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.     

High-throughput plasmid content analysis of Borrelia burgdorferi B31 by using Luminex multiplex technology

Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain B. burgdorferi B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many B. burgdorferi strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage B. burgdorferi B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease Borrelia studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease Borrelia strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.     

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.     

Sequence of a gene encoding a putative primary sigma factor from Borrelia burgdorferi strain B31

Utilizing a polymerase chain reaction-based approach, the gene (rpoD) encoding the primary sigma factor from Borrelia burgdorferi strain B31 was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1632 bp (543 amino acids (aa), 63.7 kDa). Comparison with Escherichia coli σ⁷⁰ and Bacillus subtilis σ⁴³ showed a high degree of similarity in the aa sequences, especially for the regions that are known to be required for promoter recognition and core binding.     

Bioluminescent imaging of Borrelia burgdorferi in vivo demonstrates that the fibronectin-binding protein BBK32 is required for optimal infectivity

The aetiological agent of Lyme disease, Borrelia burgdorferi, is transmitted via infected Ixodes spp. ticks. Infection, if untreated, results in dissemination to multiple tissues and significant morbidity. Recent developments in bioluminescence technology allow in vivo imaging and quantification of pathogenic organisms during infection. Herein, luciferase-expressing B. burgdorferi and strains lacking the decorin adhesins DbpA and DbpB, as well as the fibronectin adhesin BBK32, were quantified by bioluminescent imaging to further evaluate their pathogenic potential in infected mice. Quantification of bacterial load was verified by quantitative PCR (qPCR) and cultivation. B. burgdorferi lacking DbpA and DbpB were only seen at the 1 h time point post infection, consistent with its low infectivity phenotype. The bbk32 mutant exhibited a significant decrease in its infectious load at day 7 relative to its parent. This effect was most pronounced at lower inocula and imaging correlated well with qPCR data. These data suggest that BBK32-mediated binding plays an important role in B. burgdorferi colonization. As such, in vivo imaging of bioluminescent Borrelia provides a sensitive means to detect, quantify and temporally characterize borrelial dissemination in a non-invasive, physiologically relevant environment and, more importantly, demonstrated a quantifiable infectivity defect for the bbk32 mutant.     

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland. This isolate is to date the closest known relative of B. burgdorferi sensu stricto, but it is sufficiently genetically distinct from that species that it and its close relatives warrant its candidacy for new-species status. We suggest that this isolate should be named "Borrelia finlandensis."     

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.     

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas-phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI-digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc-positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5' end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.     

Analysis and expression of the Borrelia burgdorferi P/Gau fla gene: Identification of heterogeneity with the B31 strain

The flagellin gene from the P/Gau strain of Borrelia burgdorferi was cloned and sequenced. The translated P/Gau flagellin protein differed from the flagellin of the B31 strain at 13 of 336 amino acids. This includes seven differences between amino acids 190-234, an immunodominant and specific region for B. burgdorferi. The entire flagellin molecule, as well as peptides of the internal portion of the protein which is more specific for B. burgdorferi, has been expressed in Escherichia coli using a pET7HIS.2 expression system. These peptides may be of great value for the development of sensitive and specific recombinant-based serological assays.     

Borrelia burgdorferi enolase is a surface-exposed plasminogen binding protein

Borrelia burgdorferi is the causative agent of Lyme disease, the most commonly reported arthropod-borne disease in the United States. B. burgdorferi is a highly invasive bacterium, yet lacks extracellular protease activity. In order to aid in its dissemination, B. burgdorferi binds plasminogen, a component of the hosts' fibrinolytic system. Plasminogen bound to the surface of B. burgdorferi can then be activated to the protease plasmin, facilitating the bacterium's penetration of endothelial cell layers and degradation of extracellular matrix components. Enolases are highly conserved proteins with no sorting sequences or lipoprotein anchor sites, yet many bacteria have enolases bound to their outer surfaces. B. burgdorferi enolase is both a cytoplasmic and membrane associated protein. Enolases from other pathogenic bacteria are known to bind plasminogen. We confirmed the surface localization of B. burgdorferi enolase by in situ protease degradation assay and immunoelectron microscopy. We then demonstrated that B. burgdorferi enolase binds plasminogen in a dose-dependent manner. Lysine residues were critical for binding of plasminogen to enolase, as the lysine analog εaminocaproic acid significantly inhibited binding. Ionic interactions did not play a significant role in plasminogen binding by enolase, as excess NaCl had no effects on the interaction. Plasminogen bound to recombinant enolase could be converted to active plasmin. We conclude that B. burgdorferi enolase is a moonlighting cytoplasmic protein which also associates with the bacterial outer surface and facilitates binding to host plasminogen.     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking

Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease. To identify Bb motility related genes, we used a method termed `semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster. The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed. Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene. The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE. Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria. The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs). One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp. Another ORF, flbE, showed similarity to genes from both Tp and Bs. RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb. Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb. The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis.     

Regulation of G protein signaling by the 70 kDa heat shock protein

G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha_2A adrenergic receptor (α_2A-R) by the ubiquitous stress-inducible 70 kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α_2A-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α_2A-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α_2A-R-catalyzed [³⁵S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine_1A receptor (5-HT_1A-R). In heat-stressed CHO cells, the α_2A-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α_2A-R compared to the 5-HT_1A-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.     

Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi

Borrelia burgdorferi, the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surface-exposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi. Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.     

Incorporation of cysteine by Borrelia burgdorferi and Borrelia hermsii.

The growth rate of Borrelia burgdorferi and Borrelia hermsii in BSK II medium prepared with cysteine-free or cysteine-containing (0.185-5.92 mM) CMRL 1066 medium was studied. In media with cysteine-free CMRL 1066, growth of borreliae was detectable, although it was reduced by approximately 80%. Bacterial growth was maximal when the concentration of cysteine in CMRL 1066 reached 1.48 mM, which represents the standard cysteine concentrations of the medium; higher concentrations inhibited the growth of borreliae. Cysteine incorporation, measured by the uptake of radiolabeled cysteine, showed that cysteine enters B. burgdorferi and B. hermsii cells by passive diffusion. Labeling studies of borreliae with [35S]cysteine indicated that B. burgdorferi has several cysteine-containing proteins, including ones at 22, 30 (OspA), and 34 kDa (OspB), whereas B. hermsii showed only two [35S]cysteine-incorporating proteins, at 22 and 24 kDa, which were exposed onto the outer cell surface. In addition, most of the cysteine-incorporating proteins could be biosynthetically radiolabeled when bacterial cells were grown in vitro with [3H]palmitate, and the differences in cysteine incorporation observed between B. burgdorferi and B. hermsii were found to be correlated with differences in lipoproteins.     

Sequence of the complete osp operon encoding two major outer membrane proteins of a European Borrelia burgdorferi isolate (B29)

The nucleotide sequence of the operon encoding the major outer surface proteins, OspA and OspB, of a European isolate of Borrelia burgdorferi (strain B29) was determined and compared to the osp operon of the American strain, B31. An amino acid (aa) identity of 80.7% was found when comparing the OspA of B29 with that of B31, whereas the aa sequence of the OspB of B29 reveals only 61.3% identity with the OspB of B31. Thus, strains B31 and B29 can be regarded as representatives of different B. burgdorferi groups.