Identification of a novel transport system in Borrelia burgdorferi that links the inner and outer membranes.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a diderm organism that is similar to Gram-negative organisms in that it contains both an inner and outer membrane. Unlike typical Gram-negative organisms, however, B. burgdorferi lacks lipopolysaccharide (LPS). Using computational genome analyses and structural modeling, we identified a transport system containing six proteins in B. burgdorferi that are all orthologs to proteins found in the lipopolysaccharide transport (LPT) system that links the inner and outer membranes of Gram-negative organisms and is responsible for placing LPS on the surface of these organisms. While B. burgdorferi does not contain LPS, it does encode over 100 different surface-exposed lipoproteins and several major glycolipids, which like LPS are also highly amphiphilic molecules, though no system to transport these molecules to the borrelial surface is known. Accordingly, experiments supplemented by molecular modeling were undertaken to determine whether the orthologous LPT system identified in B. burgdorferi could transport lipoproteins and/or glycolipids to the borrelial outer membrane. Our combined observations strongly suggest that the LPT transport system does not transport lipoproteins to the surface. Molecular dynamic modeling, however, suggests that the borrelial LPT system could transport borrelial glycolipids to the outer membrane.

Amplification of the Specific Conformational Fluctuation of Proteins by Site-Specific Mutagenesis and Hydrostatic Pressure.

Conformational fluctuation, namely, protein interconversion between different conformations, is crucial to protein function. Outer surface protein A (OspA), comprising N- and C-terminal globular domains linked by a central ß-sheet, is expressed on the surface of Borrelia burgdorferi, the causative agent of Lyme disease, and recognizes the TROSPA receptor in the tick gut. Solution nuclear magnetic resonance studies have shown that the central ß-sheet and C-terminal domain containing TROSPA recognition sites are less stable than the N-terminal domain, revealing an intermediate conformation between the basic folded and completely unfolded proteins. We previously suggested that exposure of receptor-binding sites following denaturation of the C-terminal domain is advantageous for OspA binding to the receptor. Here, we observed amplification of a specific protein fluctuation by pressure perturbation and site-specific mutagenesis. The salt-bridge-destabilized mutant E160D and the cavity-enlarged mutant I243A favored the intermediate. The proportion of the intermediate accounted for almost 100% in E160D at 250 MPa. Strategies using a suitably chosen point mutation with high pressure are generally applicable for amplification of specific conformational fluctuation and potentially improve our understanding of the intermediate conformations of proteins. Knowledge of various conformations, including OspA intermediates, may be useful for designing a vaccine for Lyme disease.

Lyme Disease, Borrelia burgdorferi, and Lipid Immunogens.

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi, and a galactoglycerolipid (1) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3, a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography-coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1') and reveals a surprising role for C1r B loop-interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway-specific inhibition by a human pathogen.

High-resolution crystal structure of the Borreliella burgdorferi PlzA protein in complex with c-di-GMP: new insights into the interaction of c-di-GMP with the novel xPilZ domain.

In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.

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.

Molecular mechanism for rotational switching of the bacterial flagellar motor.

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

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.

Changing of the guard: How the Lyme disease spirochete subverts the host immune response.

Lyme disease, also known as Lyme borreliosis, is the most common tick-transmitted disease in the Northern Hemisphere. The disease is caused by the bacterial spirochete Borrelia burgdorferi and other related Borrelia species. One of the many fascinating features of this unique pathogen is an elaborate system for antigenic variation, whereby the sequence of the surface-bound lipoprotein VlsE is continually modified through segmental gene conversion events. This perpetual changing of the guard allows the pathogen to remain one step ahead of the acquired immune response, enabling persistent infection. Accordingly, the vls locus is the most evolutionarily diverse genetic element in Lyme disease-causing borreliae. Small stretches of information are transferred from a series of silent cassettes in the vls locus to generate an expressed mosaic vlsE gene version that contains genetic information from several different silent cassettes, resulting in ∼1040 possible vlsE sequences. Yet, despite its extreme evolutionary flexibility, the locus has rigidly conserved structural features. These include a telomeric location of the vlsE gene, an inverse orientation of vlsE and the silent cassettes, the presence of nearly perfect inverted repeats of ∼100 bp near the 5' end of vlsE, and an exceedingly high concentration of G runs in vlsE and the silent cassettes. We discuss the possible roles of these evolutionarily conserved features, highlight recent findings from several studies that have used next-generation DNA sequencing to unravel the switching process, and review advances in the development of a mini-vls system for genetic manipulation of the locus.

Molecular Bases of DNA Packaging in Bacteria Revealed by All-Atom Molecular Dynamics Simulations: The Case of Histone-Like Proteins in Borrelia burgdorferi.

DNA compaction is essential to ensure the packaging of the genetic material in living cells and also plays a key role in the epigenetic regulation of gene expression. In both humans and bacteria, DNA packaging is achieved by specific well-conserved proteins. Here, by means of all-atom molecular dynamics simulations, including the determination of relevant free-energy profiles, we rationalize the molecular bases for this remarkable process in bacteria, illustrating the crucial role played by positively charged amino acids of a small histone-like protein. We also present compelling evidence that this histone-like protein alone can induce strong bending of a DNA duplex around its core domain, a process that requires overcoming a major free-energy barrier.

Architecture and Assembly of Periplasmic Flagellum.

Periplasmic flagella are complex nanomachines responsible for distinctive morphology and motility of spirochetes. Although bacterial flagella have been extensively studied for several decades in the model systems Escherichia coli and Salmonella enterica, our understanding of periplasmic flagella in many disease-causing spirochetes remains incomplete. Recent advances, including molecular genetics, biochemistry, structural biology, and cryo-electron tomography, have greatly increased our understanding of structure and function of periplasmic flagella. In this chapter, we summarize some of the recent findings that provide new insights into the structure, assembly, and function of periplasmic flagella.

Structural insights into flagellar stator-rotor interactions.

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator-rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator-rotor interaction at an unprecedented detail. Importantly, the stator-rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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.

Linear B Cell Epitopes Derived from the Multifunctional Surface Lipoprotein BBK32 as Targets for the Serodiagnosis of Lyme Disease.

BBK32 is a multifunctional surface lipoprotein expressed by Borrelia burgdorferisensu lato, the causative agent of Lyme disease. Previous studies suggested that BBK32 could be a sensitive antigen target of new, more effective, serodiagnostic assays for the laboratory diagnosis of Lyme disease. However, nonspecific antibody binding to full-length BBK32 has hampered its use as a target in clinical assays. Specificity can be improved by the use of peptides composed of linear B cell epitopes that are unique to B. burgdorferi, eliminating cross-reactive epitopes that bind to antibodies generated by non-B. burgdorferi antigens. In this study, we identified linear B cell epitopes in 2 regions, BBK32 amino acids 16 to 30 [BBK32(16-30)] and BBK32 amino acids 51 to 80 [BBK32(51-80)], by probing overlapping peptide libraries of BBK32 with serum from patients with early Lyme disease. We screened synthetic peptides containing these epitopes using a large panel of serum (n = 355) obtained from patients with erythema migrans lesions (early Lyme disease), Lyme arthritis, syphilis, rheumatoid arthritis, or healthy volunteers. BBK32(16-30) demonstrated a nearly universal antibody binding in serum from all patients, indicating that regions of BBK32 are highly cross-reactive. BBK32(51-80) was less cross-reactive, being able to distinguish serum from Lyme disease patients from control patient serum; however, an unacceptable level of antibody binding was still observed in control samples, resulting in a reduced specificity (94.7%). These results indicate that BBK32 contains cross-reactive epitopes that make it a poor antigen target for inclusion in a serodiagnostic assay for Lyme disease and highlight the difficulties in identifying highly sensitive and specific seroassay targets.IMPORTANCE Lyme disease is an infectious disease that has the potential to cause significant morbidity with damage to nervous and musculoskeletal systems if left untreated. Appropriate antibiotic treatment during early infection prevents disease progression. Unfortunately, currently available diagnostics are suboptimal in the detection of early disease. The inability to confirm Borrelia infection using laboratory methods during early disease is, in part, responsible for much of the controversy surrounding Lyme disease today. As a result, there has been significant investment in the identification of new antigen targets to generate diagnostic assays that are more sensitive for the detection of early infection. The importance of our research is that in our evaluation of BBK32, an antigen that was previously identified as a promising target for use in serodiagnostics, we found a high degree of cross-reactivity that could compromise the specificity of assays that utilize this antigen, leading to false-positive diagnoses.

