Regulation of expression of the Borrelia burgdorferi beta(3)-chain integrin ligand,P66, in ticks and in culture

Borrelia burgdorferi is maintained in an infection cycle between mammalian and arthropod hosts. Appropriate gene expression by B. burgdorferi at different stages of this cycle is probably essential for transmission and establishment of infection. The B. burgdorferi beta(3) integrin ligand P66 is expressed by the bacteria in mammals, laboratory culture, and engorged but not unfed ticks. No in vitro culture conditions in which P66 expression reflected that in the unfed tick were found, suggesting that there are aspects of B. burgdorferi-tick interaction that remain unexplored.     

Competitive Advantage of Borrelia burgdorferi with Outer Surface Protein BBA03 during Tick-Mediated Infection of the Mammalian Host

Linear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01 to bba07 [bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07 mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03 as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03 is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03 by spirochetes in fed relative to unfed ticks. We also observed that a bba03 deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.     

Identification of Borrelia burgdorferi outer surface proteins

Several Borrelia burgdorferi outer surface proteins have been identified over the past decade that are up-regulated by temperature- and/or mammalian host-specific signals as this spirochete is transmitted from ticks to mammals. Given the potential role(s) that these differentially up-regulated proteins may play in B. burgdorferi transmission and Lyme disease pathogenesis, much attention has recently been placed on identifying additional borrelial outer surface proteins. To identify uncharacterized B. burgdorferi outer surface proteins, we previously performed a comprehensive gene expression profiling analysis of temperature-shifted and mammalian host-adapted B. burgdorferi. The combined microarray analyses revealed that many genes encoding known and putative outer surface proteins are down-regulated in mammalian host-adapted B. burgdorferi. At the same time, however, several different genes encoding putative outer surface proteins were found to be up-regulated during the transmission and infection process. Among the putative outer surface proteins identified, biochemical and surface localization analyses confirmed that seven (Bb0405, Bb0689, BbA36, BbA64, BbA66, BbA69, and BbI42) are localized to the surface of B. burgdorferi. Furthermore, enzyme-linked immunosorbent assay analysis using serum from tick-infested baboons indicated that all seven outer surface proteins identified are immunogenic and that antibodies are generated against all seven during a natural infection. Specific antibodies generated against all seven of these surface proteins were found to be bactericidal against B. burgdorferi, indicating that these newly identified outer surface proteins are prime candidates for analysis as second-generation Lyme disease vaccinogens.     

Borrelia burgdorferi bba66 Gene Inactivation Results in Attenuated Mouse Infection by Tick Transmission

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb^ΔA66, remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb^ΔA66 following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb^ΔA66-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb^ΔA66 with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.     

Borrelia burgdorferi gene expression in vivo and spirochete pathogenicity

Borrelia burgdorferi spirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate of B. burgdorferi N40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. A B. burgdorferi genomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, including dbpAB, bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required for B. burgdorferi to effectively colonize the host, evade humoral responses, and cause disease.     

Seasonal prevalence of Borrelia burgdorferi in natural populations of white-footed mice, Peromyscus leucopus

Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated from 111 of 237 Peromyscus leucopus captured during all seasons of the year. Borreliae were cultured from tissues of the spleen (101 mice), left kidney (76 mice), and right kidney (73 mice), from blood (12 mice), and from one fetus. Mice were infected during the winter, when immature Ixodes dammini were inactive. The prevalence of infection during the winter (less than or equal to 33%) was more than twofold lower than that during the summer (ca. 75%), a time when nymphal ticks are abundant. Overwintering, infected mice are reservoir hosts for subadult ticks that begin feeding in early spring. Twenty white-footed mice from which B. burgdorferi was isolated from tissues of spleen or kidney but not from blood were parasitized by larval I. dammini or Dermacentor variabilis which harbored borreliae. We conclude that these mice were infectious to feeding ticks, even though borreliae were not isolated from blood.     

Defining plasmids required by Borrelia burgdorferi for colonization of tick vector Ixodes scapularis (Acari: Ixodidae)

Maintenance in nature of Borrelia burgdorferi, the pathogenic bacterium that causes Lyme disease, requires transmission through an infectious cycle that includes a tick vector and a mammalian host. The genetic requirements for persistence in these disparate environments have not been well defined. B. burgdorferi has a complex genome composed of a chromosome and >20 plasmids. Previous work has demonstrated that B. burgdorferi requires two plasmids, lp25 and lp28-1, in the mammalian host. To investigate the requirement for these same two plasmids during tick infection, we experimentally infected larval ticks with B. burgdorferi lacking either lp25 or lp28-1 and then assessed the spirochete load in ticks at different points of the infection. Whereas plasmid lp28-1 was dispensable in ticks, plasmid lp25 was essential for tick infection. Furthermore, we investigated the requirement in ticks for the lp25 gene bbe22, which encodes a nicotinamidase that is necessary and sufficient for mammalian infection by B. burgdorferi clones lacking lp25. This gene was also sufficient in ticks to restore survival of spirochetes lacking lp25. This is the first study to investigate the requirement for specific plasmids by B. burgdorferi within the tick vector, and it begins to establish the genomic components required for persistence of this pathogen throughout its natural infectious cycle.     

