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Molecular Pathogenesis

Use of the Cre-lox Recombination System To Investigate the lp54 Gene Requirement in the Infectious Cycle of Borrelia burgdorferi

Aaron Bestor, Philip E. Stewart, Mollie W. Jewett, Amit Sarkar, Kit Tilly, Patricia A. Rosa
Aaron Bestor
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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  • For correspondence: bestora@niaid.nih.gov
Philip E. Stewart
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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Mollie W. Jewett
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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Amit Sarkar
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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Kit Tilly
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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Patricia A. Rosa
Laboratory of Zoonotic Pathogens, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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DOI: 10.1128/IAI.01059-09
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  • FIG. 1.
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    FIG. 1.

    Schematic diagram illustrating the loxP/Cre-mediated deletion of the gene encoding GFP. The introduction of Cre recombinase into B. burgdorferi containing flaBp-gfp flanked by loxP sites should result in recombination between loxP sites, the excision and loss of gfp from shuttle vector pBSV2G (15) due to the absence of replication factors in this region of the plasmid, and a loss of fluorescence by the cell.

  • FIG. 2.
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    FIG. 2.

    Loss of fluorescence by B. burgdorferi carrying loxP-flanked GFP after introduction of Cre recombinase. Strain B31-A34 was first transformed with shuttle vector pBSV2G-loxP-flaBp-gfp (Fig. 1) and subsequently transformed with the compatible shuttle vector pBSV25 or pBSV25-flgBp-cre, which encodes Cre recombinase. (Top) Presence or absence of GFP fluorescence in these strains as visualized by fluorescence microscopy. (Bottom) Cells were counterstained with the membrane stain FM4-64 to visualize all spirochetes in the same field.

  • FIG. 3.
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    FIG. 3.

    Schematic diagram of the targeted region encompassing bba01 to bba14 of lp54, extending from the left telomere to the ospAB operon. Boxed numbers beneath the diagram indicate previously identified members of paralogous gene families (11, 18). Protein designations above the diagram and gene designations below are based on previous studies and annotations (10, 11, 16, 18, 36, 37).

  • FIG. 4.
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    FIG. 4.

    Insertion of loxP into targeted lp54 loci. (A) Schematic diagram showing how a loxP site (filled arrowheads) and an adjacent selectable marker conferring resistance to streptomycin or kanamycin (aadA and Kanr, respectively) were introduced by allelic exchange into bba07 and subsequently into either bba01 or bba14 on lp54. The relevant restriction enzyme sites used in cloning allelic exchange constructs are indicated. Small arrows beneath bba07, bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm loxP insertions into these loci, as shown below (B). The designations of the resulting strains (Table 1) are shown on the left. (B) PCR amplification of targeted lp54 loci demonstrating an increase in fragment size after loxP insertion. The gene target and PCR primers are indicated above the lanes, and the source of template DNA is shown below the lanes. PCR primer positions and sequences are shown above (A) and in Table 2, respectively. The relative mobility of DNA size standards (kb) is shown on the left.

  • FIG. 5.
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    FIG. 5.

    Deletion of loxP-flanked regions of lp54 after introduction of Cre recombinase. (A and B) Schematic diagrams showing the excision of the intervening DNA between loxP sites (filled arrowheads) present in the lp54 loci bba07 and bba01 (A) or bba07 and bba14 (B) as a result of Cre-mediated recombination. A recombined loxP site and the adjacent resistance cassette are present on a nonreplicating circular DNA fragment. Small arrows beneath bba07, bba01, and bba14 indicate the positions of oligonucleotides (Table 2) used with PCR to confirm the deletion of the intervening sequences in Cre transformants, as shown below (C). The designations of the resulting strains (Table 1) are shown on the left. (C) PCR amplification of targeted lp54 regions in the wild type and deletion mutants. Smaller products spanning the deleted regions were amplified from mutant strains, whereas the larger lp54 fragments were not efficiently amplified from wild-type A3. PCR amplification of the kan and aadA genes demonstrates the presence or absence of these antibiotic resistance cassettes as a consequence of the lp54 deletions. The gene target and PCR primers are indicated above the lanes, and the template DNA is indicated below the lanes. Primer positions are indicated above (A and B), and sequences are shown in Table 2. The relative mobility of DNA size standards (kb) is shown on the left.

  • FIG. 6.
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    FIG. 6.

    Southern blots confirming the deletion of multigene segments of lp54 by loxP/Cre-mediated recombination. Genomic DNAs prepared from the wild type (B31-A3), a strain lacking lp54 (B314), and the lp54 deletion mutants (A3ΔA1-7 and A3ΔA7-14) were subjected to Southern blot analysis with probes specific to bba03 and bba10 to confirm the loss of targeted regions of lp54 containing these loci in the respective loxP/Cre deletion mutants. The source of DNA is identified above the lanes, and the probe is specified below.

  • FIG. 7.
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    FIG. 7.

