model" which proposes that maintaining high diversity of vertebrate host community may dilute the power of one host, such as the white-footed mouse by increasing the degree of specialization of ticks on inefficient hosts. This model strongly demonstrates the relationship between species diversity in the community of hosts and the risk of human exposure to Lyme disease. These ecological driving forces described in the model are useful tools in predicting the prevalence and risk of human Lyme disease.

OspC-based vaccine

An OspC-based vaccine against Lyme disease is currently being developed. Because of the variability of OspC, the recombinant OspC vaccine, targeting the antigenic site of one specific OspC type is ineffective for B. burgdorferi with different OspC types. Therefore, the development of vaccine that recognizes the antigenic determinant on the variable regions of multiple OspC types is required in order to effectively activate human immune response. Based on the mapping of epitope-containing regions from oMGs: A, B, K and D, the experiment-based tetravalent chimeric vaccine is being developed to tigger anti-ABKD response. ^[11] Taking advantage of tetravalent ABKD construct, octavalent chimeric vaccine also known as OspC-A8.1, recognizing additional epitopes of oMGs: C, E, N and K, has been tested in mice. ^[6]

OspB and Lyme Disease

OspB, along with OspA make up the major proteins found on the surface of B. burgdorferi. OspB has been found to play a vital role in the adherence of B. bugdorferi to the tick midgut, with OspB-deficient B. burgdorferi binding poorly to tick gut extracts. While the tick remains unfed, the expression of OspB, along with OspA, is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated while other proteins such as OspC, DpbA and BBK32 are upregulated ^[12]. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant ^[13].

Digestion of an antibody by Papain separates the fab reigons from the antibody

A factor contributing to the severity of Lyme disease is its ability to evade the host immune system. For example B. burgdorfer has developed resistance to a complement-dependent immune response by the evasion of the alternative complement pathway and the blocking of complement complement component 3.^[14] OspB is an important target of antibodies that can kill the bacteria without the help of the rest of the complement component of the immune system.^[1] H6831 is fragment antigen-binding (Fab fragment) IgG antibody that targets the C-terminal of OspB. A Fab consists of a heavy chain and light chain and each chain is composed of a variable and a constant region. The paratope is located in the N terminal of the variable region of the heavy and light chains of the fab. Binding of this fabs to OspB of B. burgdoferi leads to the lysis of the bacteria.^[1]

Structure of the OspB-H6831 Complex

The consist of two components, the outer surface protein and the , which is subdivided into the and the . Most hydrogen bonds and electrostatic interactions that are responsible for the binding of H6831 to OspB are between the at the C-terminal of OspB and some that include tyrosine, tryptophan, glutamate, and histidine.^[13]

The majority of hydrogen bonds and electrostatic interactions are between (residues 250-254) and the fab heavy chain. in loop 2 of OspB has a necessary and major role due to its central position in the exposed loops. A mutation at its position abrogates the binding interaction and causes the resistance of the bacteria to the bactericidal effect of the fab. Lys 253 interacts with the two aromatic residues on the fab heavy chain, tyrosine and tryptophan. It also makes hydrogen bonds with the glutamate 50 in the heavy chain of the fab and forms an ionic bond. Carbonyl in of the OspB interacts with in the fab heavy chain. of OspB interacts with fab light chain.^[13]

Structural changes to OspB in the complexed form

The binding of H6831 to OspB leads to some conformational changes in OspB compared to its . Crystallography has shown that the most significant difference is the loss of the .^[13] The loss of these β sheets may be due to conformational change as a result of the binding or a disorder that could have occurred during a crystallization of the complex. Both small positional shifts near the fab binding site and a few larger structural changes away from the binding site were observed. The largest shifts (7– 8 Å) correspond to the repositioning of a loop opposite the fab-binding site . In the free OspB structure, all regions that exhibit shifts are adjacent to the central sheet; in the OspB-H6831 complex they all shift toward, and slightly overlap the position of the missing sheet.

