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Borrelia , spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia , including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. Key points • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1 Graphical Abstract

Lyme borreliosis (LB) is the most prevalent tick-borne illness, with an estimated 700 000 cases annually in the United States and Europe. The LB diagnosis based on a two-tiered serology remains controversial due to its indirect nature and low sensitivity during the early stage of the disease. Aptamers are single-stranded DNA or RNA oligonucleotides that exhibit high selectivity and specificity for their target due to their unique three-dimensional structure. By applying cross-over-SELEX process, an enrichment of DNA oligonucleotide sequences against a surface protein of Borrelia , named CspZ, has been performed and monitored using absorbance at 260 nm, melting curves and NGS analyses. Beyond sequence enrichment, oligonucleotides binding to CspZ were observed during the selection rounds by Dot Blot and beads assays. Thirteen unique and highly redundant oligonucleotide sequences were further characterized using multiple approaches such as Dot Blot, BioLayer Interferometry and Surface Plasmon Resonance. The selected aptamers showed K D values from tens of nanomolar to the micromolar range by BLI and SPR. Two aptamers, Apta9 and Apta10, characterized by flow cytometry and epifluorescence microscopy, were able to specifically recognize Borrelia burgdorferi sensu stricto. This strategy holds promise for the development of an improved diagnostic assay. Lyme borreliosis is challenging to diagnose, particularly in its early stages. Aptamers targeting CspZ from Borrelia , selected through cross-over SELEX, bind both the recombinant protein and bacteria, demonstrating potential for enhanced diagnostics.

Background. Decorin adherence is crucial in the pathogenesis of Lyme borreliosis. Decorin binding proteins (Dbp) A and B are the adhesins that mediate this interaction. DbpA and B of Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto (ss) differ in their amino acid sequence, but little attention has been paid to the potential difference in their decorin binding. Methods. We expressed recombinant DbpA and DbpB of B. garinii, B. afzelii, and B. burgdorferi ss and studied their binding to decorin. We also generated recombinant Borrelia strains to study the role of DbpA and DbpB in the adhesion of live spirochetes to decorin and decorin-expressing cells. Results. Recombinant DbpA of B. garinii and DbpB of B. garinii and B. burgdorferi ss showed strong binding to decorin, whereas DbpA of B. burgdorferi ss and both DbpA and DbpB of B. afzelii exhibited no or only minor binding activity. DbpA and DbpB of B. garinii and B. burgdorferi ss also supported the adhesion of whole spirochetes to decorin and decorin-expressing cells, whereas DbpA and DbpB of B. afzelii did not exhibit this activity. Conclusions. Dbp A and B of B. garinii and B. burgdorferi ss mediate the interaction between the spirochete and decorin, whereas the same adhesins of B. afzelii show only negligible activity.

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.In situ cryo-ET analyses of Borrelia burgdorferi flagellar motors locked in clockwise or counterclockwise rotation provide insights into rotational switching.

Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted to vertebrate hosts by Ixodes spp. ticks. The spirochaete relies heavily on its arthropod host for basic metabolic functions and has developed complex interactions with ticks to successfully colonize, persist and, at the optimal time, exit the tick. For example, proteins shield spirochaetes from immune factors in the bloodmeal and facilitate the transition between vertebrate and arthropod environments. On infection, B. burgdorferi induces selected tick proteins that modulate the vector gut microbiota towards an environment that favours colonization by the spirochaete. Additionally, the recent sequencing of the Ixodes scapularis genome and characterization of tick immune defence pathways, such as the JAK–STAT, immune deficiency and cross-species interferon-γ pathways, have advanced our understanding of factors that are important for B. burgdorferi persistence in the tick. In this Review, we summarize interactions between B. burgdorferi and I. scapularis during infection, as well as interactions with tick gut and salivary gland proteins important for establishing infection and transmission to the vertebrate host.Borrelia burgdorferi has a complex life cycle with several different hosts, causing Lyme disease when it infects humans. In this Review, Fikrig and colleagues discuss how B. burgdorferi infects and interacts with its tick vector to ensure onward transmission.

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus.