    In vitro growth curves. Wild-type B. burgdorferi (A3) and loxP insertion mutants (A3-A07, A3-A01/A07, and A3-A07/A14) (A) or lp54 deletion mutants (A3ΔA1-7 and A3ΔA7-14) and wild-type B. burgdorferi containing Cre on a shuttle vector (A3-Cre) (B) were grown at 35°C in BSKII medium from a starting concentration of 105 spirochetes/ml. Spirochetes were counted at 24-h intervals in Petroff-Hauser chambers, and the mean number of spirochetes per ml was determined from triplicate cultures of each strain, as shown.

Tables

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  • TABLE 1.

    B. burgdorferi strains used in this study

    StrainDescriptionReference
    B314Attenuated B. burgdorferi strain B31 clone lacking all linear plasmids, including lp54 46
    B31-A34Transformable, noninfectious B31 clone lacking lp5, lp25, lp28-1, lp28-4, lp56, lp36, cp-9, and cp32-6, used for testing the Cre-lox system 26
    B31-A3Transformable, infectious B31 clone lacking cp9; background of lp54 mutants listed below 15
    A3-A07Streptomycin resistance cassette and loxP insertion into bba07This study
    A3-A01/A07Streptomycin resistance cassette and loxP insertion into bba07, kanamycin resistance cassette and loxP insertion into bba01This study
    A3-A07/A14Streptomycin resistance cassette and loxP insertion into bba07, kanamycin resistance cassette and loxP insertion into bba14This study
    A3ΔA1-7Deletion of bba01 to bba07 loci and retention of only the streptomycin resistance cassetteThis study
    A3ΔA7-14Deletion of bba07 to bba14 loci and retention of only the kanamycin resistance cassetteThis study
  • TABLE 2.

    Oligonucleotides used in this study

    PrimerDesignationSequencea
    1FlaBp-loxP 5′ATAACTTCGTATAGCATACATTATACGAAGTTATtgtctgtcgcctcttgtggc
    2GFP-loxP 3′ PstI ctgcagATAACTTCGTATAGCATACATTATACGAAGTTATttatttgtagagctcatcca
    3Cre 5′ NdeI catatgtccaatttactgaccgtac
    4Cre 3′ XbaI tctagactaatcgccatcttccagcagg
    5229F ggtaccgattaatccaagctcaataa
    61228Rggctcaatattgcgtttaag
    7LoxflgF-1 ggatccATAACTTCGTATAGCATACATTATACGAAGTTATtacccgagcttcaaggaaga
    8KanR-1 ggatccttagaaaaactcatcgagct
    94297F ggtaccggcttgcagctgccttaata
    105391Rtaaaagctgtagtcaagccc
    11LoxflaBF gagctcATAACTTCGTATAGCATACATTATACGAAGTTATctgtcgcctcttgtggcttc
    12aadaR gagctcttatttgccgactaccttgg
    138398F acgcgtcaactctggcattgacgctt
    149198Raaaaaagtcccagttttggg
    15LoxflgF-2 aagcttATAACTTCGTATAGCATACATTATACGAAGTTATtacccgagcttcaaggaaga
    16KanR-2 aagcttttagaaaaactcatcgagct
    17pBSV2* 5′ttttattatagagttcatatacatgtttttctaaatctataa
    18pBSV2* 3′acatgtatatgagttctataataaaaaatattctagtgaggg
    19pncA+RBS EcoRI 5′ccggaattcttgtatcaagggatattgcc
    20pncA XbaI 3′gctctagattatatattaagcttactttggctg
    21Cre+RBS XbaI 5′gctctagataagaacaataaaaaggaaaatttatgtccaatttactgaccgtacacc
    22cre SphI 3′acatgcatgcctaatcgccatcttccagcagg
    23pncA prom 5′ EcoRIcggaattcctatgctatccccttgttcaagtc
    24pncA prom 3′ EcoRIcggaattccttttgtcaataattatatatttggg
    • ↵ a Restriction enzyme sites are indicated in boldface type, and the loxP sequences are in uppercase type.

  • TABLE 3.

    Plasmid constructs used in this study

    PlasmidDescriptionReference(s)
    pBSV2G-loxP-flaBp-gfpGFP expression vector with loxP sites flanking flaBp-gfp 10, 15; this study
    pBSV25-flgBp-creCre expression vector used to excise flaBp-gfp in pilot expt 58; this study
    pABA07Suicide vector used for inserting loxP-Strr into bba07This study
    pABA01Suicide vector used for inserting loxP-Kanr into bba01This study
    pABA14Suicide vector used for inserting loxP-Kanr into bba14This study
    pBSV2*Created by disruption of the EcoRI site found at bp 1852 on pBSV2 59; this study
    pBSV2G-pncAp-pncA-creCre expression vector used to excise targeted regions of lp54 15; this study
  • TABLE 4.