Potential Mechanism of Lysis

The fab binding destabilizes the outer membrane (OM) of B. burdorferi, with subsequent formation of spheroplasts. It has been observed that the bactericidal action, but not the binding, requires the presence of divalent cations (Mg²⁺ and Ca²⁺), and fab is unable to clear bacteria in the absence of these cations.^[15] It is speculated that OspB-Cb2 (a fab similar to H6831) complexes could lead to the lysis of the cell by creating physical openings in the OM, allowing for rapid infusion of electrolytes and increasing the osmolarity of the periplasm.^[16]

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of B. burgdorferi ^[13]. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons^[17]. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB ^[13]. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB ^[16]. In and forms of OspB, some changes result from proteolysis near the N terminus ^[13]. Residues 157 - 201 on OspB contain the , shown in pink.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain.

Potential Catalytic Triad

Comparison of catalytic triads

The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 ^[18].

It is possible that OspB performs an autoproteolysis. There is a found on OspB that resembles the catalytic triad of Serine_Proteases. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease trypsin, which are Ser-195, His-57, Asp-102 ^[19].

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. forms an H-bond with and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease.

Potential Oxidative Mechanism

It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement.

OspA and Lyme Disease

Outer Surface Protein A (OspA), together with OspB make up the major proteins found on the surface of B. burgdorferi. It has been used as a target in the development of a vaccine for Lyme disease. OspA's expression is differentially regulated over the B. burgdorferi infection cycle. It is expressed while the bacteria resides in the midgut of the tick, downregulated while the tick feeds on its host, and then upregulated in the host's cerebrospinal fluid (CSF), which may induce an inflammatory response resulting in acute Lyme neuroborreliosis.

OspA is involved in attachment of B. burgdorferi to the tick gut by interacting with tick gut protein, TROSPA. ^[20] When a tick feeds, OspA is downregulated, releasing the B. burgdorferi from the gut wall and allowing its migration into the tick's salivary glands and into the host through the tick bite. The downregulation of OspA during transmission is evidenced by the fact that patients with Lyme disease do not possess OspA antibodies in the early stages of the disease.^[1][21]

Structure of OspA

OspA is made up of 273 residues over 21 anti-parallel β-sheets and a single α-helix. It's folded conformation is divided into three main sections: a N-terminus "sandwich," a central region comprising of several β-sheets and a C-terminus "barrel" domain.^[15] The folded regions at its ends are connected by a single β-sheet layer in the middle, giving the protein the unique shape of a dumbell.^[22] There are at the C-terminus of OspA that are important in binding with the LA-2 Fab antibody, whose interactions provide great insight into vaccine research and effectiveness. These three loops are linearly arranged and form protruding ridge at the C-terminus of OspA. Within these loops, there are where there are distinct variations between the different strains of B. burgdorferi and serve as potential targets for the creation of a broader vaccine.^[15]

, (residues 203-220), is important in showing variation amongst the different strains of B. burgdorferi as well as being optimally conformed for binding without steric hindrance. (residues 224-233) and (residues 246-257) are more strongly conserved than Loop 1 but also help to show some variation amongst strains. The LA-2 Fab antibody readily recognizes OspA from B. burgdorferi, but does not recognize that from B. afzelii or B. garinii. Between B. burgdorferi and B. afzelii genetic sequences are generally invariant, but two residues change between the species: in B. burgdorferi is a Glutamine (Gln) in B. afzelii, and in B. burgdorferi is an Alanine (Ala) in B. afzelii. B. garinii has more variation and in addition to the previous two differences, having at least one more difference, where in B. burgdorferi is a Lysine (Lys), and sometimes also has a deletion at B. burgdorferi’s Alanine 208. LA-2 and OspA of B. burgdorferi form a tight interface when binding, and the longer Glutamine (Gln) sidechain found in B. afzelii and B. garinii is more difficult to accommodate, causing less binding. A chimera that was weakly recognized by LA-2 was made with parts of loop 1 from B. burgdorferi, and loops 2 and 3 from B. garinii.^[15] Recently, a different kind of chimera has been made which combined the proximal region of B. burgdorferi and distal region of B. afzelii, and was able to successfully protect mice from both species.^[23]

