Identification of loci critical for replication and compatibility of a <i>Borrelia burgdorferi</i> cp32 plasmid and use of a cp32‐based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete

Eggers, Christian H.; Caimano, Melissa J.; Clawson, Marion; Miller, William G.; Samuels, D. Scott; Radolf, Justin D.

doi:10.1046/j.1365-2958.2002.02758.x

Cited by 133 publications

(222 citation statements)

References 58 publications

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“…The positive charges of the lysines apparently contribute to the binding interaction. These could involve not only lysine residues in regions III-V, but the Lys 41 and Lys 43 residues in the N-terminal part close to region I. This part contains the sequence KIKNK that was deleted from the nonfunctional N-terminal mutants of Metts et al (21), but not from our P21⌬1-35 mutant that was functionally active.…”

Section: Discussionmentioning

confidence: 90%

Lysine-Dependent Multipoint Binding of theBorrelia burgdorferiVirulence Factor Outer Surface Protein E to the C Terminus of Factor H

Alitalo

Meri

Chen

et al. 2004

The Journal of Immunology

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Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18–20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala151-Lys166) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.

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Section: Discussionmentioning

confidence: 90%

Lysine-Dependent Multipoint Binding of theBorrelia burgdorferiVirulence Factor Outer Surface Protein E to the C Terminus of Factor H

Alitalo

Meri

Chen

et al. 2004

The Journal of Immunology

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“…Borrelial cultures were passaged no more than 3 times prior to experimental manipulations. The plasmid content of each isolate was monitored by PCR as previously described (88). To obtain temperature-shifted organisms, B. burgdorferi clones were grown at 23°C to a density of 2 to 5 × 10 6 spirochetes/ml, diluted into fresh BSK-II to a density of 1 × 10 3 spirochetes/ml, and incubated at 37°C until mid-log phase (~5 × 10 7 spirochetes/ml).…”

Section: Methodsmentioning

confidence: 99%

Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks

et al. 2009

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Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination "adherence-mediated migration" and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease-causing spirochetes within ticks is exclusively motility driven.

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“…The 360 bp product from pPflaB was gel extracted and ligated into the gel extracted fragment of pRecA.1 to generate pRecA.2. The flaB promoter-recA fusion was removed from pRecA.2 by digestion with KpnI and XbaI (sites in pCR2.1 flanking the TA insertion site), separated by gel electrophoresis, gel extracted and cloned into like sites in pCE320 (Eggers et al 2002) (a B. burgdorferi shuttle vector with a circular plasmid 32 (cp32) origin for replication that confers kanamycin resistance) to generate pRecA.3. The complementation construct pRecA.3 was concentrated and transformed into JA10 as described above.…”

Section: Complementation Of the Reca Mutantmentioning

confidence: 99%

Adaptation to Glucosamine Starvation in Borrelia burgdorferi is Mediated by recA

Rhodes¹,

Atoyan²,

Nelson³

2012

Lyme Disease

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Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32‐based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete

Cited by 133 publications

References 58 publications

Lysine-Dependent Multipoint Binding of theBorrelia burgdorferiVirulence Factor Outer Surface Protein E to the C Terminus of Factor H

Lysine-Dependent Multipoint Binding of theBorrelia burgdorferiVirulence Factor Outer Surface Protein E to the C Terminus of Factor H

Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks

Adaptation to Glucosamine Starvation in Borrelia burgdorferi is Mediated by recA

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