lacZ Reporter System for Use in Borrelia burgdorferi

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.Borrelia burgdorferi sensu lato, the pathogen that causes Lyme disease (7), alternates between two distinct environments, an arthropod vector and a vertebrate host. As B. burgdorferi moves from one milieu to the other, its ability to adapt and survive requires dramatic changes in gene expression. Many studies have shown that different B. burgdorferi gene products are upregulated or downregulated at specific times during the infectious cycle (19, 31) and in response to host and environmental signals (6, 8a, 15, 24, 25). Although it is clear that B. burgdorferi alters gene expression to adapt to different environments, the genetic tools for studying gene regulation in B. burgdorferi are limited.Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10, 14) and techniques for basic genetic manipulation of B. burgdorferi became available (5, 11, 13, 27-29, 36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9, 11, 30) as reporter systems in B. burgdorferi followed. Although these systems have value, there are limitations with each. β-Galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in Escherichia coli and is still applicable to a broad range of organisms, both prokaryotic and eukaryotic, but has not yet been used with B. burgdorferi. β-Galactosidase activity can be monitored easily and quickly by simple colorimetric assays in both liquid and solid media, neither of which require expensive or specialized equipment. Additionally, a wide variety of substrates for β-galactosidase allow for different levels of sensitivity in either in vitro or in vivo detection formats (17). Having lacZ available as a genetic tool for B. burgdorferi would enhance investigation of the complex regulatory events that are integral to the spirochete''s infectious cycle. To this end, we developed lacZ as a reporter gene in B. burgdorferi and demonstrated its utility.     

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.     

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.     

Regulation of expression of the fibronectin-binding protein BBK32 in Borrelia burgdorferi

The BBK32 protein binds to host extracellular ligand fibronectin and contributes to the pathogenesis of Borrelia burgdorferi. Here we showed that expression of the BBK32 gene is influenced by multiple environmental factors and that its regulation is governed by the response regulator Rrp2 and RpoN-RpoS (σ⁵⁴-σ^S) sigma cascade in B. burgdorferi.     

Chemotaxis in Borrelia burgdorferi

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.     

Molecular cloning and characterization of Borrelia burgdorferi rpoB

Borrelia burgdorferi rpoB, the gene encoding the β-subunit of RNA polymerase, has been cloned and sequenced. The full-length gene encodes a protein of 1154 amino acids with a calculated molecular mass of 129.8 kDa. The amino-acid sequence is 49% identical to the corresponding protein from Escherichia coli. B. burgdorferi rpoB is a component of a gene cluster, which includes rplJ, rplL and rpoC. A temperature-sensitive E. coli rpoB mutant could be complemented by introduction of the B. burgdorferi gene, indicating that the B. burgdorferi rpoB is expressed in E. coli and the β-subunit can be assembled into functional holoenzyme. The wild-type amino-acid sequence of the B. burgdorferi β-subunit is consistent with those of spontaneously arising rifampicin-resistant mutants of E. coli and Mycobacterium tuberculosis at certain critical residues. This suggests that the natural resistance of B. burgdorferi to rifampicin may be due to the primary amino-acid sequence of its β-subunit.     

Characterization of the ATP2B gene family in blood pressure

The 12q21 locus, which lies near the plasma membrane calcium-transporting ATPase 1 gene (ATP2B1), has one of the strongest associations with blood pressure and hypertension in Europeans and Asians. We performed an association analysis of the ATP2B isomers ATP2B2, ATP2B3, and ATP2B4 with blood pressure and hypertension in 7,551 Korean individuals and observed a link with ATP2B2 and ATP2B4. To examine the regulation of blood pressure by ATP2B, ATP2B1 and ATP2B4 mRNA was reduced in vascular smooth muscle cells by siRNA. The expression pattern of 23 ATP2B-related genes was analyzed by quantitative real-time PCR, which differed between treatment with ATP2B1 and ATP2B4 siRNA. The reduction inATP2B1 mRNA induced a significant change in mRNA levels in the calcium pump RYR1 and the ATP2B-binding protein HOMER1. Conversely, the decrease in ATP2B4 mRNA significantly altered mRNA expression of the calcium pump SLC8A1 and the ATP2B-binding proteins CASK and DLG4. These results suggest that blood pressure is differentially regulated through calcium signaling between ATP2B1 and ATP2B4.     

Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.     

