Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

Solution NMR structure of Borrelia burgdorferi outer surface lipoprotein BBP28, a member of the mlp protein family.

Lyme disease is the most widespread vector-transmitted disease in North America and Europe, caused by infection with Borrelia burgdorferi sensu lato complex spirochetes. We report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBP28, a member of the multicopy lipoprotein (mlp) family. The structure comprises a tether peptide, five α-helices and an extended C-terminal loop. The fold is similar to that of Borrelia turicatae outer surface protein BTA121, which is known to bind lipids. These results contribute to the understanding of Lyme disease pathogenesis by revealing the molecular structure of a protein from the widely found mlp family.

Structural analysis of the outer surface proteins from Borrelia burgdorferi paralogous gene family 54 that are thought to be the key players in the pathogenesis of Lyme disease.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. Through a complex enzootic cycle, the bacteria transfer between two different hosts: Ixodes ticks and mammalian organisms. At the start of the tick blood meal, the spirochetes located in the tick gut upregulate the expression of several genes, mainly coding for outer surface proteins. Outer surface proteins belonging to the paralogous gene family 54 (PFam54) have been shown to be the most upregulated among the other borrelial proteins and the results clearly point to the potential importance of these proteins in the pathogenesis of Lyme disease. The significance of PFam54 proteins is confirmed by the fact that of all ten PFam54 proteins, BBA64 and BBA66 are necessary for the transfer of B. burgdorferi from infected Ixodes ticks to mammalian hosts. To enhance the understanding of the pathogenesis of Lyme disease and to promote the development of novel therapies against Lyme disease, we solved the crystal structure of the PFam54 member BBA65. Additionally, we report the structure of the B. burgdorferi BBA64 orthologous protein from B. spielmanii. Together with the previously determined crystal structures of five PFam54 members and several related proteins, we performed a comprehensive structural analysis for this important group of proteins. In addition to revealing the molecular aspects of the proteins, the structural data analysis suggests that the gene families PFam54 and PFam60, which have long been referred to as separate paralogous families, should be merged into one and designated as PFam54_60.

Crystal structures of the Erp protein family members ErpP and ErpC from Borrelia burgdorferi reveal the reason for different affinities for complement regulator factor H.

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH.

Structural and functional analysis of BB0689 from Borrelia burgdorferi, a member of the bacterial CAP superfamily.

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Structure of the Borrelia burgdorferi ATP-dependent metalloprotease FtsH in its functionally relevant hexameric form.

ATP-dependent proteases FtsH are conserved in bacteria, mitochondria, and chloroplasts, where they play an essential role in degradation of misfolded/unneeded membrane and cytosolic proteins. It has also been demonstrated that the FtsH homologous protein BB0789 is crucial for mouse and tick infectivity and in vitro growth of the Lyme disease-causing agent Borrelia burgdorferi. This is not surprising, considering B. burgdorferi complex life cycle, residing in both in mammals and ticks, which requires a wide range of membrane proteins and short-lived cytosolic regulatory proteins to invade and persist in the host organism. In the current study, we have solved the crystal structure of the cytosolic BB0789166-614, lacking both N-terminal transmembrane α-helices and the small periplasmic domain. The structure revealed the arrangement of the AAA+ ATPase and the zinc-dependent metalloprotease domains in a hexamer ring, which is essential for ATPase and proteolytic activity. The AAA+ domain was found in an ADP-bound state, while the protease domain showed coordination of a zinc ion by two histidine residues and one aspartic acid residue. The loop region that forms the central pore in the oligomer was poorly defined in the crystal structure and therefore predicted by AlphaFold to complement the missing structural details, providing a complete picture of the functionally relevant hexameric form of BB0789. We confirmed that BB0789 is functionally active, possessing both protease and ATPase activities, thus providing novel structural-functional insights into the protein, which is known to be absolutely necessary for B. burgdorferi to survive and cause Lyme disease.