Acute Lyme Neuroborreliosis (LNB)

Acute Lyme Neuroborreliosis (LNB) is part of the second stage of Lyme disease in which the spirochete invades the peripheral and central nervous systems (CNS). Symptoms of LNB include: meningoradiculitis with inflammation of the nerve roots and radiculitis (Bannwarth’s syndrome), lymphocytic meningitis, and cranial and peripheral neuritis. In Europe, the strain predominantly found in the CSF of patients with Bannwarth's syndrome is B. garinii. However, in the United States, Bannwarth's syndrome is rare and the most common manifestations of Lyme neuroborreliosis is meningitis, caused by B. burgdorferi. The presence of OspA in the cerebrospinal fluid (CSF) is responsible for this complex inflammatory response in the brain that leads to the neuroborreliosis.^[21]

It is not fully understood how B. burgdorferi get past the blood-brain barrier, though some researchers suggest a paracellular route, which involves a process using transient tether-type associations, short-term dragging interactions, and stationary adhesion. There is evidence that B. burgdorferi utilizes OspA in the transient tethering stage. The blood-brain barrier is composed of brain microvascular endothelial cells, astrocytes, a basement membrane, pericytes, and neurons. OspA is a major adherent molecule to brain microvascular cells by binding to the CD40 receptors outside, which results in events that are typically seen when leukocytes cross the blood brain barrier.

Activation of CD40 receptors leads to the production of proinflammatory cytokines and enhanced expression of ICAM-1, E-selectin and VCAM-1, resulting in increased cell binding, and the formation of fenestrations due to increased vascular endothelial growth factor, and vascular permeability factor. OspA might be mimicking leukocytes in order to cross the blood-brain barrier. However not all strains of B. burgdorferi can utilize OspA to do this, OspA only contributes about 70% to adherence, and other B. burgdorferi proteins are also needed in this process. It has also been seen that OspA mediates the adhesion of B. burgdorferi to murine neural and glial cell lines. ^[24]

Role of OspA in Inflammation

Mechanism of the host inflammatory response to OspA

There are six steps involved in the host's inflammatory response to OspA: ^[21]

1. When B. burgdorferi enter the host’s CNS they encounter several different types of immune cells such as monocytes, macrophages, and dendritic cells. While in the CSF, outer surface protein A (OspA) is upregulated and it’s increased expression promotes recognition by a specific receptor on a monocyte.
2. The OspA-bound monocyte then releases proinflammatory cytokines (i.e. interferon), as well as chemokines, such as CXCL13. In patients with LNB, there is an observed increase in the levels of these cytokines and chemokines in their CSF. The production of chemokines leads to the recruitment of other immune cells to the site of infection.
3. B-lymphocytes respond to the new concentration gradient of CXCL13 between the blood and CSF and migrate into the CSF.
4. B-lymphocytes undergo receptor-mediated endocytosis, consuming the OspA antigens present in the CSF, thereby triggering its activation. The B-lymphocytes then are able to differentiate and mature into plasma cells.
5. The plasma cells create large quantities of anti-OspA antibodies specific to this strain of B. burgdorferi and release them into the CSF.
6. The anti-OspA antibodies will then bind to the OspA on the spirochete’s membrane, thus killing the B. burgdorferi.

This process is two-sided in the sense that the OspA aids in the pathogenesis of new symptoms (neuroborreliosis) through the chemokine’s actions, as well as initiating the signaling cascade to destroy itself.

