Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.
Background. Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow. Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient. Methods. We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells. Results. Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii. Conclusions. The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells.
Summary Systemic dissemination of microbial pathogens permits microbes to spread from the initial site of infection to secondary target tissues and is responsible for most mortality due to bacterial infections. Dissemination is a critical stage of disease progression by the L yme spirochaete, B orrelia burgdorferi . However, many mechanistic features of the process are not yet understood. A key step is adhesion of circulating microbes to vascular surfaces in the face of the shear forces present in flowing blood. Using real‐time microscopic imaging of the L yme spirochaete in living mice we previously identified the first bacterial protein ( B . burgdorferi BBK 32) shown to mediate vascular adhesion in vivo. Vascular adhesion is also dependent on host fibronectin ( Fn ) and glycosaminoglycans ( GAG s). In the present study, we investigated the mechanisms of BBK 32‐dependent vascular adhesion in vivo . We determined that BBK 32– Fn interactions (tethering) function as a molecular braking mechanism that permits the formation of more stable BBK 32– GAG interactions (dragging) between circulating bacteria and vascular surfaces. Since BBK 32‐like proteins are expressed in a variety of pathogens we believe that the vascular adhesion mechanisms we have deciphered here may be critical for understanding the dissemination mechanisms of other bacterial pathogens.
Significance Invariant natural killer T cells (iNKT) have been found primarily patrolling inside blood vessels in the liver, where they respond to bacterial glycolipids presented by CD1d on liver macrophages. We show joint iNKT cells are localized outside of blood vessels and respond directly to the joint-homing pathogen, Borrelia burgdorferi , which causes Lyme borreliosis using multichannel spinning-disk intravital microscopy. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted its dissemination attempts into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway. These results suggest a critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier.
Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme. Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme. Replication and protection of telomeric DNA are required to ensure the genomic stability of all organisms with linear replicons. Until quite recently, it was assumed that linearity is a property confined to the replicons of eukaryotes and certain primarily eukaryotic viruses. However, a growing body of evidence indicates that linear DNA is also found in a broad range of bacteriophages (1.Mellado R.P. Peñalva M.A. Inciarte M.R. Salas M. 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EMBO J. 2001; 20: 3229-3237Crossref PubMed Scopus (76) Google Scholar, 18.Chaconas G. Mol. Microbiol. 2005; 58: 625-635Crossref PubMed Scopus (50) Google Scholar). Resolution of the linear chromosome and plasmids in Borrelia species and of the linear plasmid prophages from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca is performed by telomere resolvases (also referred to as protelomerases) (5.Hertwig S. Klein I. Lurz R. Lanka E. Appel B. Mol. Microbiol. 2003; 48: 989-1003Crossref PubMed Scopus (64) Google Scholar, 19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 20.Deneke J. Ziegelin G. Lurz R. Lanka E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7721-7726Crossref PubMed Scopus (74) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar). A growing number of candidate telomere resolvases have been identified in the genomes of eukaryotic viruses, phages, and bacteria (22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23.Oakey H.J. Cullen B.R. Owens L. J. Appl. Microbiol. 2002; 93: 1089-1098Crossref PubMed Scopus (75) Google Scholar). Telomere resolvases are DNA cleavage and rejoining enzymes related to tyrosine recombinases and type 1B topoisomerases (19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar, 22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 24.Deneke J. Ziegelin G. Lurz R. Lanka E. J. Biol. 