Defining the plasmid-borne restriction-modification systems of the Lyme disease spirochete Borrelia burgdorferi

The restriction-modification (R-M) systems of many bacteria present a barrier to the stable introduction of foreign DNA. The Lyme disease spirochete Borrelia burgdorferi has two plasmid-borne putative R-M genes, bbe02 and bbq67, whose presence limits transformation by shuttle vector DNA from Escherichia coli. We show that both the bbe02 and bbq67 loci in recipient B. burgdorferi limit transformation with shuttle vector DNA from E. coli, irrespective of its dam, dcm, or hsd methylation status. However, plasmid DNA purified from B. burgdorferi transformed naïve B. burgdorferi much more efficiently than plasmid DNA from E. coli, particularly when the bbe02 and bbq67 genotypes of the B. burgdorferi DNA source matched those of the recipient. We detected adenine methylation of plasmid DNA prepared from B. burgdorferi that carried bbe02 and bbq67. These results indicate that the bbe02 and bbq67 loci of B. burgdorferi encode distinct R-M enzymes that methylate endogenous DNA and cleave foreign DNA lacking the same sequence-specific modification. Our findings have basic implications for horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. Further characterization and identification of the nucleotide sequences recognized by BBE02 and BBQ67 will facilitate efficient genetic manipulation of this pathogenic spirochete.Borrelia burgdorferi sensu lato is a zoonotic pathogen whose natural infectious cycle alternates between a tick vector and rodent or bird reservoir hosts (1, 7, 8, 14, 32, 33, 36). Transmission of B. burgdorferi to humans occurs through the bite of an infected tick and can lead to Lyme disease, which is a major public health concern in areas of North America and Europe where B. burgdorferi is endemic (8, 53).The genomic structure of the spirochete B. burgdorferi is unique, consisting of a linear chromosome of approximately 900 kb and more than 20 linear (lp) and circular (cp) plasmids, ranging in size from ∼5 kb to 56 kb, in the type strain B31 (9, 10, 11, 19, 42). The plasmids of B. burgdorferi are present at unit copy number relative to the chromosome (22), and some are relatively unstable during in vitro propagation (52, 57). The loss of linear plasmids lp25, lp28-1, and lp36 by strain B31 was found to correlate with the loss of infectivity in mice (20, 31, 45, 56), leading to the identification of genes carried on these plasmids that are dispensable in vitro but required in vivo during an experimental infectious cycle (21, 26, 35, 44, 47). The loss of two linear plasmids, lp25 and lp56, was shown to correlate with enhanced shuttle vector transformation, suggesting that specific lp25 and lp56 gene products present a barrier to stable introduction of foreign DNA (34). Further studies linked the transformation phenotype of B. burgdorferi strain B31 with the bbe02 and bbq67 genes on lp25 and lp56, respectively, and the putative restriction-modification (R-M) enzymes that they encode (11, 27, 29, 34). The recent demonstration by Chen and colleagues of enhanced transformation of B. burgdorferi following in vitro methylation of DNA (13) further supports the hypothesis that these B. burgdorferi plasmids encode R-M enzymes that degrade foreign DNA lacking the appropriate modification.The barrier to foreign DNA presented by the bbe02 and bbq67 loci of B. burgdorferi implies that genomic DNA should be modified in spirochetes carrying these plasmid genes. To test this hypothesis, we compared the transformation of B. burgdorferi with shuttle vector DNA isolated from either Escherichia coli or B. burgdorferi, as outlined in Fig. ​Fig.1.1. We also examined whether and how the presence of putative R-M genes in either the donor or recipient B. burgdorferi strain influenced transformation. Finally, we analyzed the type of modification present on DNA isolated from B. burgdorferi with different plasmid or gene contents. Our data indicate that the bbe02 and bbq67 loci of B. burgdorferi encode enzymes that both methylate endogenous DNA and restrict foreign DNA lacking these modifications. These findings have basic implications regarding horizontal gene transfer among B. burgdorferi strains with distinct plasmid contents. These results also help elucidate the molecular mechanisms underlying the relative inefficiency of genetic transformation of B. burgdorferi and suggest ways in which genetic manipulation of this pathogenic spirochete could be enhanced.Open in a separate windowFIG. 1.Shuttle vector transformations. Schematic representation of the various DNA sources, strains and methods used to assess the contributions of bbe02 and bbq67 to the restriction-modification (R-M) systems of B. burgdorferi.