Antigenic composition of Borrelia burgdorferi during infection of SCID mice

Abstract

The general concept that during infection of mice the Borrelia burgdorferi surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.

FIG. 1.

Antigenic profiles of IVCB and HAB detected with IRS. Triton X-114 detergent-phase tissue samples from infected C3H SCID mice and detergent-phase IVCB controls were analyzed by SDS-PAGE and immunoblotting with IRS. A preparation containing 1 × 10⁵ detergent-phase B31 cells was used for comparison.

FIG. 2.

Antigenic profiles of IVCB and HAB detected with monospecific antisera. Triton X-114 detergent-phase tissue samples from infected C3H SCID mice and detergent-phase IVCB controls were analyzed by SDS-PAGE and immunoblotted with the following monospecific antisera: VlsE (A), OspC (B), and DbpA (C). Preparations containing 5 × 10⁶ and 5 × 10⁵ detergent-phase B31 cells were used for comparison. In panel A the additional heart lane on the right is an overexposure.

FIG. 3.

Multiple sizes and isoforms of VlsE are present in IVCB and B31-infected C3H SCID mice. Triton X-114 detergent-phase tissue samples from infected C3H SCID mice and IVCB were analyzed by IPG-2DE and immunoblotted with monospecific VlsE antiserum. (A) Ear; (B) knee; (C) ankle; (D) heart; (E) 1 × 10⁹ B31 detergent-phase cells; (F) 1 × 10⁹ B31 whole organisms; (G) 1 × 10⁹ ME3-2 detergent-phase cells; (H) ear, probed with CMS.

FIG. 4.

Amounts of vlsE, dbpA, and ospC transcripts in infected SCID mouse tissues and IVCB. RT-PCR was performed to identify the transcripts. IVCB controls were normalized to provide an amount of flaB transcripts approximately equal to the amount in each infected tissue sample. (A) Ear; (B) ankle; (C) knee; (D) heart. Lanes 1, IVCB control; lanes 2, infected tissue. An asterisk indicates that 45 cycles were used; 40 cycles were used for the other samples.

FIG. 5.

Antigenic composition of HAB. Triton X-114 detergent-phase samples derived from the tissues of infected C3H SCID mice and IVCB were analyzed by using IPG-2DE and immunoblotting with IRS. The arrows indicate antigens that appear to be upregulated in tissue. The asterisks indicate unidentified proteins found in both tissue and IVCB.