    Mouse infectivity of loxP insertion mutants by needle inoculation

    B. burgdorferi strainNo. of infected mice/no. of mice injected with 5 × 103 spirochetesaNo. of tissue reisolates/no. of injected mice (ear, bladder, joint)b
    A33/33/3, 3/3, 3/3
    A3-A073/33/3, 3/3, 3/3
    A3-A01/A073/33/3, 3/3, 3/3
    A3-A07/A143/33/3, 3/3, 3/3
    • ↵ a Number of infected mice/number of mice injected with 5 × 103 spirochetes (4 × 103 spirochetes intraperitoneally and 1 × 103 spirochetes subcutaneously). Seroconversion was determined by immunoblot analysis with B. burgdorferi lysate 3 weeks postinoculation.

    • ↵ b Number of tissue reisolates/number of injected mice, for ear, bladder, and joint. Mice were euthanized and tissues were cultured at 4 weeks postinjection.

  • TABLE 5.

    Mouse infectivity of lp54 deletion mutants by needle inoculation

    B. burgdorferi strainNo. of infected mice/no. of mice injected with 5 × 103 spirochetesaNo. of tissue reisolates/no. of injected mice (ear, bladder, joint)bNo. of spirochetes/larval tick (mouse 1; mouse 2)c
    A33/43/4, 3/4, 3/42,500; 933
    A3ΔA1-74/44/4, 4/4, 4/427,332; 866
    A3ΔA7-144/44/4, 4/4, 4/46,333; 4
    • ↵ a Number of infected mice/number of mice injected with 5 × 103 spirochetes (4 × 103 spirochetes intraperitoneally and 1 × 103 spirochetes subcutaneously). Seroconversion was determined by immunoblot analysis with B. burgdorferi lysate 3 weeks postinoculation.

    • ↵ b Number of tissue reisolates/number of injected mice, for ear, bladder, and joint at 5 weeks postinoculation. All three tissues from infected mice were positive.

    • ↵ c Larval ticks were fed to repletion on two seropositive mice for each strain. Ten days postfeeding, three fed larvae from each mouse were pooled, crushed, and plated to determine the number of viable spirochetes. The average number of spirochetes per tick was calculated from the total number of colonies obtained per pool of three larvae for each mouse. Mean numbers of spirochetes per tick for the A3ΔA1-7 and A3ΔA7-14 mutants were not significantly different from those of the wild type (A3) as determined by a two-tailed, unpaired t test (P = 0.3 and P = 0.7, respectively), performed by use of GraphPad Prism 5 software.

  • TABLE 6.

    Mouse infectivity of lp54 deletion mutants by tick transmission

    B. burgdorferi strainNo. of infected mice/no. of mice fed on by 20 infected nymphsaNo. of tissue reisolates/no. of mice fed on by infected ticks (ear, bladder, joint)bNo. of spirochetes/nymphal tick (mouse 1; mouse 2; mouse 3)c
    A33/33/3, 3/3, 3/3100,000; 130,000; 140,000
    A3ΔA1-74/74/7, 4/7, 4/7100,000; 140,000; 130,000
    A3ΔA7-143/33/3, 3/3, 3/3140,000; 120,000; 140,000
    • ↵ a Seroconversion was determined by immunoblot analysis with B. burgdorferi lysate 3 weeks postfeeding. The number of mice infected by either A3- or A3ΔA1-7-infected ticks was not significantly different using 20 ticks per mouse (P = 0.48, performed by Fisher's two-tailed exact-probability test).

    • ↵ b Number of tissue reisolates/number of mice fed on by infected ticks, for ear, bladder, and joint. All three tissues from infected mice were positive.

    • ↵ c Twenty infected nymphal ticks were fed to repletion on three naïve mice per strain. Ten days postfeeding, three fed nymphs from each mouse were pooled, crushed, and plated to determine the number of viable spirochetes. The average number of spirochetes per tick was calculated from the total number of colonies obtained per pool of three nymphs. Mean numbers of spirochetes per tick were not significantly different between strains as determined by one-way analysis of variance (ANOVA) (P = 0.75), performed by use of GraphPad Prism 5 software.

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Use of the Cre-lox Recombination System To Investigate the lp54 Gene Requirement in the Infectious Cycle of Borrelia burgdorferi
Aaron Bestor, Philip E. Stewart, Mollie W. Jewett, Amit Sarkar, Kit Tilly, Patricia A. Rosa
Infection and Immunity May 2010, 78 (6) 2397-2407; DOI: 10.1128/IAI.01059-09

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Use of the Cre-lox Recombination System To Investigate the lp54 Gene Requirement in the Infectious Cycle of Borrelia burgdorferi
Aaron Bestor, Philip E. Stewart, Mollie W. Jewett, Amit Sarkar, Kit Tilly, Patricia A. Rosa
Infection and Immunity May 2010, 78 (6) 2397-2407; DOI: 10.1128/IAI.01059-09
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KEYWORDS

Bacterial Proteins
Borrelia burgdorferi
Ixodes
plasmids
Recombination, Genetic
virulence factors

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