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Concept: Borrelia burgdorferi

174

Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or (3)H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

Concepts: Archaea, Bacteria, Eukaryote, Cell membrane, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaete

170

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in Lyme disease endemic and non-endemic European and North American regions revealed close relatedness of geographically distinct populations. OspC alleles A, B and L were detected on both continents in vectors and hosts including humans. Six ospC alleles, A, B, L, Q, R and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3 and M, were identified in the far-western U.S.A. Four ospC alleles, B, G, H and L, were abundant in the southeastern U.S.A. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern U.S.A with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in endemic European and North American regions were detected in B. burgdorferi strains isolated from non-human biting tick Ixodes affinis and rodent hosts in southeastern U.S.A. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern U.S.A., a finding that confirms the hypothesis of trans-oceanic migration of Borrelia species.

Concepts: Bacteria, Lyme disease, Ixodes scapularis, North America, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer

169

ABSTRACT The rodent Peromyscus leucopus is a major natural reservoir for the Lyme disease agent Borrelia burgdorferi and a host for its vector Ixodes scapularis. At various locations in northeastern United States 10 to 15 B. burgdorferi strains coexist at different prevalences in tick populations. We asked whether representative strains of high or low prevalence differed in their infections of P. leucopus. After 5 weeks of experimental infection of groups with each of 6 isolates, distributions and burdens of bacteria in tissues were measured by quantitative PCR, and antibodies to B. burgdorferi were evaluated by immunoblotting and protein microarray. All groups of animals were infected in their joints, ears, tails, and hearts, but overall spirochete burdens were lower in animals infected with low-prevalence strains. Animals were similar regardless of the infecting isolate in their levels of antibodies to whole cells, FlaB, BmpA, and DbpB proteins, and the conserved N-terminal region of the serotype-defining OspC proteins. But there were strain-specific antibody responses to full-length OspC and to plasmid-encoded VlsE, BBK07, and BBK12 proteins. Sequencing of additional VlsE genes revealed substantial diversity within some pairs of strains but near-identical sequences within other pairs, which otherwise differed in their ospC alleles. The presence or absence of full-length bbk07 and bbk12 genes accounted for the differences in antibody responses. We propose that for B. burgdorferi, there is selection in reservoir species for (i) sequence diversity, as for OspC and VlsE, and (ii) the presence or absence of polymorphisms, as for BBK07 and BBK12. IMPORTANCE Humans are dead-end hosts for Borrelia agents of Lyme disease (LD), and, thus, irrelevant for the pathogens' maintenance. Many reports of human cases and laboratory mouse infections exist, but less is known about infection and immunity in natural reservoirs, such as the rodent Peromyscus leucopus. We observed that high- and low-prevalence strains of Borrelia burgdorferi were capable of infecting P. leucopus but elicited different patterns of antibody responses. Antibody reactivities to the VlsE protein were as type-specific as previously characterized reactivities to serotype-defining OspC proteins. In addition, the low-prevalence strains lacked full-length genes for two proteins that (i) are encoded by a virulence-associated plasmid in some high-prevalence strains and (ii) LD patients and field-captured rodents commonly have antibodies to. Immune selection against these genes may have led to null phenotype lineages that can infect otherwise immune hosts but at the cost of reduced fitness and lower prevalence.

Concepts: Immune system, Antibody, Protein, Bacteria, Infection, Lyme disease, Borrelia burgdorferi, Borrelia

168

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). Recent studies have shown that recognition of the spirochete is mediated mainly by TLR2 and NOD2. The latter receptor has been associated with the induction of the intracellular degradation process called autophagy. The present study demonstrated for the first time the induction of autophagy by exposure to B. burgdorferi and that autophagy modulates the B. burgdorferi-dependent cytokine production. Human PBMCs treated with autophagy inhibitors showed an increased IL-1β and IL-6 production in response to the exposure of the spirochete, while TNFα production was unchanged. Autophagy induction against B. burgdorferi was dependent on reactive oxygen species (ROS) since cells from patients with chronic granulomatous disease (CGD), which are defective in ROS production, also produced elevated IL-1β. Further, the enhanced production of the pro-inflammatory cytokines was because of the elevated mRNA expression in the absence of autophagy. Our results thus demonstrate the induction of autophagy, which in-turn modulates cytokine production, by B. burgdorferi for the first time.