Structural studies of chromosomally encoded outer surface lipoprotein BB0158 from Borrelia burgdorferi sensu stricto.

Lyme disease, or also known as Lyme borreliosis, is caused by the spirochetes belonging to the Borrelia burgdorferi sensu lato complex, which can enter the human body following the bite of an infected tick. Many membrane lipid-bound proteins, also known as lipoproteins, are located on the surface of B. burgdorferi sensu lato and play a crucial role in the spirochete to interact with its environment, whether in ticks or mammals. Since the spirochete needs to perform various tasks, such as resisting the host's immune system or spreading throughout the organism, it is not surprising that numerous surface proteins have been found to be essential for B. burgdorferi sensu lato complex bacteria in causing Lyme disease. In this study, we have determined (at 2.4 Å resolution) and characterized the 3D structure of BB0158, one of the few chromosomally encoded outer surface proteins from B. burgdorferi sensu stricto. BB0158 belongs to the paralogous gene family 44 (PFam44), consisting of four other members (BB0159, BBA04, BBE09 and BBK52). The characterization of BB0158, which appears to form a domain-swapped dimer, in conjunction with the characterization of the corresponding PFam44 members, certainly contribute to our understanding of B. burgdorferi sensu stricto proteins.

Members of the paralogous gene family 12 from the Lyme disease agent Borrelia burgdorferi are non-specific DNA-binding proteins.

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria.

Structure of an outer surface lipoprotein BBA64 from the Lyme disease agent Borrelia burgdorferi which is critical to ensure infection after a tick bite.

Lyme disease is a tick-borne infection caused by the transmission of Borrelia burgdorferi from infected Ixodes ticks to a mammalian host during the blood meal. Previous studies have shown that the expression of B. burgdorferi surface-localized lipoproteins, which include BBA64, is up-regulated during the process of tick feeding. Although the exact function of BBA64 is not known, this lipoprotein is critical for the transmission of the spirochete from the tick salivary glands to the mammalian organism after a tick bite. Since the mechanism of development of the disease and the functions of the surface lipoproteins associated with borreliosis are still poorly understood, the crystal structure of the B. burgdorferi outer surface lipoprotein BBA64 was solved at 2.4 Å resolution in order to obtain a better insight into the pathogenesis of B. burgdorferi and to promote the discovery of novel potential preventive drugs against Lyme disease. In this study, the crystal structure of BBA64 was also compared with that of the paralogous protein CspA (also referred to as BbCRASP-1, CRASP-1 or BBA68). CspA is the complement regulator-acquiring surface protein-1 of B. burgdorferi; its structure is known, but its function apparently differs from that of BBA64. It is demonstrated that unlike the homologous CspA, BBA64 does not form a homodimer. Their differences in function could be explained by divergence in their amino-acid sequences, electrostatic surface potentials and overall tertiary structures. The C-terminal part of BBA64 has a different conformation to that of CspA; the conformation of this region is essential for the proper function of CspA.

Structural characterization of the Borrelia burgdorferi outer surface protein BBA73 implicates dimerization as a functional mechanism.

Borrelia burgdorferi, which is the causative agent of Lyme disease, is transmitted from infected Ixodes ticks to a mammalian host following a tick bite. Upon changing the host organism from an Ixodes tick to a warm-blooded mammal, the spirochete must adapt to very different conditions, which is achieved by altering the expression of several genes in response to a changing environment. Recently, considerable attention has been devoted to several outer surface proteins, including BBA73, that undergo dramatic upregulation during the transmission of B. burgdorferi from infected Ixodes ticks to mammals and that are thought to be important for the establishment and maintenance of the infection. These upregulated proteins could reveal the mechanism of pathogenesis and potentially serve as novel drug targets to prevent the transmission of the pathogenic bacteria. To promote effective treatments for Lyme disease and to gain better insight into B. burgdorferi pathogenesis, we have determined the crystal structure of the upregulated outer surface protein BBA73 at 2.0 Å resolution. We observed that the BBA73 protein exists as a homodimer both in the crystal and in solution. The monomers interact with their N-terminal α-helices and form a cleft that could potentially serve as a ligand or receptor binding site. To confirm that the protein dimerizes through the interaction of the N-terminal regions, we produced an N-terminal deletion mutant of BBA73 to disrupt dimerization, and we determined the crystal structure of the truncated BBA73 protein at 1.9 Å resolution. The truncated protein did not form a homodimer, and the crystal structure confirmed that the overall fold is identical to that of the native BBA73 protein. Notably, a paralogous protein CspA from B. burgdorferi with known crystal structure also forms a homodimer, albeit through an entirely different interaction between the monomers.

