Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Borrelia burgdorferi sensu lato, the agent of lyme borreliosis: life in the wilds

In Europe, Borrelia burgdorferi sensu lato (sl) the agent of Lyme borreliosis circulates in endemic areas between Ixodes ricinus ticks and a large number of vertebrate hosts upon which ticks feed. Currently, at least 12 different Borrelia species belonging to the complex B. burgdorferi sl have been identified among which seven have been detected in I. ricinus: B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. valaisiana, B. spielmanii and B. bissettii. A few dozens of vertebrate hosts have been identified as reservoirs for these Borrelia species. Specific associations were rather early observed between hosts, ticks and borrelia species, like for example between rodents and B. afzelii and B. burgdorferi ss, and between birds and B. garinii and B. valaisiana. The complement present in the blood of the hosts is the active component in the Borrelia host specificity. Recent studies confirmed trends toward specific association between Borrelia species and particular host, but also suggested that loose associations may be more frequent in transmission cycles in nature than previously thought.     

Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.     

Microarray analyses of inflammation response of human dermal fibroblasts to different strains of Borrelia burgdorferi sensu stricto

In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease. Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans). The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-κB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated. In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts. Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs.     

Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.     

Host association of Borrelia burgdorferi sensu lato--the key role of host complement

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.     

Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic

Emerging zoonotic pathogens are a constant threat to human health throughout the world. Control strategies to protect public health regularly fail, due in part to the tendency to focus on a single host species assumed to be the primary reservoir for a pathogen. Here, we present evidence that a diverse set of species can play an important role in determining disease risk to humans using Lyme disease as a model. Host-targeted public health strategies to control the Lyme disease epidemic in North America have focused on interrupting Borrelia burgdorferi sensu stricto (ss) transmission between blacklegged ticks and the putative dominant reservoir species, white-footed mice. However, B. burgdorferi ss infects more than a dozen vertebrate species, any of which could transmit the pathogen to feeding ticks and increase the density of infected ticks and Lyme disease risk. Using genetic and ecological data, we demonstrate that mice are neither the primary host for ticks nor the primary reservoir for B. burgdorferi ss, feeding 10% of all ticks and 25% of B. burgdorferi-infected ticks. Inconspicuous shrews feed 35% of all ticks and 55% of infected ticks. Because several important host species influence Lyme disease risk, interventions directed at a multiple host species will be required to control this epidemic.     

Evaluation of a genotyping method based on the ospA gene to detect Borrelia burgdorferi sensu lato in multiple samples of lyme borreliosis patients

In this study we have developed a new Restriction-Fragment-Length-Polymorphism (RFLP) genotyping method for rapid detection and identification of Borrelia genospecies present as unique species or as co-infection in multiple specimens obtained simultaneously from 29 individual patients affected by early or late Lyme borreliosis (LB). The target of the RFLP-genotyping was the heterogeneous plasmid located ospA gene, thus we developed a method able to detect and differentiate between six clinically relevant Borrelia genospecies circulating in Europe, B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. bissettii and B. spielmanii. In this study Borrelia DNA could be detected by PCR in at least one specimen of each patient, except in one case of neuroborreliosis (NB); blood samples gave the highest sensitivity in all patient groups. The genotyping indicated that B. afzelii was present in 8 patients with skin involvement, B. garinii in 2 cases of NB and 4 cases with skin involvement, B. burgdorferi sensu stricto was detected in one patient with skin involvement and another with Lyme arthritis. Different Borrelia species in distinct specimens were identified in one patient with EM. The RFLP analysis of 11 patients revealed mixed patterns, which suggested pluri-infection with different Borrelia species.     

Fundamental processes in the evolutionary ecology of Lyme borreliosis

The evolutionary ecology of many emerging infectious diseases, particularly vector-borne zoonoses, is poorly understood. Here, we aim to develop a biological, process-based framework for vector-borne zoonoses, using Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis in humans, as an example. We explore the fundamental biological processes that operate in this zoonosis and put forward hypotheses on how extrinsic cues and intrinsic dynamics shape B. burgdorferi s.l. populations. Additionally, we highlight possible epidemiological parallels between B. burgdorferi s.l. and other vector-borne zoonotic pathogens, including West Nile virus.     

Preparation and purification of recombinant outer surface protein A (rOspA) of Borrelia burgdorferi sensu stricto and Borrelia afzelii

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitating disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recombinant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.     

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance of Borreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferi sensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15-20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.     

Distinct combinations of Borrelia burgdorferi sensu lato genospecies found in individual questing ticks from Europe

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.     

Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.     

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.     

Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi

Abstract The expression of outer surface protein C (OspC) was determined for North American Borrelia burgdorferi isolates HB19, DN127c19-2, 25015 and both low and high culture passage B31. A monoclonal antibody detected the presence of OspC protein in only two isolates, while polyclonal antiserum identified this protein in all five isolates. The ospC gene was cloned and sequenced for isolates HB19, DN127c19-2 and 25015, and compared with the published ospC sequences of other Lyme disease spirochetes. Bothe the nucleotide and amino acid sequences were found to vary as much among isolates from the same geographic area as between isolates of different species.