Targeted mutation of the outer membrane protein P66 disrupts attachment of the Lyme disease agent, Borrelia burgdorferi, to integrin alphavbeta3

Abstract

Borrelia burgdorferi, the agent of Lyme disease, expresses several adhesion molecules that are probably required for initial establishment of infection in mammalian hosts, and for colonization of various tissues within the host. The B. burgdorferi outer membrane protein P66 was previously identified as a ligand for beta3-chain integrins by using a variety of biochemical approaches. Although the earlier data suggested that P66 is an adhesin that mediates B. burgdorferi attachment to beta3-chain integrins, lack of genetic systems in B. burgdorferi precluded definitive demonstration of a role for P66 in beta3 integrin attachment by intact borreliae. Recent advances in the genetic manipulation of B. burgdorferi have now made possible the targeted disruption of the p66 gene. Mutants in p66 show dramatically reduced attachment to integrin alphavbeta3. This is, to our knowledge, the first description of the targeted disruption of a candidate B. burgdorferi virulence factor with a known biochemical function that can be quantified, and demonstrates the importance of B. burgdorferi P66 in the attachment of this pathogenic spirochete to a human cell-surface receptor.

Fig. 1.

Schematic representation of the plasmids and oligonucleotides used to generate and analyze mutants in B. burgdorferi (not drawn to scale). (Upper) The B. burgdorferi p66 locus (BB0603 in the Institute for Genomic Research (TIGR) sequence of strain B31). Amino acids 203–209, implicated as being important in integrin-binding activity, are denoted by the gray stippled region. Although the strain HB19 locus has not been sequenced in its entirety, the PCR primers shown amplify fragments of the sizes that are expected from the B31 sequence. The structures of the mutagenic cassettes generated in the pTAkanA vector are depicted below. The amino-terminal portion of P66 is truncated at amino acid 260 in p66KO2 and at amino acid 150 in p66KO4. Numbered arrows depict oligonucleotides. Corresponding sequences, which were based on the strain B31 sequence, are given in Table 1.

Fig. 2.

Comparison of protein expression by B. burgdorferi wild type and mutants. (A) The total protein contents of ≈2.5 × 10⁷ B. burgdorferi cells were solubilized and fractionated by electrophoresis through replicate 12.5% polyacrylamide gels under denaturing conditions. One gel was stained with Coomassie brilliant blue (Left) and the second was transferred to a poly(vinylidene difluoride) (PVDF) membrane and probed with a polyclonal rabbit antiserum directed against the integrin-binding domain of P66 (Right). Wild-type B. burgdorferi is shown in comparison to a KO1 mutant, a KO2 mutant, and a KO4 mutant. Positions of markers are shown in kilodaltons. (B) Silver-stained two-dimensional gels of the KO1 and KO4 mutants with the first dimension isoelectric focusing (IEF) gels run by using ampholines (pH 5–7). The images were cropped to focus on the region expected to contain P66. The pH gradient (left to right for each panel) is ≈5.0–5.9. (C) Silver-stained two-dimensional gels of the KO1 and KO4 mutants, with the IEF first dimension performed by using ampholines (pH 3.5–10); the second-dimension gels were 10% polyacrylamide. The entire gels are shown. Arrows indicate the position of P66.

Fig. 3.

Growth of wild-type and mutant B. burgdorferi in vitro. B. burgdorferi cultures at densities of 300–400 bacteria per high-power field (hpf) (late-logarithmic phase) in MKP medium were diluted into fresh medium to a density of ≈1 per hpf and the cultures were placed at 33°C. Growth was followed by dark-field microscopy through the time course indicated on the x axis. Shown are the means and standard deviations of four independent growth experiments for each strain. Several fields were counted for each sample, and at high spirochete densities, quarter fields were counted. The clones used for this experiment were the same as those analyzed in Fig. 2.

Fig. 4.

Attachment of wild-type and P66-deficient B. burgdorferi to purified α_vβ₃ and to epithelial cells. Purified α_vβ₃ was plated at 5 μg/ml; buffer alone was plated as a control. Cells were plated to achieve at least 95% coverage of the well area on the day of the assay; medium alone served as the control. Wells were probed with B. burgdorferi wild-type or mutant clones and attachment was quantified as described in Materials and Methods. For the experiment on the right, antibodies were used at 10 μg/ml and preincubated with the cells for 30 min before the addition of the bacteria. Shown are the means and standard deviations of four replicates. *, P ≤ 0.05 in comparison to wild type.

Fig. 5.

Binding of wild-type and P66-deficient B. burgdorferi to purified α_vβ₃ in the presence of RGD and RGE peptides. α_vβ₃ was plated at 1 μg/ml and attachment was quantified as described in Materials and Methods. The B. burgdorferi clones used for this experiment were the same as those analyzed in Figs. 2 and 3. The RGD and RGE peptides were used at a concentration of 0.1 mg/ml. Shown are the means and standard deviations of four replicates. *, P ≤ 0.05 in comparison to wild type, all in the absence of peptides. In the presence of RGD, but not RGE peptide, binding was significantly different (P < 0.05) from the control without peptide for each strain.