Concepts: Bacteria, Cytokine, Reactive oxygen species, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete

167

Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.

Concepts: Immune system, Antibody, Bacteria, Lyme disease, Antigen, Epitope, Borrelia burgdorferi, Borrelia

164

Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

Concepts: Immune system, DNA, Gene, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaete

151

Ticks carry and transmit a remarkable array of pathogens including bacteria, protozoa and viruses, which may be of veterinary and/or of medical significance. With little to no information regarding the presence of tick-borne zoonotic pathogens or their known vectors in southern Africa, the aim of our study was to screen for Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Rickettsia species and Ehrlichia ruminantium in ticks collected and identified from ruminants in the Eastern Cape, Free State, KwaZulu-Natal and Mpumalanga Provinces of South Africa. The most abundant tick species identified in this study were Rhipicephalus evertsi evertsi (40%), Rhipicephalus species (35%), Amblyomma hebraeum (10%) and Rhipicephalus decoloratus (14%). A total of 1,634 ticks were collected. DNA was extracted, and samples were subjected to PCR amplification and sequencing. The overall infection rates of ticks with the target pathogens in the four Provinces were as follows: A. phagocytophilum, 7%; C. burnetii, 7%; E. ruminantium, 28%; and Rickettsia spp., 27%. The presence of B. burgdorferi could not be confirmed. The findings of this study show that zoonotic pathogens are present in ticks in the studied South African provinces. This information will aid in the epidemiology of tick-borne zoonotic diseases in the country as well as in raising awareness about such diseases in the veterinary, medical and tourism sectors, as they may be the most affected.

Concepts: Bacteria, Africa, Coxiella burnetii, Q fever, Lyme disease, South Africa, Borrelia burgdorferi, Eastern Cape

143

Lyme disease is a tickborne illness that generates controversy among medical providers and researchers. One of the key topics of debate is the existence of persistent infection with the Lyme spirochete, Borreliaburgdorferi, in patients who have been treated with recommended doses of antibiotics yet remain symptomatic. Persistent spirochetal infection despite antibiotic therapy has recently been demonstrated in non-human primates. We present evidence of persistent Borrelia infection despite antibiotic therapy in patients with ongoing Lyme disease symptoms.

Concepts: Medicine, Bacteria, Lyme disease, Borrelia burgdorferi, Borrelia, Spirochaetes, Spirochaete, Tick-borne disease

85

Non-human primates currently serve as the best experimental model for Lyme disease due to their close genetic homology with humans and demonstration of all three phases of disease following infection with Borreliella (Borrelia) burgdorferi (Bb). We investigated the pathology associated with late disseminated Lyme disease (12 to 13 months following tick inoculation) in doxycycline-treated (28 days; 5mg/kg, oral, 2x/day) and untreated rhesus macaques (Rm). Minimal to moderate lymphoplasmacytic inflammation, with a predilection for perivascular spaces and collagenous tissues, was observed in multiple tissues including the cerebral leptomeninges, brainstem, peripheral nerves from both fore and hind limbs, stifle synovium and perisynovial adipose tissue, urinary bladder, skeletal muscle, myocardium, and visceral pericardium. Indirect immunofluorescence assays (IFA) combining monoclonal (outer surface protein A) and polyclonal antibodies were performed on all tissue sections containing inflammation. Rare morphologically intact spirochetes were observed in the brains of two treated Rm, the heart of one treated Rm, and adjacent to a peripheral nerve of an untreated animal. Borrelia antigen staining of probable spirochete cross-sections was also observed in heart, skeletal muscle, and near peripheral nerves of both treated and untreated animals. These findings support the notion that chronic Lyme disease symptoms can be attributable to residual inflammation in and around tissues that harbor a low burden of persistent host-adapted spirochetes and/or residual antigen.

Concepts: Antibody, Muscle, Lyme disease, Macaque, Primate, Rhesus Macaque, Borrelia burgdorferi, Borrelia

73

Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets.

Concepts: DNA, Medicine, Gene, Gene expression, Bacteria, Medical terms, Lyme disease, Borrelia burgdorferi