Borrelia burgdorferi

Borrelia burgdorferi OM and PCs were isolated as previously described (Skare et al., 1995 (link); Mulay et al., 2007 (link)) with minor modifications. Five hundred millilitres to 1 L cultures of B. burgdorferi B31-A3-LK, flacp-795-LK (in 0.05 mM or 1 mM IPTG), B31cF and 795-cF were grown in complete BSK-II to late-log phase and harvested by centrifugation at 5800 g for 20 min. After one wash in PBS (pH 7.4) containing 0.1% BSA, the pellet (corresponding to 5.7 × 10¹⁰ spirochetes for B31-A3-LK and flacp-795-LK, or 9.4 × 10¹⁰ spirochetes for B31cF and 795-cF) was resuspended in 90 ml of ice-cold 25 mM citrate buffer (pH 3.2) containing 0.1% BSA. The spirochetes were agitated at room temperature for 2 h with a 1 min vortex every 30 min, followed by centrifugation at 20 000 g for 20 min. The resulting pellet, containing the OM and PC fractions, was resuspended in 5.5 ml of 25 mM citrate buffer (pH 3.2) containing 0.1% BSA and layered onto a discontinuous sucrose gradient in 25 mM citrate buffer (pH 3.2) composed of 5 ml of 56% (wt/wt), 15.5 ml of 42% (wt/wt) and 12.5 ml of 25% (wt/wt) sucrose. The discontinuous gradient was centrifuged overnight at 100 000 g, and the buoyant OM band was removed using a Gradient Station 153 fraction collector (BioComp Instruments, New Brunswick, Canada). The heavier band containing the PC material was collected and diluted eightfold in PBS (pH 7.4), pelleted at 10 000 g for 20 min and resuspended in 1–1.5 ml of PBS (pH 7.4) prior to storage at −80°C for subsequent analyses. The OMs isolated from the discontinuous gradient were resuspended in PBS (pH 7.4), divided between two 14 × 89 mm centrifuge tubes (Beckman Instruments) and subjected to centrifugation at 141 000 g for 4 h at 5°C. Following centrifugation, the OM pellets were combined and resuspended in 1 ml of 25 mM citrate buffer (pH 3.2). Next, a 10–41% (wt/wt) continuous sucrose gradient, in 25 mM citrate buffer (pH 3.2), was prepared using a Gradient Master 107 gradient maker according to manufacturer’s instructions (BioComp Instruments). For further OM purification, the 1 ml of OM sample was layered onto the prechilled continuous gradient and centrifuged overnight at 100 000 g at 5°C. The resultant membrane band was collected and pelleted as described above, and the final OM pellet was resuspended in 150–350 μl of PBS containing 1 mM PMSF and stored at −80°C. To ensure proper fractionation technique and to verify that the OM and PC fractions were not cross contaminated, all OM and PC fractions were immunoblotted with antisera to the known inner membrane-anchored lipoprotein OppAIV (Bono et al., 1998 (link); Nowalk et al., 2006 (link); Mulay et al., 2007 (link)).

Residual clinical specimens [whole blood (EDTA), synovial fluid, cerebrospinal fluid (CSF), and tissue] submitted to Mayo Clinic from health care providers nationwide for patients suspected of having a tickborne illness [i.e. testing by either Tick Borne Pathogen PCR Panel (Babesia, Ehrlichia/Anaplasma) or Lyme (Borrelia burgdorferi sensu lato) PCR] and the accompanying nucleic acid extract were stored at 4°C or −70°C, de-identified and shipped to the Minnesota Department of Health (MDH). Synovial fluid, CSF, and tissue specimens were originally submitted for Lyme PCR, whereas blood specimens were submitted for either Tick Borne Pathogen PCR Panel or Lyme PCR. Aliquots of each clinical specimen were prepared, frozen at −70°C, and shipped to the Centers for Disease Control and Prevention, Fort Collins, CO. Associated patient information included specimen type, originating state of the ordering provider, patient age, and sex. As travel history of patients was not available, the state of the ordering provider does not necessarily correlate to the patient’s state of residence or exposure. Analysis of de-identified specimens was approved by the Institutional Review Board at Mayo Clinic (Protocol ID: 14-001148). Review at MDH and CDC determined the protocol to be non-human subjects research.