Evasion and the Extracellular Matrix

The B. burgdorferi are able to hide in the extracellular matrix, allowing it to survive by avoiding leukocytes circulating in the bloodstream. OspA can rapidly bind to plasminogen, which becomes plasmin once activated, and degrades the extracellular matrix. By binding to plasminogen, B. burgdorferi could be exploiting its function and utilizing it to invade the extracellular matrix. However, due to the fact that OspA is downregulated during feeding, and stays unexpressed, a different mechanism may be used instead. Additionally, B. burgdorferi induces the local upregulation of matrix metalloproteinase-9, causing the digestion of the surrounding extracellular matrix. B. burgdorferi can also bind to several proteins in the extracellular matrix, such as fibronectin, integrins or decorin, which can aid in the spread and survival of the spirochetes in these tissues.^[21]

Antibodies to OspA

Interaction between OspA and LA-2

LA-2 is an IgM murine monoclonal antibody that interacts with on the C-terminal of OspA. These interactions include eight direct hydrogen bonds, four solvent-bridged hydrogen bonds, three ion pairs, and numerous van der Waals interactions.^[15]

Structural changes to OspA in the complexed form

Conformational changes upon the binding of OspA and LA-2 show that LA-2 recognition of OspA involves an induced fit mechanism where the conformations of loops 1-3 shift to optimize complementarity to the antigen-combining site.^[15] The overall structure of the C-terminal of OspA is unchanged upon the binding of LA-2 with comparison to the free OspA. The maximum atomic shift is 4.7Å at the site of .^[15]

OspA-based Vaccine

Risk of developing Lyme disease can be mitigated by staying clear of areas with populations of ticks, wearing proper attire to minimize easily bitten areas of the body, and using insect repellents containing DEET (N,N-diethy-m-toluamide). However, another effective means for prevention could be possible by using an outer surface protein from B. burgdorferi in the creation of a vaccine.^[25]

The membrane composition of B. burgdorferi is abundant in both OspA and OspB, and the two proteins share a 53% similarity in their primary sequences, however, OspB has greater variability than OspA.^[13]. Of the three exposed loops found on OspA, only loop 1 is variable while loops 2 and 3 are conserved. This makes OspA a more consistent antigen (compared to OspB and OspC) for the immune system to target and usable as a vaccine to Lyme disease.^[15] The first vaccine developed against Lyme disease, Lymerix, used a purified recombinant form of OspA and functioned in blocking transmission of the spirochetes expressing OspA from tick to host during feeding, killing them while still attached to the tick's gut.^[1][26] The vaccine was 76% to 92% effective in separate clinical trials in which patients were treated for two years following a three-dose schedule. However, the vaccine was suspended from use in 2002 when opponents claimed the IgG antibodies for OspA were associated with the onset of severe chronic arthritis, as well as other side effects affecting immunity.^[1][27] This claim, in conjunction with the desire for a more widespread vaccine treating multiple strains of B. burgdorferi, has spurred research towards a new vaccine. One goal is to develop a vaccine with broader protection, creation of a chimera, mixing the OspA of different strains of B. burgdorferi.

Study of the epitope of and its with the murine monoclonal antibody have proved useful in determining effectiveness of a given vaccine trial as high levels of antibodies in test sera compete against LA-2 for binding with OspA. LA-2 makes direct contact with three exposed loops of the C-terminus of OspA. The recognition of OspA by LA-2 requires an induced fit mechanism where these three loops undergo conformational changes to optimize their interaction in the complex. ^[15]

VlsE and Lyme Disease

Variable Major Protein -like sequence Expressed (VlsE) is a surface lipoprotein of Borrelia burgdorferi. It undergoes antigenic variation seemingly important in evasion of the host’s immune system. In addition, the protein is used for Lyme disease diagnosis.