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Cleavage is accompanied by the formation of a 3′-phosphotyrosyl protein-DNA linkage. Subsequent nucleophilic attack on opposing strands by the free 5′-OH groups in the nicked substrate creates covalently closed hairpin telomeres. A recent crystal structure of the Klebsiella phage telomere resolvase (TelK) in complex with its substrate identified the residues involved in catalysis (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar); all but one of these residues are conserved in all telomere resolvases (22.Deneke J. Burgin A.B. Wilson S.L. Chaconas G. J. Biol. Chem. 2004; 279: 53699-53706Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), implying that the basic catalytic mechanism underlying telomere resolution is conserved. However, telomere resolvase sequences vary substantially outside of the central catalytic region (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar), and the enzymes characterized to date demonstrate important differences in substrate usage that likely reflect functionally distinct mechanisms of substrate interaction. The Borrelia burgdorferi telomere resolvase, ResT, appears to be particularly divergent. It is substantially smaller than phage telomere resolvases, and unlike its phage counterparts (5.Hertwig S. Klein I. Lurz R. Lanka E. Appel B. Mol. Microbiol. 2003; 48: 989-1003Crossref PubMed Scopus (64) Google Scholar, 20.Deneke J. Ziegelin G. Lurz R. Lanka E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7721-7726Crossref PubMed Scopus (74) Google Scholar, 21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar), it cannot efficiently resolve negatively supercoiled DNA (19.Kobryn K. Chaconas G. Mol. Cell. 2002; 9: 195-201Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27.Bankhead T. Kobryn K. Chaconas G. Mol. Microbiol. 2006; 62: 895-905Crossref PubMed Scopus (24) Google Scholar), presumably reflecting differences in the substrates resolved by phage and Borrelia telomere resolvases in vivo. On the other hand, B. burgdorferi ResT can fuse hairpin telomeres in a reversal of the resolution reaction (28.Kobryn K. Chaconas G. Mol. Cell. 2005; 17: 783-791Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), a function that is not shared with the phage telomere resolvase TelK (25.Aihara H. Huang W.M. Ellenberger T. Mol. Cell. 2007; 27: 901-913Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It can also synapse replicated telomeres and catalyze the formation of Holliday junctions (29.Kobryn K. Briffotaux J. Karpov V. Mol. Microbiol. 2009; 71: 1117-1130Crossref PubMed Scopus (13) Google Scholar). The ability of ResT to promote hairpin fusion has been proposed as the mechanism underlying the ongoing genetic rearrangements that are a prominent feature of the B. burgdorferi genome (18.Chaconas G. Mol. Microbiol. 2005; 58: 625-635Crossref PubMed Scopus (50) Google Scholar, 28.Kobryn K. Chaconas G. Mol. Cell. 2005; 17: 783-791Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Finally, B. burgdorferi ResT can tolerate a surprising amount of variation in its substrate (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), a feature that is not shared by phage telomere resolvases (21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar). Although B. burgdorferi ResT appears to be more permissive with a greater scope of activities than other telomere resolvases, the sequences mediating most of its unique properties have not yet been identified. The B. burgdorferi genome contains a total of 19 distinct hairpin sequences, all of which must be resolved by ResT (31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). These sequences can be classified into three groups based on the presence and positioning of the box 1 motif, which is a critical determinant of activity in phage and Borrelia telomere resolvases (see Fig. 1A) (21.Huang W.M. Joss L. Hsieh T. Casjens S. J. Mol. Biol. 2004; 337: 77-92Crossref PubMed Scopus (30) Google Scholar, 24.Deneke J. Ziegelin G. Lurz R. Lanka E. J. Biol. Chem. 2002; 277: 10410-10419Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar). A box 1-like motif is also found in many of the hairpin telomeres sequenced to date (6.Casjens S.R. Gilcrease E.B. Huang W.M. Bunny K.L. Pedulla M.L. Ford M.E. Houtz J.M. Hatfull G.F. Hendrix R.W. J. Bacteriol. 2004; 186: 1818-1832Crossref PubMed Scopus (82) Google Scholar, 14.González A. Talavera A. Almendral J.M. Viñuela E. Nucleic Acids Res. 1986; 14: 6835-6844Crossref PubMed Scopus (101) Google Scholar, 32.Hinnebusch J. Barbour A.G. J. Bacteriol. 1991; 173: 7233-7239Crossref PubMed Scopus (109) Google Scholar, 33.Casjens S. Curr. Opin. Microbiol. 1999; 2: 529-534Crossref PubMed Scopus (70) Google Scholar, 34.Rybchin V.N. Svarchevsky A.N. 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B. burgdorferi ResT can resolve telomeres in which box 1 is located at positions 1 and 4 nucleotides away from the axis of symmetry (Type 1 and 2 telomeres, respectively), as well as AT-rich telomeres without a box 1 sequence (Type 3 telomeres) (see Fig. 1A) (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). B. burgdorferi ResT cleaves telomeres at a fixed position relative to the axis of symmetry, independent of the location of box 1 (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar). Positioning of the enzyme for cleavage in all telomere types is most likely driven by sequence-specific interactions between ResT domains 2 (catalytic) and/or 3 (C-terminal) and a fixed element upstream of box 1 that is positioned 14 nucleotides from the axis of symmetry in all Borrelia telomeres (box 3 and adjacent nucleotides) (see Figs. 1A and 2) (26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar, 30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In contrast, box 1 and axis-flanking nucleotides are not involved in high affinity and/or sequence-specific interactions with ResT and require the ResT N-terminal domain for full protection in DNase footprinting assays (26.Tourand Y. Lee L. Chaconas G. Mol. Microbiol. 