Fibronectin-binding nanoparticles for intracellular targeting addressed by B. burgdorferi BBK32 protein fragments.

Virus-like particles (VLPs) are created by the self-assembly of multiple copies of envelope and/or capsid proteins from many viruses, mimicking the conformation of a native virus. Such noninfectious nanostructures are mainly used as antigen-presenting platforms, especially in vaccine research; however, some of them recently were used as scaffolds in biotechnology to produce targeted nanoparticles for intracellular delivery. This study demonstrates the creation of fusion VLPs using hepatitis B core protein-based system maintaining a fibronectin-binding property from B. burgdorferi BBK32 protein, including the evidence of particles' transmission to BHK-21 target cells via caveolae/rafts endocythosis. These results make this construct to be an attractive model in development of HBc-based nanoparticles for cellular targeting applications and highlights the fragment of B. burgdorferi BBK32 as a novel cellular uptake-promoting peptide. FROM THE CLINICAL EDITOR: This paper discusses the nanotechnology-based application of self-assembling viral-like peptides (VLP-s) for targeted delivery using a hepatitis B core protein based system. Creating fusion VLPs may be an attractive model for cellular targeting applications.

A unique borrelial protein facilitates microbial immune evasion.

IMPORTANCE: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi, which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi, partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection.

Structural Analysis of the Outer Membrane Lipoprotein BBA14 (OrfD) and the Corresponding Paralogous Gene Family 143 (PFam143) from Borrelia burgdorferi.

Lyme disease is caused by the spirochete Borrelia burgdorferi, which can be transmitted to a mammalian host when infected Ixodes ticks feed. B. burgdorferi has many unique characteristics, such as the presence of at least 130 different lipoproteins, which is considerably more than any other known bacterium. Moreover, the B. burgdorferi genome is relatively small (1.5 Mbp) but at the same time it is quite complicated because it comprises a chromosome and 21 linear and circular plasmids. B. burgdorferi is also rich in paralogous proteins; in total, there are approximately 150 paralogous gene families. Equally important is the fact that there is still no vaccine against the Lyme disease. To better understand the role of lipoproteins in this unique bacterium, we solved the crystal structure of the outer membrane lipoprotein BBA14, which is coded on the relatively stable linear plasmid 54 (lp54). BBA14 does not share sequence identity with any other known proteins, and it is one of the ten members of the paralogous gene family 143 (PFam143). PFam143 members are known as orfD proteins from a genetic locus, designated 2.9. The obtained crystal structure revealed similarity to the antitoxin from the epsilon/zeta toxin-antitoxin system. The results of this study help to characterize BBA14 and to clarify the role of PFam143 in the lifecycle of B. burgdorferi.

Structural and Functional Analysis of BBA03, Borrelia burgdorferi Competitive Advantage Promoting Outer Surface Lipoprotein.

BBA03 is a Borrelia burgdorferi outer surface lipoprotein encoded on one of the most conserved plasmids in Borrelia genome, linear plasmid 54 (lp54). Although many of its genes have been identified as contributing or essential for spirochete fitness in vivo, the majority of the proteins encoded on this plasmid have no known function and lack homologs in other organisms. In this paper, we report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBA03, which is known to provide a competitive advantage to the bacteria during the transmission from tick vector to mammalian host. BBA03 shows structural homology to other outer surface lipoproteins reflecting their genetic and evolutionary relatedness. Analysis of the structure reveals a pore in BBA03, which could potentially bind lipids.

Structural analysis of Borrelia burgdorferi periplasmic lipoprotein BB0365 involved in Lyme disease infection.

The periplasmic lipoprotein BB0365 of the Lyme disease agent Borrelia burgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG subunits of the NADH:quinone and ferredoxin:NAD+ sodium-translocating oxidoreductase complexes, which points to a potential role for BB0365 as a component of the sodium pump. Additionally, BB0365 coordinated Zn2+ by the His51, His55, His140 residues, and the Zn2+ -binding site indicates that BB0365 could act as a potential metalloenzyme; therefore, this structure narrows down the potential functions of BB0365, an essential protein for B. burgdorferi to cause Lyme disease.

BBE31 from the Lyme disease agent Borrelia burgdorferi, known to play an important role in successful colonization of the mammalian host, shows the ability to bind glutathione.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determined the crystal structures of the key outer surface protein BBE31 from B. burgdorferi and its orthologous protein BSE31 (BSPA14S_RS05060 gene product) from B. spielmanii. BBE31 is known to be important for the transfer of B. burgdorferi from the gut to the hemolymph in the tick after a tick bite. While BBE31 exerts its function by interacting with the Ixodes scapularis tick gut protein TRE31, structural and mass spectrometry data revealed that BBE31 has a glutathione (GSH) covalently attached to Cys142 suggesting that the protein may have acquired some additional functions in contrast to its orthologous protein BSE31, which lacks any interactions with GSH. In the current study, in addition to analyzing the potential reasons for GSH binding, the three-dimensional structure of BBE31 provides new insights into the molecular details of the transmission process as the protein plays an important role in the initial phase before the spirochete is physically transferred to the new host. This knowledge will be potentially used for the development of new strategies to fight against Lyme disease.

Crystal structure of the N-terminal domain of the major virulence factor BB0323 from the Lyme disease agent Borrelia burgdorferi.

Lyme disease is an infection caused by the spirochete Borrelia burgdorferi after it is transmitted to a mammalian organism during a tick blood meal. B. burgdorferi encodes at least 140 lipoproteins located on the outer or inner membrane, thus facing the surroundings or the periplasmic space, respectively. However, most of the predicted lipoproteins are of unknown function, and only a few proteins are known to be essential for the persistence and virulence of the pathogen. One such protein is the periplasmic BB0323, which is indispensable for B. burgdorferi to cause Lyme disease and the function of which is associated with cell fission and outer membrane integrity. After expression and transport to the periplasm, BB0323 is cleaved into C-terminal and N-terminal domains by the periplasmic serine protease BB0104. The resulting N-terminal domain is sufficient to ensure the survival of B. burgdorferi throughout the mouse-tick infection cycle. The crystal structure of the N-terminal domain of BB0323 was determined at 2.35 Šresolution. The overall fold of the protein belongs to the spectrin superfamily, with the characteristic interconnected triple-helical bundles known as spectrin repeats that function as linkers between different cell components in other organisms. Overall, the reported three-dimensional structure of the N-terminal domain of BB0323 not only reveals the molecular details of a protein that is essential for B. burgdorferi membrane integrity, cell fission and infectivity, but also suggests that spectrin repeats in bacteria are not limited to the EzrA proteins.

Crystal structure of Borrelia burgdorferi outer surface protein BBA69 in comparison to the paralogous protein CspA.