Structure of VlsE

VlsE is composed of four similar subunits each possessing two invariable domains and one variable domain.^[28] The variable domain contains six variable regions (VR₁-VR₆), and six invariable regions (IR₁-IR₆). Research suggests that the protein may exist as a dimer where each monomeric C & N termini neighbor each other forming the membrane proximal portion of the protein, and the variable regions form the membrane distal portion.^{[29] [30]} The invariable regions are largely embedded in the protein and remain relatively unchanged within the host and across strains. The variable regions encompass 37% of the VlsE’s exposed surface area despite comprising only 25% of the protein.^{[28] [29]} However, 50% of the VR surface area is exposed while IR₆, a strong antigen, exposes just 13.7% of its surface area. This leaves only of the antigenic IR₆ unprotected: lysine-276, glutamine-279, lysine-291, and lysine-294. Thus, it is almost entirely embedded in the protein and .^[29]

Antigenicity

The variable regions undergo a recombination event stimulated by the host’s cytokines and absence of those cytokines results in a decreased bacterial burden.^[31] This leads to variation with an estimated 10³⁰ possible combinations, far exceeding the number of antibodies found in the human immune system. While the VR does exhibit antigenicity, this recombination makes it unlikely that a sufficient amount of a single VR variation will be present in large enough supply to lead to an immunodominant variable region.^[32] IR₆, however, exhibits immunodominance while IR₁-IR₅ are primarily nonantigenic in humans. Thus, shielding of the immunodominant IR₆ by VR regions not subject to antibody response allows for IR₆ to elicit an immune response while remaining inaccessible to antibody binding. Research suggests that the 26 amino residues of may function as a single epitope with a central alpha helical core.^{[29] [31] [33]}

Function in Immune System Evasion

VlsE is essential to the persistence and virulence of Lyme disease and is upregulated under humoral immune pressure.^{[34] [35]} While the exact mechanism for immune evasion remains unknown, several theories have been put forth. One popular theory maintains that VlsE masks other surface antigens by coating the surface of the bacteria, thereby sterically blocking the antigens from antibody binding. This is similar to other pathogens with variable regions, such as Trypanosoma brucei, the protozoa responsible for African sleeping sickness and Neisseria gonorrhea, the bacterial cause of gonorrhea. However, recent studies have cast doubt on this theory. An alternate theory provides that VlsE directly stimulates B cell antibody production independent of T-cells. The robust response elicited is thought to override antibody production against other antigens.^[34]

C₆ Diagnostic Testing

Throughout the course of the disease, IR₆ produces a strong antibody response that can be identified from early to late phases. Applications in diagnostic testing have been identified as a result of this strong immune response and IR₆’s relative invariability across strains.^{[36] [33]} A C₆ ELISA test has been developed which uses a 26 amino acid synthetic peptide, C₆, containing the IR₆ sequence. Results show 99% specificity and 100% precision with high sensitivity. In fact, OspA vaccination does not influence C6 specificity; therefore, C₆ ELISA tests are valuable diagnostic tools.^[36] The CDC currently recommends a two-step test incorporating first a polyvalent, whole-cell sonicate (WCS) immunofluorescent assay. If results are positive, this is followed by IgG and IgM WCS Western blots to eliminate false positives.^[37] Therefore, this one-step ELISA test presents an accurate and economical alternative to the current two-step model.^[36]