2007; 64: 580-590Crossref PubMed Scopus (19) Google Scholar, 27.Bankhead T. Kobryn K. Chaconas G. Mol. Microbiol. 2006; 62: 895-905Crossref PubMed Scopus (24) Google Scholar). The most likely candidate for interactions with box 1 and axis-flanking nucleotides is a Borrelia-specific hairpin-binding region in the N terminus, which is thought to promote a pre-hairpinning step involving strand opening at the axis (38.Bankhead T. Chaconas G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13768-13773Crossref PubMed Scopus (35) Google Scholar). ResT from the relapsing fever Borrelia species Borrelia hermsii exhibits a more restricted substrate usage pattern in vitro when compared with ResT from the Lyme disease pathogen B. burgdorferi (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar). Specifically, B. hermsii ResT is unable to efficiently resolve a Type 2 telomere. Therefore, B. burgdorferi ResT appears to be a more permissive enzyme than its relapsing fever counterpart. In this study, we investigated the basis for permissive substrate usage by B. burgdorferi ResT. Using a library of chimeric B. hermsii/B. burgdorferi ResTs, we mapped the sequence determinants in B. burgdorferi ResT that confer the ability to resolve multiple Type 2 telomeres. Surprisingly, this approach indicated that Type 2 telomere resolution was crucially regulated by a single proline residue located in a small Borrelia-specific insertion in the central catalytic region of ResT. The proline at this position was conserved in the ResTs from all Lyme disease Borrelia species but in none of the ResTs from relapsing fever Borrelia species, which were unable to efficiently resolve Type 2 telomeres in vitro. This study has identified a specific residue in ResT responsible for permissive substrate usage patterns. All relapsing fever and avian Borrelia strains were generously provided by Tom Schwan. ResT coding sequences were cloned from B. anserina strain BA.2 (GCB802), B. parkeri strain RML (GCB803), B. recurrentis strain number 132, P6 (GCB804), and B. turicatae strain 91E135 (GCB801). GenBankTM accession numbers for ResT coding sequences are as follows: B. anserina (FJ882620), B. parkeri (FJ882621), B. recurrentis (FJ882622), and B. turicatae (FJ882623). ResT coding sequences were amplified from the appropriate genomic DNAs using primers containing NdeI and BamH1 sites (described in supplemental Table S2). PCR conditions for the 50-μl reactions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs, Pickering, Ontario, Canada), 3% DMSO 3The abbreviation used is: DMSOdimethyl sulfoxide., 0.2 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.5 pmol/μl F primer, 0.5 pmol/μl R primer, 3 ng/μl genomic DNA template. PCR cycling conditions were as follows: 98 °C 45 s followed by 30 cycles of 98 °C, 10 s; 57 °C, 30 s; and 72 °C, 45 s and then 72 °C for 10 min. Appropriately sized PCR products were gel-purified using a Qiagen gel purification kit (Qiagen, Mississauga, Ontario, Canada) and then cloned using the ZeroBlunt®TOPO®PCR cloning kit (Invitrogen, Burlington, Ontario, Canada), all according to the manufacturer's instructions. Inserts were cut out of TOPO clones using NdeI/BamH1 (New England Biolabs) and cloned into NdeI/BamHI-digested pET15b. dimethyl sulfoxide. Constructs encoding chimeric proteins were built using site-directed mutagenesis or overlap extension PCR. Site-directed mutagenesis was used to introduce amino acid substitutions or to introduce unique restriction sites that were subsequently used for swapping sequences between B. burgdorferi and relapsing fever ResTs. The methods, templates, and primers used to build each construct are described in supplemental Table S2, and primer sequences are provided in supplemental Table S3. All constructs were sequenced before expression in E. coli. PCR conditions for the 50-μl reactions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs), 3% DMSO, 0.1 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.4 pmol/μl F primer, 0.4 pmol/μl R primer, 1.5 ng/μl plasmid DNA template. The first three PCR cycles were performed separately for each primer (e.g. one tube for F primer, one tube for R primer). After the third cycle the contents of the F and R primer tubes were mixed, and PCR cycling was continued to completion. PCR cycling conditions were