Analysis of a flagellar filament cap mutant reveals that HtrA serine protease degrades unfolded flagellin protein in the periplasm of Borrelia burgdorferi.

Unlike external flagellated bacteria, spirochetes have periplasmic flagella (PF). Very little is known about how PF are assembled within the periplasm of spirochaetal cells. Herein, we report that FliD (BB0149), a flagellar cap protein (also named hook-associated protein 2), controls flagellin stability and flagellar filament assembly in the Lyme disease spirochete Borrelia burgdorferi. Deletion of fliD leads to non-motile mutant cells that are unable to assemble flagellar filaments and pentagon-shaped caps (10 nm in diameter, 12 nm in length). Interestingly, FlaB, a major flagellin protein of B. burgdorferi, is degraded in the fliD mutant but not in other flagella-deficient mutants (i.e., in the hook, rod, or MS-ring). Biochemical and genetic studies reveal that HtrA, a serine protease of B. burgdorferi, controls FlaB turnover. Specifically, HtrA degrades unfolded but not polymerized FlaB, and deletion of htrA increases the level of FlaB in the fliD mutant. Collectively, we propose that the flagellar cap protein FliD promotes flagellin polymerization and filament growth in the periplasm. Deletion of fliD abolishes this process, which leads to leakage of unfolded FlaB proteins into the periplasm where they are degraded by HtrA, a protease that prevents accumulation of toxic products in the periplasm.

Predicting the ligand-binding properties of Borrelia burgdorferi s.s. Bmp proteins in light of the conserved features of related Borrelia proteins.

Bacteria of the genus Borrelia cause vector-borne infections like the most important hard tick-borne disease in the northern hemisphere, Lyme borreliosis (LB), and soft tick or louse transmitted relapsing fevers (RF), prevalent in temperate and tropical areas. Borrelia burgdorferi sensu lato (s.l.) includes several genospecies and causes LB in humans. In infected patients, Borrelia burgdorferi sensu stricto (s.s.) expresses the BmpA, BmpB, BmpC and BmpD proteins. The role of these proteins in the pathogenesis of LB remains incompletely characterized, but they are, however, closely related to Treponema pallidum PnrA (Purine nucleoside receptor A), a substrate-binding lipoprotein of the ATP-binding cassette (ABC) transporter family preferentially binding purine nucleosides. Based on 3D homology modeling, the Bmp proteins share the typical fold of the substrate-binding protein family and the ligand-binding properties of BmpA, BmpB and BmpD are highly similar, whereas those of BmpC differ markedly. Nevertheless, these residues are highly conserved within the genus Borrelia and the inferred phylogenetic tree also reveals that the RF Borrelia lack BmpB proteins but has an additional Bmp protein (BmpA2) missing in LB-causing Borrelia burgdorferi s.l. Our results indicate that the Bmp proteins could bind nucleosides, although BmpC might have a different ligand-binding specificity and, therefore, a distinct function. Furthermore, the work provides a means for classifying the Bmp proteins and supports further elucidation of the roles of these proteins.

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior.

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a ß-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer ß-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer ß-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer ß-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.

Analysis of Lipids and Lipid Rafts in Borrelia.

Lipid rafts are membrane microdomains that are involved in cellular processes such as protein trafficking and signaling processes, and which play a fundamental role in membrane fluidity and budding. The lipid composition of the membrane and the biochemical characteristics of the lipids found within rafts define the ability of cells to form microdomains and compartmentalize the membrane. In this chapter, we describe the biophysical, biochemical, and molecular approaches used to define and characterize lipid rafts in the Lyme disease agent, Borrelia burgdorferi.