Borrelia burgdorferi RevA Significantly Affects Pathogenicity and Host Response in the Mouse Model of Lyme Disease

The Lyme disease spirochete, Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, with increased fibrotic collagen deposition in tibiotarsal joints. The mutants also induced increased levels of the chemokine CCL2, a monocyte chemoattractant, in serum, and this increase was abolished in the complemented strain. Therefore, while revA is not absolutely essential for infection, deletion of revA had distinct effects on dissemination, arthritis severity, and host response.     

Quantitative Detection of Borrelia burgdorferi by Real-Time PCR

Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect B. burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of B. burgdorferi was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the B. burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of B. burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.     

Lack of Serum Antibodies against Borrelia burgdorferi in Children with Autism

It has been proposed that Borrelia burgdorferi infection is present in ∼25% of children with autism spectrum disorders. In this study, antibodies against Borrelia burgdorferi were assessed in autistic (n = 104), developmentally delayed (n = 24), and healthy control (n = 55) children. No seropositivity against Borrelia burgdorferi was detected in the children with and without autism. There was no evidence of an association between Lyme disease and autism.     

Antibodies to Borrelia burgdorferi in rodents in the eastern and southern United States.

Serologic studies were conducted to determine whether white-footed mice (Peromyscus leucopus) and cotton mice (Peromyscus gossypinus) contained serum antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Enzyme-linked immunosorbent assays detected antibodies to this spirochete in 35.7 and 27.3% of 56 P. leucopus and 535 P. gossypinus serum samples, respectively, collected in Connecticut, North Carolina, South Carolina, Georgia, Florida, Alabama, and Mississippi. Antibody titers ranged from 1:160 to greater than or equal to 1:40,960. On the basis of adsorption tests, the antibodies detected appeared to be specific to Borrelia spirochetes. Seropositive rodents in the eastern and southern United States, areas where human cases of Lyme borreliosis have been reported, indicate a widespread geographic distribution of B. burgdorferi or a closely related spirochete.     

Detection of established virulence genes and plasmids to differentiate Borrelia burgdorferi strains

Borrelia burgdorferi sensu stricto is the major causative agent of Lyme disease in the United States, while B. garinii and B. afzelii are more prevalent in Europe. The highly complex genome of B. burgdorferi is comprised of a linear chromosome and a large number of variably sized linear and circular plasmids. Many plasmids of this spirochete are unstable during its culture in vitro. Given that many of the B. burgdorferi virulence factors identified to date are plasmid encoded, spirochetal plasmid content determination is essential for genetic analysis of Lyme pathogenesis. Although PCR-based assays facilitate plasmid profiling of sequenced B. burgdorferi strains, a rapid genetic content determination strategy for nonsequenced strains has not yet been described. In this study, we combined pulsed-field gel electrophoresis (PFGE) and Southern hybridization for detection of genes encoding known virulence factors, ribosomal RNA gene spacer restriction fragment length polymorphism types (RSTs), ospC group determination, and sequencing of the variable dbpA and ospC genes. We show that two strains isolated from the same tick and both originally named N40 are in fact very distinct. Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin, in one "N40" strain. Thus, two distinct strains that show different plasmid profiles, as determined by PFGE and PCR, were isolated from the same tick and vary in their ospC and dbpA sequences. However, both belong to group RST3B.     

Heat Shock Protein 70 of the Agent of Human Granulocytic Ehrlichiosis Binds to Borrelia burgdorferi Antibodies

We describe a patient with human granulocytic ehrlichiosis (HGE), a diagnosis confirmed by PCR and immunoblot analysis. Unexpectedly, immunoglobulin G (IgG) directed towards an 80-kDa ehrlichial antigen (without detectable IgM) was present in the patient’s serum in the first week of illness. Lyme disease immunoblots were reactive for IgG (but not IgM), a result indicative of prior exposure to the Lyme disease spirochete. Amino-terminal sequencing revealed that the 80-kDa ehrlichial antigen was an HSP-70 homolog similar to Borrelia burgdorferi HSP-70. We conclude that antibodies against B. burgdorferi HSP-70 may cross-react with the ehrlichial heat shock protein and that this possibility must be considered when serologic test results for HGE and Lyme disease are interpreted.     

Detection of Borrelia burgdorferi using the polymerase chain reaction.

Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.     