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis - the most common tick-borne disease in Europe and the United States. Spirochetes are transmitted from infected Ixodes ticks to the mammalian host when the ticks feed. In general, the transfer process of the borreliae is quite complicated, as the environments in the tick and the new mammalian host differs significantly. Therefore, Borrelia changes the expression profile of dozens of proteins, mainly outer surface proteins, to adapt to the new tasks and needs in the new organism. In the transfer process from the tick to the mammalian host, spirochetes that cause Lyme disease show the strongest upregulation of members of paralogous gene family 54 (PFam54). PFam54 members encode 10 proteins, and BBA69 is one of its members. Although several PFam54 members play an important role in the pathogenesis of Lyme disease, the exact function has been determined only for CspA, which binds complement regulator factor H (CFH) and factor H-like protein 1 (FHL-1); thus, CspA is essential to resist the vertebrate host's immune response. In the current study, we determined the crystal structure of BBA69 at a 2.25 Ǻ resolution. The BBA69 structure revealed a seven α-helical BbCRASP-1 fold previously found only in PFam54 member proteins. Among the PFam54 members, BBA69 shares the highest sequence identity (61%) and 3-D similarity with CspA. Although none of the PFam54 members besides CspA bind CFH and FHL-1, in the current study, we investigated the structural differences accounting for the divergence in the functions of these proteins. The results clearly indicated that the C-terminal α-helix is the main determinant of this functional divergence. The results provide better insight into the PFam54 proteins that play an important role in the pathogenesis of Lyme disease.

Crystal structure of the membrane attack complex assembly inhibitor BGA71 from the Lyme disease agent Borrelia bavariensis.

Borrelia (B.) bavariensis, B. burgdorferi, B. afzelii, B. garinii, B. spielmanii, and B. mayonii are the causative agents in Lyme disease. Lyme disease spirochetes reside in infected Ixodes ticks and are transferred to mammalian hosts during tick feeding. Once transmitted, spirochetes must overcome the first line of defense of the innate immune system either by binding complement regulators or by terminating the formation of the membrane attack complex (MAC). In B. bavariensis, the proteins BGA66 and BGA71 inhibit complement activation by interacting with the late complement components C7, C8, and C9, as well as with the formed MAC. In this study, we have determined the crystal structure of the potent MAC inhibitor BGA71 at 2.9 Ǻ resolution. The structure revealed a cysteine cross-linked homodimer. Based on the crystal structure of BGA71 and the structure-based sequence alignment with CspA from B. burgdorferi, we have proposed a potential binding site for C7 and C9, both of which are constituents of the formed MAC. Our results shed light on the molecular mechanism of immune evasion developed by the human pathogenic Borrelia species to overcome innate immunity. These results will aid in the understanding of Lyme disease pathogenesis and pave the way for the development of new strategies to prevent Lyme disease.

Crystal structure of the infectious phenotype-associated outer surface protein BBA66 from the Lyme disease agent Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host organisms by infected Ixodes ticks. Transfer of the spirochaetal bacteria from Ixodes ticks to the warm-blooded mammalian organism provides a challenge for the bacteria to adapt and survive in the different environmental conditions. B. burgdorferi has managed to differentially express genes in response to the encountered changes such as temperature and pH variance or metabolic rate to survive in both environments. In recent years, much interest has been turned on genes that are upregulated during the borrelial transfer to mammalian organisms as this could reveal the proteins important in the pathogenesis of Lyme disease. BBA66 is one of the upregulated outer surface proteins thought to be important in the pathogenesis of B. burgdorferi as it has been found out that BBA66 is necessary during the transmission and propagation phase to initiate Lyme disease. As there is still little known about the pathogenesis of B. burgdorferi, we have solved the crystal structure of the outer surface protein BBA66 at 2.25Å resolution. A monomer of BBA66 consists of 6 α-helices packed in a globular domain, and the overall folding is similar to the homologous proteins BBA64, BBA73, and CspA. Structure-based sequence alignment with the homologous protein BBA64 revealed that the conserved residues are mainly located inwards the core region of the protein and thus may be required to maintain the overall fold of the protein. Unlike the other homologous proteins, BBA66 has an atypically long disordered region at the N terminus thought to act as a "tether" between the structural domain and the cell surface.