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5} Connolly SE, Benach JL. The versatile roles of antibodies in Borrelia infections. Nat Rev Microbiol. 2005 May;3(5):411-20. PMID:15864264 doi:10.1038/nrmicro1149
2. ↑ Templeton TJ. Borrelia outer membrane surface proteins and transmission through the tick. J Exp Med. 2004 Mar 1;199(5):603-6. Epub 2004 Feb 23. PMID:14981110 doi:10.1084/jem.20040033
3. ↑ Templeton TJ. Borrelia outer membrane surface proteins and transmission through the tick. J Exp Med. 2004 Mar 1;199(5):603-6. Epub 2004 Feb 23. PMID:14981110 doi:10.1084/jem.20040033
4. ↑ ^{4.0 4.1 4.2} Kumaran D, Eswaramoorthy S, Luft BJ, Koide S, Dunn JJ, Lawson CL, Swaminathan S. Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi. EMBO J. 2001 Mar 1;20(5):971-8. PMID:11230121 doi:http://dx.doi.org/10.1093/emboj/20.5.971
5. ↑ ^{5.0 5.1 5.2 5.3} Brisson D, Dykhuizen DE. A modest model explains the distribution and abundance of Borrelia burgdorferi strains. Am J Trop Med Hyg. 2006 Apr;74(4):615-22. PMID:16606995
6. ↑ ^{6.0 6.1} Earnhart CG, Marconi RT. An octavalent lyme disease vaccine induces antibodies that recognize all incorporated OspC type-specific sequences. Hum Vaccin. 2007 Nov-Dec;3(6):281-9. Epub 2007 Jul 2. PMID:17921702
7. ↑ ^{7.0 7.1} Eicken C, Sharma V, Klabunde T, Owens RT, Pikas DS, Hook M, Sacchettini JC. Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi. J Biol Chem. 2001 Mar 30;276(13):10010-5. Epub 2001 Jan 3. PMID:11139584 doi:10.1074/jbc.M010062200
8. ↑ Earnhart C, LeBlanc D, Alix K, Desrosiers D, Radolf J, and Marconi R. 2010. Identification of residues within ligand-binding domain 1 (LBD1) of the Borrelia burgdorferi OspC protein required for function in the mammalian environment. Molecular Microbiology 76(2): 393-408. DOI: 10.1111/j.1365-2958.2010.07103.x
9. ↑ LoGiudice K, Ostfeld RS, Schmidt KA, Keesing F. The ecology of infectious disease: effects of host diversity and community composition on Lyme disease risk. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):567-71. Epub 2003 Jan 13. PMID:12525705 doi:10.1073/pnas.0233733100
10. ↑ Brisson D, Dykhuizen DE. ospC diversity in Borrelia burgdorferi: different hosts are different niches. Genetics. 2004 Oct;168(2):713-22. PMID:15514047 doi:10.1534/genetics.104.028738
11. ↑ Christopher G. Earnhart, Eric L. Buckles, Richard T. Marconi. "Development of an OspC-based tetravalent, recombinant, chimeric vaccinogen that elicits bactericidal antibody against diverse Lyme disease spirochete strains, Vaccine." 25(3) 466-480 (2007). DOI: 10.1016/j.vaccine.2006.07.052
12. ↑ Neelakanta G, Li X, Pal U, Liu X, Beck DS, DePonte K, Fish D, Kantor FS, Fikrig E. Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks. PLoS Pathog. 2007 Mar;3(3):e33. PMID:17352535 doi:10.1371/journal.ppat.0030033
13. ↑ ^{13.0 13.1 13.2 13.3 13.4 13.5 13.6 13.7} Becker M, Bunikis J, Lade BD, Dunn JJ, Barbour AG, Lawson CL. Structural investigation of Borrelia burgdorferi OspB, a bactericidal Fab target. J Biol Chem. 2005 Apr 29;280(17):17363-70. Epub 2005 Feb 15. PMID:15713683 doi:10.1074/jbc.M412842200
14. ↑ LaRocca TJ, Benach JL. The important and diverse roles of antibodies in the host response to Borrelia infections. Curr Top Microbiol Immunol. 2008;319:63-103. PMID:18080415
15. ↑ ^{15.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8} Ding W, Huang X, Yang X, Dunn JJ, Luft BJ, Koide S, Lawson CL. Structural identification of a key protective B-cell epitope in Lyme disease antigen OspA. J Mol Biol. 2000 Oct 6;302(5):1153-64. PMID:11183781 doi:10.1006/jmbi.2000.4119
16. ↑ ^{16.0 16.1} Escudero R, Halluska ML, Backenson PB, Coleman JL, Benach JL. Characterization of the physiological requirements for the bactericidal effects of a monoclonal antibody to OspB of Borrelia burgdorferi by confocal microscopy. Infect Immun. 1997 May;65(5):1908-15. PMID:9125579
17. ↑ Schwan TG, Schrumpf ME, Karstens RH, Clover JR, Wong J, Daugherty M, Struthers M, Rosa PA. Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California. J Clin Microbiol. 1993 Dec;31(12):3096-108. PMID:8308101
18. ↑ Benjamin DC, Williams DC Jr, Smith-Gill SJ, Rule GS. Long-range changes in a protein antigen due to antigen-antibody interaction. Biochemistry. 1992 Oct 13;31(40):9539-45. PMID:1382591