Understanding Barriers to Borrelia burgdorferi Dissemination during Infection Using Massively Parallel Sequencing

Borrelia burgdorferi is an invasive spirochete that can cause acute and chronic infections in the skin, heart, joints, and central nervous system of infected mammalian hosts. Little is understood about where the bacteria encounter the strongest barriers to infection and how different components of the host immune system influence the population as the infection progresses. To identify population bottlenecks in a murine host, we utilized Tn-seq to monitor the composition of mixed populations of B. burgdorferi during infection. Both wild-type mice and mice lacking the Toll-like receptor adapter molecule MyD88 were infected with a pool of infectious B. burgdorferi transposon mutants with insertions in the same gene. At multiple time points postinfection, bacteria were isolated from the mice and the compositions of the B. burgdorferi populations at the injection site and in distal tissues determined. We identified a population bottleneck at the site of infection that significantly altered the composition of the population. The magnitude of this bottleneck was reduced in MyD88^−/− mice, indicating a role for innate immunity in limiting early establishment of B. burgdorferi infection. There is not a significant bottleneck during the colonization of distal tissues, suggesting that founder effects are limited and there is not a strict limitation on the number of organisms able to initiate populations at distal sites. These findings further our understanding of the interactions between B. burgdorferi and its murine host in the establishment of infection and dissemination of the organism.     

伯氏疏螺旋体与嗜吞噬细胞无浆体混合感染的实验研究

目的应用动物模型探讨蜱传病原体伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染对组织螺旋体载量、莱姆关节炎严重性、宿主免疫功能的影响。方法建立伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染和伯氏疏螺旋体单一感染小鼠模型，在不同时间点研究各组小鼠组织中的螺旋体载量、关节炎严重性和血清IgG效价，并对数据进行统计学分析。结果与单一感染组相比，在螺旋体感染后18d和30d，混合感染组小鼠组织螺旋体载量显著增高，关节炎明显严重，血清IgC效价显著偏低。结论伯氏疏螺旋体和嗜吞噬细胞无浆体混合感染显著增加小鼠组织螺旋体载量、加重关节炎症状和病理改变，且抑制体液免疫功能。     

Detection of Immune Complexes Is Not Independent of Detection of Antibodies in Lyme Disease Patients and Does Not Confirm Active Infection with Borrelia burgdorferi

The Borrelia burgdorferi-specific immune complex (IC) test, which uses polyethylene glycol (PEG) precipitation to isolate ICs from serum, has been used as a research test in the laboratory diagnosis of early Lyme disease (LD) and has been proposed as a marker of active infection. We examined whether B. burgdorferi-specific antibodies were present within PEG-precipitated ICs (PEG-ICs) in patients with LD, posttreatment Lyme disease syndrome, and controls, including individuals who received the outer surface protein A (OspA) vaccine. Using a B. burgdorferi whole-cell enzyme-linked immunosorbent assay (ELISA), we obtained positive PEG-IC results not only in patients with a history of LD, but also in individuals vaccinated with OspA vaccine. The frequency of positive PEG-IC ELISAs in OspA vaccinees was significantly higher with ELISA-reactive than with ELISA-negative unprocessed serum samples (P = 0.001), demonstrating dependency between the tests. Similar results were found using samples from rhesus macaques infected with B. burgdorferi, uninfected macaques vaccinated with OspA, and controls. Therefore, testing for the presence of antibodies against B. burgdorferi in PEG-IC preparations is not more likely to reflect active infection than testing in unprocessed serum and should not be used in individuals who received the OspA vaccine.     

Sensitive Detection of Borrelia burgdorferi Sensu Lato DNA and Differentiation of Borrelia Species by LightCycler PCR

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.     

Linear chromosome of Borrelia burgdorferi

The DNA organization of several European and American isolates of Borrelia burgdorferi, the aetiological agent of Lyme disease, was analysed in pulse-field agarose gel electrophoresis. The results of in situ cell lysis in agarose plugs demonstrated a unique arrangement for the DNA of this spirochete. The chromosome of Borrelia behaved as a eukaryotic linear chromosome with a size of around 1,000 kb. The genome also comprised several circular and linear plasmids which varied in size from 15 to 60 kb.     

Binding of human plasminogen to Borrelia burgdorferi.

We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in the binding of Glu- or Lys-plasminogen to B. burgdorferi. Binding of plasminogen was similar in low-passage (infectious) and high-passage (noninfectious) isolates of B. burgdorferi. Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen ligand blots of borrelial membrane proteins demonstrated two prominent binding proteins at approximately 70 and approximately 30 kDa. By Western blot (immunoblot), the 30-kDa protein was found to be outer surface protein A (Osp A) of B. burgdorferi. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. By scanning densitometry, the 70-kDa band bound > 10 time more 125I-labeled plasminogen than did Osp A. An Osp A-deficient mutant of B. burgdorferi and wild-type B. burgdorferi bound equal amounts of 125I-labeled plasminogen. Ligand blots of membrane proteins from an Osp A-deficient mutant showed association of 125I-labeled plasminogen at only the 70-kDa protein. Two-dimensional gel electrophoresis showed that the 70-kDa protein had a pI of approximately 5.3, clearly separable from Osp A. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.     

Specificities and Sensitivities of Four Monoclonal Antibodies for Typing of Borrelia burgdorferi Sensu Lato Isolates

Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.     

Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies

Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpA(N40) were found to be inferior to secreted lipoprotein DbpA(N40) in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.