Temporal analysis of Borrelia burgdorferi Erp protein expression throughout the mammal-tick infectious cycle

Previous immunological studies indicated that the Lyme disease spirochete, Borrelia burgdorferi, expresses Erp outer surface proteins during mammalian infection. We conducted analyses of Erp expression throughout the entire tick-mammal infectious cycle, which revealed that the bacteria regulate Erp production in vivo. Bacteria within unfed nymphal ticks expressed little to no Erp proteins. However, as infected ticks fed on mice, B. burgdorferi increased production of Erp proteins, with essentially all transmitted bacteria expressing these proteins. Mice infected with B. burgdorferi mounted rapid IgM responses to all tested Erp proteins, followed by strong immunoglobulin G responses that generally increased in intensity throughout 11 months of infection, suggesting continued exposure of Erp proteins to the host immune system throughout chronic infection. As naive tick larvae acquired B. burgdorferi by feeding on infected mice, essentially all transmitted bacteria produced Erp proteins, also suggestive of continual Erp expression during mammalian infection. Shortly after the larvae acquired bacteria, Erp production was drastically downregulated. The expression of Erp proteins on B. burgdorferi throughout mammalian infection is consistent with their hypothesized function as factor H-binding proteins that protect the bacteria from host innate immune responses.     

Ecology of Borrelia burgdorferi in ticks (Acari: Ixodidae), rodents, and birds in the Sierra Nevada foothills, Placer County, California

This study examined the prevalence of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner in host-seeking adult and nymphal Ixodes pacificus Cooley & Kohls and estimated the I. pacificus infestation and B. burgdorferi infection of rodent and avian hosts in the western Sierra Nevada foothills of northern California. Additionally, we identified species likely to participate in an enzootic cycle for B. burgdorferi in this yellow pine transition habitat. Evidence of infection with B. burgdorferi was identified in 7.3 and 5.4% of host-seeking I. pacificus adults and nymphs, respectively. Mean numbers of I. pacificus observed on rodents were 1.15 for Neotoma fuscipes Baird and 0.18 for Peromyscus spp. One of 104 ear punch tissues obtained from woodrats and none from 49 Peromyscus spp. yielded B. burgdorferi. A total of 291 collected birds representing 34 species had a mean of 0.27 I. pacificus per bird. The mean I. pacificus infestation of ground-dwelling birds was 2.5 ticks per bird. Forty-nine of 92 (53%) blood smears collected from birds were reactive to a B. burgdorferi specific antibody. This study presents the identification of a B. burgdorferi-like spirochete in birds in western North America. The tick burden and spirochete infection of birds suggests that birds may be involved in a local B. burgdorferi enzootic cycle and likely participate in the transport of ticks and spirochetes to other locations while rodents from this site do not appear to be major contributors.     

A surface enolase participates in Borrelia burgdorferi-plasminogen interaction and contributes to pathogen survival within feeding ticks

Borrelia burgdorferi, a tick-borne bacterial pathogen, causes a disseminated infection involving multiple organs known as Lyme disease. Surface proteins can directly participate in microbial virulence by facilitating pathogen dissemination via interaction with host factors. We show here that a fraction of the B. burgdorferi chromosomal gene product BB0337, annotated as enolase or phosphopyruvate dehydratase, is associated with spirochete outer membrane and is surface exposed. B. burgdorferi enolase, either in a recombinant form or as a membrane-bound native antigen, displays enzymatic activities intrinsic to the glycolytic pathway. However, the protein also interacts with host plasminogen, potentially leading to its activation and resulting in B. burgdorferi-induced fibrinolysis. As expected, enolase displayed consistent expression in vivo, however, with a variable temporal and spatial expression during spirochete infection in mice and ticks. Despite an extracellular exposure of the antigen and a potential role in host-pathogen interaction, active immunization of mice with recombinant enolase failed to evoke protective immunity against subsequent B. burgdorferi infection. In contrast, enolase immunization of murine hosts significantly reduced the acquisition of spirochetes by feeding ticks, suggesting that the protein could have a stage-specific role in B. burgdorferi survival in the feeding vector. Strategies to interfere with the function of surface enolase could contribute to the development of novel preventive measures to interrupt the spirochete infection cycle and reduce the incidences of Lyme disease.