19. ↑ Hedstrom L. Serine protease mechanism and specificity. Chem Rev. 2002 Dec;102(12):4501-24. PMID:12475199
20. ↑ Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, Desilva AM, Bao F, Yang X, Pypaert M, Pradhan D, Kantor FS, Telford S, Anderson JF, Fikrig E. TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell. 2004 Nov 12;119(4):457-68. PMID:15537536 doi:10.1016/j.cell.2004.10.027
21. ↑ ^{21.0 21.1 21.2 21.3} Rupprecht TA, Koedel U, Fingerle V, Pfister HW. The pathogenesis of lyme neuroborreliosis: from infection to inflammation. Mol Med. 2008 Mar-Apr;14(3-4):205-12. PMID:18097481 doi:10.2119/2007-00091.Rupprecht
22. ↑ Makabe K, Tereshko V, Gawlak G, Yan S, Koide S. Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering. Protein Sci. 2006 Aug;15(8):1907-14. Epub 2006 Jul 5. PMID:16823038 doi:10.1110/ps.062246706
23. ↑ Livey I, O'Rourke M, Traweger A, Savidis-Dacho H, Crowe BA, Barrett PN, Yang X, Dunn JJ, Luft BJ. A new approach to a Lyme disease vaccine. Clin Infect Dis. 2011 Feb;52 Suppl 3:s266-70. PMID:21217174 doi:10.1093/cid/ciq118
24. ↑ Pulzova L, Kovac A, Mucha R, Mlynarcik P, Bencurova E, Madar M, Novak M, Bhide M. OspA-CD40 dyad: ligand-receptor interaction in the translocation of neuroinvasive Borrelia across the blood-brain barrier. Sci Rep. 2011;1:86. Epub 2011 Sep 8. PMID:22355605 doi:10.1038/srep00086
25. ↑ Nigrovic LE, Thompson KM. The Lyme vaccine: a cautionary tale. Epidemiol Infect. 2007 Jan;135(1):1-8. Epub 2006 Aug 8. PMID:16893489 doi:10.1017/S0950268806007096
26. ↑ Battisti JM, Bono JL, Rosa PA, Schrumpf ME, Schwan TG, Policastro PF. Outer surface protein A protects Lyme disease spirochetes from acquired host immunity in the tick vector. Infect Immun. 2008 Nov;76(11):5228-37. Epub 2008 Sep 8. PMID:18779341 doi:10.1128/IAI.00410-08
27. ↑ Plotkin SA. Correcting a public health fiasco: The need for a new vaccine against Lyme disease. Clin Infect Dis. 2011 Feb;52 Suppl 3:s271-5. PMID:21217175 doi:10.1093/cid/ciq119
28. ↑ ^{28.0 28.1} Liang FT, Philipp MT. Analysis of antibody response to invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi. Infect Immun. 1999 Dec;67(12):6702-6. PMID:10569796
29. ↑ ^{29.0 29.1 29.2 29.3} Eicken C, Sharma V, Klabunde T, Lawrenz MB, Hardham JM, Norris SJ, Sacchettini JC. Crystal structure of Lyme disease variable surface antigen VlsE of Borrelia burgdorferi. J Biol Chem. 2002 Jun 14;277(24):21691-6. Epub 2002 Mar 28. PMID:11923306 doi:10.1074/jbc.M201547200
30. ↑ Jones K, Guidry J, Wittung-Stafshede P. Characterization of surface antigen from Lyme disease spirochete Borrelia burgdorferi. Biochem Biophys Res Commun. 2001 Nov 30;289(2):389-94. PMID:11716485 doi:10.1006/bbrc.2001.5983
31. ↑ ^{31.0 31.1} Anguita J, Thomas V, Samanta S, Persinski R, Hernanz C, Barthold SW, Fikrig E. Borrelia burgdorferi-induced inflammation facilitates spirochete adaptation and variable major protein-like sequence locus recombination. J Immunol. 2001 Sep 15;167(6):3383-90. PMID:11544329
32. ↑ Liang FT, Alvarez AL, Gu Y, Nowling JM, Ramamoorthy R, Philipp MT. An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi. J Immunol. 1999 Nov 15;163(10):5566-73. PMID:10553085
33. ↑ ^{33.0 33.1} Liang FT, Philipp MT. Epitope mapping of the immunodominant invariable region of Borrelia burgdorferi VlsE in three host species. Infect Immun. 2000 Apr;68(4):2349-52. PMID:10722641
34. ↑ ^{34.0 34.1} Bankhead T, Chaconas G. The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens. Mol Microbiol. 2007 Sep;65(6):1547-58. Epub 2007 Aug 21. PMID:17714442 doi:10.1111/j.1365-2958.2007.05895.x
35. ↑ Liang FT, Yan J, Mbow ML, Sviat SL, Gilmore RD, Mamula M, Fikrig E. Borrelia burgdorferi changes its surface antigenic expression in response to host immune responses. Infect Immun. 2004 Oct;72(10):5759-67. PMID:15385475 doi:10.1128/IAI.72.10.5759-5767.2004
36. ↑ ^{36.0 36.1 36.2} Liang FT, Steere AC, Marques AR, Johnson BJ, Miller JN, Philipp MT. Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE. J Clin Microbiol. 1999 Dec;37(12):3990-6. PMID:10565920
37. ↑ Branda JA, Linskey K, Kim YA, Steere AC, Ferraro MJ. Two-tiered antibody testing for Lyme disease with use of 2 enzyme immunoassays, a whole-cell sonicate enzyme immunoassay followed by a VlsE C6 peptide enzyme immunoassay. Clin Infect Dis. 2011 Sep;53(6):541-7. PMID:21865190 doi:10.1093/cid/cir464