as follows: 98 °C 45 s followed by 25–30 cycles of 98 °C, 20 s; 65–68 °C, 15 s; 70–72 °C, 3 min and 30 s and then 72 °C for 7 min. PCR products were purified using the QIAQuick PCR purification kit, according to the manufacturer's instructions (Qiagen), and then template DNA was digested with DpnI (New England Biolabs). Purified, digested DNA was used directly to transform chemically competent DH5α. A description of the three-step PCR reaction and primer design is provided in supplemental Fig. S3. In the first step (25 PCR cycles), primers A and B were used to amplify the first approximately one-half of the chimera, and primers C and D were used to amplify the second half (e.g. B. burgdorferi ResT), in separate 50-μl reactions. Primers A and D contained 5′-NdeI and -BamHI sites, respectively. Primers B and C were partially complementary to one another and contained sequences that annealed to both B. burgdorferi and B. hermsii ResT coding sequences (60 °C of sequence complementarity for each). In the second PCR step (six PCR cycles), 2.5 μl of each of the two PCR products from Step 1 were mixed together directly (5-μl final reaction volume) and extended by DNA polymerase already present in the PCR mixture to generate a small amount of chimeric product containing all 1,347 nucleotides of the ResT coding sequence. In Step 3 (25 PCR cycles), 2.5 μl of full-length chimeric Step 2 product were amplified in a 50-μl PCR reaction, using primers A and D. PCR reaction conditions were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs), 3% DMSO, 0.1 mm dNTPs, 0.02 units/μl Phusion DNA polymerase (New England Biolabs), 0.1 pmol/μl F primer, 0.1 pmol/μl R primer, 1.5 ng/μl plasmid DNA template. PCR cycling conditions were as follows: 98 °C 3 min followed by 25 cycles of 98 °C, 15 s; 63 °C, 15 s; 72 °C, 30 s and then 72 °C for 7 min. Following PCR, products were gel-purified from agarose gels using the QIAQuick gel extraction kit (Qiagen). Cleaned products were cloned into pJET1/blunt using the GeneJET PCR cloning kit, according to the manufacturer's instructions (MBI Fermentas, Burlington, Ontario, Canada). NdeI/BamHI-flanked ResT coding sequences were cloned into NdeI/BamHI-digested pET15b. ResT expression and purification were performed as described previously (38.Bankhead T. Chaconas G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13768-13773Crossref PubMed Scopus (35) Google Scholar) with the following exceptions. Bacteria were collected by centrifugation at 6,000 × g for 15 min at 4 °C. Pellets were resuspended in an EDTA-free buffer (25 mm Hepes-NaOH, pH 7.6). Resuspended cells were subjected to three freeze-thaw cycles following lysozyme treatment followed by ultracentrifugation for 45 min at 100,000 × g 4 °C. Columns for His tag affinity purification were prepared using 1 ml of nickel-nitrilotriacetic acid slurry (Qiagen). All purification buffers contained 0.5 m NaCl. Fractions were diluted to 50 ng/μl in elution buffer, and dithiothreitol was added to a final concentration of 1 mm. Previously described telomere substrates (30.Tourand Y. Kobryn K. Chaconas G. Mol. Microbiol. 2003; 48: 901-911Crossref PubMed Scopus (35) Google Scholar, 31.Tourand Y. Deneke J. Moriarty T.J. Chaconas G. J. Biol. Chem. 2009; 284: 7264-7272Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) were prepared from Qiagen midipreps (Qiagen), linearized with PstI (New England Biolabs), and cleaned by phenol extraction/ethanol precipitation. Twenty-μl telomere resolution reactions contained 25 mm Tris-HCl (pH 8.5), 100 mm NaCl, 1 mm EDTA, 100 μg/ml bovine serum albumin, 5 mm spermidine, 10 ng/μl PstI-linearized substrate DNA, and 10 ng/μl ResT. Time course reactions (140 μl) were incubated at 30 °C with 20-μl aliquots removed at 0, 1, 2, 4, 8, and 16 min. Reactions were performed in duplicate or triplicate and stopped by the addition of SDS to a final concentration of 0.2%. Samples were resolved on 20-cm 1% agarose gels in 1× Tris-acetate-EDTA (TAE) buffer at 100 V for 2 h. The gels were stained with ethidium bromide, and fluorescence of DNA bands was quantified using the AlphaInnotech software. The percentage of telomere resolution was determined by dividing the fluorescence of the reaction products by the total fluorescence (products plus substrate). Initial velocity values were calculated from the graphical plots of reaction kinetics. Statistical analyses of data were performed using Microsoft Excel and a two-tailed Student's t test with unequal variance. The methods used to identify and align telomere resolvase sequences are described in the legends for Fig. 2 and supplemental Fig. S2. The method used to thread ResT sequences onto the TelK structure is described in the legend for Fig. 4. Our previous work showed that B. burgdorferi (Lyme disease) and B. hermsii (relapsing fever) ResTs exhibit differences in substrate usage in vitro (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar). Specifically, B. hermsii ResT resolves a Type 2 telomere very inefficiently when compared with B. burgdorferi, whereas both enzymes can resolve a Type 1 telomere efficiently (Fig. 1, A and B.) The basis for this species-specific difference in substrate usage is unknown. To determine whether ResTs from other relapsing fever Borrelia exhibit similar Type 2 telomere resolution defects, we cloned the ResT coding sequences from three other relapsing fever strains, B. parkeri, B. recurrentis, and B. turicatae, as well as the avian Borrelia species B. anserina. These sequences are shown in Fig. 2, together with the ResT sequences from other Lyme disease (Borrelia afzelii, Borrelia spielmanii, Borrelia garinii, and B. burgdorferi) and relapsing fever (B. hermsii and Borrelia duttonii) Borrelia species (39.Tourand Y. Bankhead T. Wilson S.L. Putteet-Driver A.D. Barbour A.G. Byram R. Rosa P.A. Chaconas G. J. Bacteriol. 2006; 188: 7378-7386Crossref PubMed Scopus (28) Google Scholar, 40.Fraser C.M. Casjens S. Huang W.M. Sutton G.G. Clayton R. Lathigra R. White O. Ketchum K.A. Dodson R. Hickey E.K. Gwinn M. Dougherty B. Tomb J.F. Fleischmann R.D. Richardson D. Peterson J. Kerlavage A.R. Quackenbush J. Salzberg S. Hanson M. van Vugt R. Palmer N. Adams M.D. Gocayne J. Weidman J. Utterback T. Watthey L. McDonald L. Artiach P. Bowman C. Garland S. Fuji C. Cotton M.D. Horst K. Roberts K. Hatch B. Smith H.O. Venter J.C. Nature. 1997; 39
Lyme disease is caused by members of the Borrelia burgdorferi sensu lato species complex. Arthritis is a well-known late-stage pathology of Lyme disease, but the effects of B. burgdorferi infection on bone at sites other than articular surfaces are largely unknown. In this study, we investigated whether B. burgdorferi infection affects bone health in mice. In mice inoculated with B. burgdorferi or vehicle (mock infection), we measured the presence of B. burgdorferi DNA in bones, bone mineral density (BMD), bone formation rates, biomechanical properties, cellular composition, and two- and three-dimensional features of bone microarchitecture. B. burgdorferi DNA was detected in bone. In the long bones, increasing B. burgdorferi DNA copy number correlated with reductions in areal and trabecular volumetric BMDs. Trabecular regions of femora exhibited significant, copy number-correlated microarchitectural disruption, but BMD, microarchitectural, and biomechanical properties of cortical bone were not affected. Bone loss in tibiae was not due to increased osteoclast numbers or bone-resorbing surface area, but it was associated with reduced osteoblast numbers, implying that bone loss in long bones was due to impaired bone building. Osteoid-producing and mineralization activities of existing osteoblasts were unaffected by infection. Therefore, deterioration of trabecular bone was not dependent on inhibition of osteoblast function but was more likely caused by blockade of osteoblastogenesis, reduced osteoblast survival, and/or induction of osteoblast death. Together, these data represent the first evidence that B. burgdorferi infection induces bone loss in mice and suggest that this phenotype results from inhibition of bone building rather than increased bone resorption.
Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high-fat diet-induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil- and macrophage-based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi-infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high-fat diet, toll-like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow-derived macrophages from obese, B. burgdorferi-infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.
SUMMARY Similar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” to specific vascular sites and tissues during hematogenous dissemination 1–5 . For many pathogens, the “postal codes” and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction properties measured by live cell imaging and particle tracking in aorta, bladder, brain, joint and skin microvascular flow chamber models predict strain- and tissue-specific dissemination patterns in vivo These studies identified strain- and endothelial cell type-specific interaction properties that accurately predicted in vivo dissemination of B. burgdorferi to bladder, brain, joint and skin but not aorta, and indicated that dissemination mechanisms in all of these tissues are distinct. Thus, the ability to interact with vascular surfaces under physiological shear stress is a key determinant of tissue-specific tropism for Lyme disease bacteria. The methods and model systems reported here will be invaluable for identifying and characterizing the diverse, largely undefined molecules and mechanisms supporting dissemination of Lyme disease bacteria. These methods and models may be useful for studying tissue tropism and vascular dissemination mechanisms of other blood-borne microbes.
