The Borrelia burgdorferi outer-surface protein ErpX binds mammalian laminin

Abstract

The Lyme disease spirochaete, Borrelia burgdorferi, can invade and persistently infect its hosts' connective tissues. We now demonstrate that B. burgdorferi adheres to the extracellular matrix component laminin. The surface-exposed outer-membrane protein ErpX was identified as having affinity for laminin, and is the first laminin-binding protein to be identified in a Lyme disease spirochaete. The adhesive domain of ErpX was shown to be contained within a small, unstructured hydrophilic segment at the protein's centre. The sequence of that domain is distinct from any previously identified bacterial laminin adhesin, suggesting a unique mode of laminin binding.

Fig. 1.

Adherence of intact B. burgdorferi to laminin. Bars indicate 1 standard deviation from the mean. Asterisks indicate statistically significant differences between adherence to laminin and BSA (P<0.001). (A) Microscopical enumeration of bacteria adhering to slides coated with laminin or BSA at initial concentrations of 5 μg protein ml⁻¹. (B) Results of ELISA-based studies of live bacteria binding to fixed laminin, means of 30 assays. Assays without added bacteria served as negative controls.

Fig. 2.

Ligand-affininty blot analyses of laminin binding to components of outer-membrane-enriched Triton X-114 extract of cultured B. burgdorferi B31-MI-16. Arrows to the right indicate bacterial proteins that bound laminin. Positions of molecular mass standards are indicated to the left.

Fig. 3.

Ligand-affininty blot analyses of laminin binding by polyhistidine-tagged recombinant Erp proteins. (A) Recombinant Erp proteins of B. burgdorferi strain B31. BSA was included as a control for non-specific protein–laminin interactions. (B) Recombinant ErpX from strain B31 and Erp26 from strain N40. Positions of molecular mass standards are indicated between the two panels.

Fig. 4.

(A) Amino acid sequences of recombinant ErpX (rErpX) and the truncated derivatives of ErpX used in these studies. The 55 amino acids deleted from rErpX17 are indicated by dots. (B) Predicted order/disorder of wild-type ErpX. A PONDR score above 0.5 predicts protein disorder; a score below 0.5 predicts order. The laminin-binding region in the centre of ErpX, as identified through use of variant proteins rErpX15, rErpX16 and rErpX17, is predicted to be extremely disordered in the absence of bound ligand.

Fig. 5.

Ligand-affininty blot analysis of laminin binding by recombinant ErpX (rErpX) and truncated derivatives thereof. (A) Full-length ErpX and three of the examined truncations. The rightmost lane contained an equal molar content of BSA, to serve as a control for non-specific laminin binding. (B) The upper panel shows results of laminin-binding analysis of truncated proteins rErpX12, rErpX13, rErpX15, rErpX16 and rErpX14, and the lower panel shows the same amounts of each protein in a gel stained with Coomassie brilliant blue. Note that even though relatively less rErpX15 appears to have been loaded onto this gel, the laminin-binding signal for that protein was comparable to those from ErpX12 and rErpX13. (C) The upper panel illustrates results of laminin-binding analysis of proteins rErpX3 and rErpX17, and the lower panel shows the proteins stained with Coomassie brilliant blue. Migration positions of molecular mass standards are indicated on the left of each panel. Highly charged proteins such as ErpX often exhibit aberrant SDS-PAGE mobilities (Stevenson et al., 1998): note that rErpX-5 migrated at a rate suggestive of a molecular mass greater than rErpX-3, even though rErpX-5 is actually the smaller protein.

Fig. 6.

Effects of recombinant proteins on adherence of B. burgdorferi to laminin, as measured by ELISA. Results for each protein are illustrated relative to control experiments lacking added protein. All added ErpX derivatives significantly affected adherence to laminin: rErpX-17 (P=0.0009), and rErpX, rErpX-12, rErpX-13 and rErpX-15 (all P<0.0001). Bb, B. burgdorferi.