Teaching at Stony Brook University

This Proteopedia page is the product of a new introductory biology laboratory started in the spring of 2012 at Stony Brook University. Undergraduates model and print tactile 3-dimensional proteins involved in Lyme disease in order to understand and interpret contemporary structural biology research. The best student-authored summaries from spring and summer 2012 were selected for this Proteopedia page, thereby connecting students to scientists and facilitating further research experiences.

Author contributions:
OspC: Irene Chen, Khine Tun
Antibodies to Osp A and B: Safa Abdelhakim, Alexandros Konstantinidis, Philip J. Pipitone, Christopher Smilios
OspA: Jenny Kim Kim, Cara Lin, Andrea Mullen, Kimberly Slade
OspB: Olivia Cheng, Stephanie Maung, Ying Zhao
VlsE: Frank J. Albergo, Rachel Cirineo, Tanya Turkewitz
Editors, teachers: Jeff Ecklund, Joan M. Miyazaki, Christopher Morales, Carol Nicosia, Deborah A. Spikes, Raymond Suhandynata, La Zhong
Technical support: Nancy A. Black, Jameson T. Crowley
Collaborating research scientists, editors: Jorge L. Benach, Timothy J. LaRocca
Course co-developer, writer, editor: Niamh B. O'Hara
Course director, course co-developer, writer, editor: Marvin H. O'Neal III
Supported by: Howard Hughes Medical Institute 52006940

Proteopedia Page Authors

Safa Abdelhakim, Frank J. Albergo, Irene Chen, Olivia Cheng, Rachel Cirineo, Jenny Kim Kim, Alexandros Konstantinidis, Cara Lin, Stephanie Maung, Christopher Morales, Andrea Mullen, Niamh B. O'Hara, Marvin H. O'Neal III, Philip J. Pipitone, Kimberly Slade, Christopher Smilios, Raymond Suhandynata, Khine Tun, Tanya Turkewitz, Ying Zhao, La Zhong.