Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

Genetic diversity of Borrelia burgdorferi sensu stricto in Peromyscus leucopus, the primary reservoir of Lyme disease in a region of endemicity in southern Maryland

In the north central and northeastern United States, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease (LD), is maintained in an enzootic cycle between the vector, Ixodes scapularis, and the primary reservoir host, Peromyscus leucopus. Genetic diversity of the pathogen based on sequencing of two plasmid-located genes, those for outer surface protein A (ospA) and outer surface protein C (ospC), has been examined in both tick and human specimens at local, regional, and worldwide population scales. Additionally, previous studies have only been conducted with tick or human specimens at the local population level in areas with high LD transmission rates. This study examined the genetic diversity of circulating borreliae in the reservoir population from a large region of the western coastal plains of southern Maryland, where moderate numbers of human LD cases are reported. Six ospA mobility classes, including two that were not previously described, and eight ospC groups were found among the P. leucopus samples. Twenty-five percent of all specimens were infected with more than one ospA or ospC variant. The frequency distribution of variants was homogeneous, both locally and spatially. The spirochete diversity found in Maryland was not as high as that observed among northern tick populations, yet similar genotypes were observed in both populations. These results also show that mice are important for maintaining Borrelia variants, even rare variants, and that reservoir populations should therefore be considered when assessing the diversity of B. burgdorferi.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.     

Functional analysis of the Borrelia burgdorferi bba64 gene product in murine infection via tick infestation

Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans from the bite of Ixodes spp. ticks. During the borrelial tick-to-mammal life cycle, B. burgdorferi must adapt to many environmental changes by regulating several genes, including bba64. Our laboratory recently demonstrated that the bba64 gene product is necessary for mouse infectivity when B. burgdorferi is transmitted by an infected tick bite, but not via needle inoculation. In this study we investigated the phenotypic properties of a bba64 mutant strain, including 1) replication during tick engorgement, 2) migration into the nymphal salivary glands, 3) host transmission, and 4) susceptibility to the MyD88-dependent innate immune response. Results revealed that the bba64 mutant's attenuated infectivity by tick bite was not due to a growth defect inside an actively feeding nymphal tick, or failure to invade the salivary glands. These findings suggested there was either a lack of spirochete transmission to the host dermis or increased susceptibility to the host's innate immune response. Further experiments showed the bba64 mutant was not culturable from mouse skin taken at the nymphal bite site and was unable to establish infection in MyD88-deficient mice via tick infestation. Collectively, the results of this study indicate that BBA64 functions at the salivary gland-to-host delivery interface of vector transmission and is not involved in resistance to MyD88-mediated innate immunity.     

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.     

Genetic variation at the vlsE locus of Borrelia burgdorferi within ticks and mice over the course of a single transmission cycle

The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. The vlsE locus represents the best-studied example of antigenic variation in B. burgdorferi. During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population of B. burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts. Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles. Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Proteome analysis of Borrelia burgdorferi response to environmental change

We examined global changes in protein expression in the B31 strain of Borrelia burgdorferi, in response to two environmental cues (pH and temperature) chosen for their reported similarity to those encountered at different stages of the organism's life cycle. Multidimensional nano-liquid chromatographic separations coupled with tandem mass spectrometry were used to examine the array of proteins (i.e., the proteome) of B. burgdorferi for different pH and temperature culture conditions. Changes in pH and temperature elicited in vitro adaptations of this spirochete known to cause Lyme disease and led to alterations in protein expression that are associated with increased microbial pathogenesis. We identified 1,031 proteins that represent 59% of the annotated genome of B. burgdorferi and elucidated a core proteome of 414 proteins that were present in all environmental conditions investigated. Observed changes in protein abundances indicated varied replicon usage, as well as proteome functional distributions between the in vitro cell culture conditions. Surprisingly, the pH and temperature conditions that mimicked B. burgdorferi residing in the gut of a fed tick showed a marked reduction in protein diversity. Additionally, the results provide us with leading candidates for exploring how B. burgdorferi adapts to and is able to survive in a wide variety of environmental conditions and lay a foundation for planned in situ studies of B. burgdorferi isolated from the tick midgut and infected animals.     

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.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Adaptation of Borrelia burgdorferi in the tick and the mammalian host

Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete.