Middle region of the Borrelia burgdorferi surface‐located protein 1 (Lmp1) interacts with host chondroitin‐6‐sulfate and independently facilitates infection

Borrelia burgdorferi surface‐located membrane protein 1, also known as Lmp1, has been shown to play critical roles in pathogen evasion of host‐acquired immune defences, thereby facilitating persistent infection. Lmp1 possesses three regions representing potentially discrete domains: Lmp1N, Lmp1M and Lmp1C. Because of its insignificant homology to known proteins, how Lmp1 or its specific regions contribute to microbial biology and infection remains enigmatic. Here, we show that distinct from Lmp1N and Lmp1C, Lmp1M is composed of at least 70% alpha helices and completely lacks recognizable beta sheets. The region binds to host glycosaminoglycan chondroitin‐6‐sulfate molecules and facilitates mammalian cell attachment, suggesting an adhesin function of Lmp1M. Phenotypic analysis of the Lmp1‐deficient mutant engineered to produce Lmp1M on the microbial surface suggests that Lmp1M can independently support B. burgdorferi infectivity in murine hosts. Further exploration of functions of Lmp1 distinct regions will shed new light on the intriguing biology and infectivity of spirochetes and help develop novel interventions to combat Lyme disease.     

Borrelia burgdorferi surface‐located Lmp1 protein processed into region‐specific polypeptides that are critical for microbial persistence

One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface‐exposed, conserved, and potential multi‐domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host–pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region‐specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N‐terminal and middle regions and a combined middle and C‐terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain‐specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N‐terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.     

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.     

Strain-Specific Variation of the Decorin-Binding Adhesin DbpA Influences the Tissue Tropism of the Lyme Disease Spirochete

Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.     

A Chitin Deacetylase-Like Protein Is a Predominant Constituent of Tick Peritrophic Membrane That Influences the Persistence of Lyme Disease Pathogens within the Vector

Ixodes scapularis is the specific arthropod vector for a number of globally prevalent infections, including Lyme disease caused by the bacterium Borrelia burgdorferi. A feeding-induced and acellular epithelial barrier, known as the peritrophic membrane (PM) is detectable in I. scapularis. However, whether or how the PM influences the persistence of major tick-borne pathogens, such as B. burgdorferi, remains largely unknown. Mass spectrometry-based proteome analyses of isolated PM from fed ticks revealed that the membrane contains a few detectable proteins, including a predominant and immunogenic 60 kDa protein with homology to arthropod chitin deacetylase (CDA), herein termed I. scapularis CDA-like protein or IsCDA. Although IsCDA is primarily expressed in the gut and induced early during tick feeding, its silencing via RNA interference failed to influence either the occurrence of the PM or spirochete persistence, suggesting a redundant role of IsCDA in tick biology and host-pathogen interaction. However, treatment of ticks with antibodies against IsCDA, one of the most predominant protein components of PM, affected B. burgdorferi survival, significantly augmenting pathogen levels within ticks but without influencing the levels of total gut bacteria. These studies suggested a preferential role of tick PM in limiting persistence of B. burgdorferi within the vector. Further understanding of the mechanisms by which vector components contribute to pathogen survival may help the development of new strategies to interfere with the infection.     

Decorin Binding Proteins of Borrelia burgdorferi Promote Arthritis Development and Joint Specific Post-Treatment DNA Persistence in Mice

Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.     

Differential Transmission of the Genospecies of Borrelia burgdorferi Sensu Lato by Game Birds and Small Rodents in England

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.     

Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d ^-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.     

Borrelia burgdorferi protein interactions critical for microbial persistence in mammals

Borrelia burgdorferi is the causative agent of Lyme disease that persists in a complex enzootic life cycle, involving Ixodes ticks and vertebrate hosts. The microbe invades ticks and vertebrate hosts in spite of active immune surveillance and potent microbicidal responses, and establishes long‐term infection utilising mechanisms that are yet to be unravelled. The pathogen can cause multi‐system disorders when transmitted to susceptible mammalian hosts, including in humans. In the past decades, several studies identified a limited number of B. burgdorferi gene‐products critical for pathogen persistence, transmission between the vectors and the host, and host–pathogen interactions. This review will focus on the interactions between B. burgdorferi proteins, as well as between microbial proteins and host components, protein and non‐protein components, highlighting their roles in pathogen persistence in the mammalian host. A better understanding of the contributions of protein interactions in the microbial virulence and persistence of B. burgdorferi would support development of novel therapeutics against the infection.     

Borrelia burgdorferi Sensu Stricto Is Clonal in Patients with Early Lyme Borreliosis

Lyme borreliosis, the most commonly reported vector-borne disease in North America, is caused by the spirochete Borrelia burgdorferi. Given the extensive genetic polymorphism of B. burgdorferi, elucidation of the population genetic structure of the bacterium in clinical samples may be relevant for understanding disease pathogenesis and may have applicability for the development of diagnostic tests and vaccine preparations. In this investigation, the genetic polymorphism of the 16S-23S rRNA (rrs-rrlA) intergenic spacer and ospC was investigated at the sequence level in 127 clinical isolates obtained from patients with early Lyme borreliosis evaluated in suburban New York City. Sixteen distinct rrs-rrlA and 16 distinct ospC alleles were identified, representing virtually all of the genotypes previously found in questing Ixodes scapularis nymphs in this region. In addition, a new ospC group was identified in a single patient. The strong linkage observed between the chromosome-located rrs-rrlA and plasmid-borne ospC genes suggests a clonal structure of B. burgdorferi in these isolates, despite evidence of recombination at ospC.     