Flagella propel pathogens through their environments, yet are expensive to synthesize and are immunogenic. Thus, complex hierarchical regulatory networks control flagellar gene expression. Spirochetes are highly motile bacteria, but peculiarly, the archetypal flagellar regulator σ 28 is absent in the Lyme spirochete Borrelia burgdorferi . Here, we show that gene bb0268 ( flgV ) in B. burgdorferi , previously and incorrectly annotated to encode the RNA-binding protein Hfq, is instead a structural flagellar component that modulates flagellar assembly. The flgV gene is broadly conserved in the flagellar superoperon alongside σ 28 in many Spirochaetae , Firmicutes and other phyla, with distant homologs in Epsilonproteobacteria . We find that B. burgdorferi FlgV is localized within flagellar basal bodies, and strains lacking flgV produce fewer and shorter flagellar filaments and are defective in cell division and motility. During the enzootic cycle, flgV -deficient B. burgdorferi survive and replicate in Ixodes ticks but are attenuated for infection and dissemination in mice. Our work defines infection timepoints when spirochete motility is most crucial and implicates FlgV as a broadly distributed structural flagellar component that modulates flagellar assembly. The control of flagellar synthesis and function in the Lyme spirochete Borrelia burgdorferi is poorly understood, as this pathogen lacks the typical flagellar sigma factor and transcriptional regulators. Here, the authors identify a broadly conserved structural flagellar component that modulates flagellar assembly and is important for cell division, motility and virulence.

Lyme disease is caused by the host-adapted spirochete Borrelia burgdorferi . With a genome of only 1.5 mbp, B. burgdorferi is dependent on metabolites scavenged from their vertebrate and invertebrate hosts for growth. These scavenged nutrients include several lipid precursors: the spirochete is auxotrophic for fatty acids and cholesterol, and also accumulates environmental phospholipids. Comprehensive lipidomic analysis of B. burgdorferi by LC MS/MS was used to identify previously undescribed membrane components. These include some likely scavenged from the culture medium and some which may be synthesized de novo via unknown pathways. Changes in fatty acid composition as cells enter stationary phase suggest that scavenging of environmental lipids is preferential to de novo synthesis, while transcriptomics suggests that this may be due to the energetic cost of synthesizing glycerol phosphate precursors. In media supplemented with excess phospholipids, scavenged lipids can be found at high concentrations in cells, suggesting that the membranes of infecting bacteria are likely to be partly shaped by the host environment. Transcriptomic analysis also show a link between environmental lipids and the expression of virulence-associated surface lipoproteins including reciprocal regulation of ospA and ospC . Given that borrelial membrane lipids are known to be antigenic during infection, these findings identify potential new targets for the development of diagnostic tests or vaccines.

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PG Bb ) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PG Bb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PG Bb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PG Bb fragments using a mass spectrometry-based approach. By characterizing the l , d -carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PG Bb turnover largely occurs at sites of PG Bb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PG Bb fragments with 1,6-anhydro bond on their N -acetylmuramyl residues. Stimulation of human cell lines with various synthetic PG Bb fragments revealed that 1,6-anhydromuramyl-containing PG Bb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PG Bb isolated from dead bacteria. We also showed that the activity of the human N -acetylmuramyl- l -alanine amidase PGLYRP2, which reduces the immunogenicity of PG Bb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PG Bb fragments and that PG Bb -induced immunopathology likely results from host sensing of PG Bb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals.

Many spirochetes cause serious human diseases. They are well recognized by their distinct morphology and motility. Spirochetes are a remarkable group of bacteria with distinct morphology and periplasmic flagella that enable motility in viscous environments, such as host connective tissues. The collar, a spirochete-specific complex of the periplasmic flagellum, is required for this unique spirochete motility, yet it has not been clear how the collar assembles and enables spirochetes to transit between complex host environments. Here, we characterize the collar complex in the Lyme disease spirochete Borrelia burgdorferi . We discover as well as delineate the distinct functions of two novel collar proteins, FlcB and FlcC, by combining subtractive bioinformatic, genetic, and cryo-electron tomography approaches. Our high-resolution in situ structures reveal that the multiprotein collar has a remarkable structural plasticity essential not only for assembly of flagellar motors in the highly curved membrane of spirochetes but also for generation of the high torque necessary for spirochete motility. IMPORTANCE Many spirochetes cause serious human diseases. They are well recognized by their distinct morphology and motility. Spirochete motility is driven by a periplasmic flagellum, which possesses a unique collar essential for flagellar assembly and spirochete motility. Here, we discover two novel collar proteins in the Lyme disease spirochete Borrelia burgdorferi . We demonstrate, for the first time, that the collar is a multiprotein complex with a remarkable plasticity that enables the motor to accommodate the highly curved membrane of spirochetes and generate the high torque necessary for spirochete motility.