Outer surface protein C is a dissemination-facilitating factor of Borrelia burgdorferi during mammalian infection

Background

The Lyme disease spirochete Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection.

Methodology/Principal Findings

In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn''t significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice.

Conclusions/Significance

The study indicates that OspC plays an important role in dissemination of B. burgdorferi during mammalian infection.     

Genome-Wide Transposon Mutagenesis of Borrelia burgdorferi for Identification of Phenotypic Mutants

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.     

An investigation of binding ability of Ixodes persulcatus Schulze Salp15 with Lyme disease spirochetes

Salp15, a 15-kDa tick salivary gland protein, has several suppressive modes of activity against host immunity and plays a critical role in the transmission of Lyme disease spirochetes in Ixodes scapularis and Ixodes ricinus, major vectors of Lyme disease in North America and Western Europe. Salp15 adheres to Borrelia burgdorferi and specifically interacts with its outer surface protein C (OspC), protecting the spirochete from antibody-mediated cytotoxicity and facilitating infection in the mice. Recently, we identified two Salp15 homologues, IperSalp15-1 and IperSalp15-2, in Ixodes persulcatus, a vector for Lyme disease in Japan. Here we describe the function of IperSalp15 in the transmission of Lyme borreliosis. To investigate the function of IperSalp15, recombinant IperSalp15-1 and IperSalp15-2 were prepared in bacterial and insect cells. Both were identified in the sera of tick-immunized hamsters, indicating that these are secretory proteins in exposed host animals. Solid-phase overlay and indirect fluorescence assays showed that IperSalp15 binds to OspC from B. burgdorferi, Borrelia garinii, and Borrelia afzelii. Importantly, this binding likely protected the spirochete from antibody-mediated cytotoxicity in vitro. In addition, IperSalp15 tended to facilitate infection in mice. Thus, further characterization of tick molecules, including IperSalp15, could lead to the development of new strategies to prevent the transmission of tick-borne diseases.     

Characterization of 6S RNA in the Lyme disease spirochete

6S RNA binds to RNA polymerase and regulates gene expression, contributing to bacterial adaptation to environmental stresses. In this study, we examined the role of 6S RNA in murine infectivity and tick persistence of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. B. burgdorferi 6S RNA (Bb6S RNA) binds to RNA polymerase, is expressed independent of growth phase or nutrient stress in culture, and is processed by RNase Y. We found that rny (bb0504), the gene encoding RNase Y, is essential for B. burgdorferi growth, while ssrS, the gene encoding 6S RNA, is not essential, indicating a broader role for RNase Y activity in the spirochete. Bb6S RNA regulates expression of the ospC and dbpA genes encoding outer surface protein C and decorin binding protein A, respectively, which are lipoproteins important for host infection. The highest levels of Bb6S RNA are found when the spirochete resides in unfed nymphs. ssrS mutants lacking Bb6S RNA were compromised for infectivity by needle inoculation, but injected mice seroconverted, indicating an ability to activate the adaptive immune response. ssrS mutants were successfully acquired by larval ticks and persisted through fed nymphs. Bb6S RNA is one of the first regulatory RNAs identified in B. burgdorferi that controls the expression of lipoproteins involved in host infectivity.     

Treatment of Lyme borreliosis

Borrelia burgdorferi sensu lato is the causative agent of Lyme borreliosis in humans. This inflammatory disease can affect the skin, the peripheral and central nervous system, the musculoskeletal and cardiovascular system and rarely the eyes. Early stages are directly associated with viable bacteria at the site of inflammation. The pathogen-host interaction is complex and has been elucidated only in part. B. burgdorferi is highly susceptible to antibiotic treatment and the majority of patients profit from this treatment. Some patients develop chronic persistent disease despite repeated antibiotics. Whether this is a sequel of pathogen persistence or a status of chronic auto-inflammation, auto-immunity or a form of fibromyalgia is highly debated. Since vaccination is not available, prevention of a tick bite or chemoprophylaxis is important. If the infection is manifest, then treatment strategies should target not only the pathogen by using antibiotics but also the chronic inflammation by using anti-inflammatory drugs.     

What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.     

The role of host immune cells and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> antigens in the etiology of Lyme disease

Lyme disease is a zoonosis caused by infection with bacteria belonging to the Borrelia burgdorferi species after the bite of an infected tick. Even though an infection by this bacterium can be effectively treated with antibiotics, when the infection stays unnoticed B. burgdorferi can persist and chronic post-treatment Lyme disease syndrome is able to develop. Although a cellular and humoral response is observed after an infection with the Borrelia bacteria, these pathogens are still capable to stay alive. Several immune evasive mechanisms have been revealed and explained and much work has been put into the understanding of the contribution of the innate and adaptive immune response. This review provides an overview with the latest findings regarding the cells of the innate and adaptive immune systems, how they recognize contribute and mediate in the killing of the B. burgdorferi spirochete. Moreover, this review also elaborates on the antigens that are expressed by on the spirochete. Since antigens drive the adaptive and, indirectly, the innate response, this review will discuss briefly the most important antigens that are described to date. Finally, there will be a brief elaboration on the escape mechanisms of B. burgdorferi with a focus on tick salivary proteins and spirochete antigens.