P66 porins are present in both Lyme disease and relapsing fever spirochetes: A comparison of the biophysical properties of P66 porins from six Borrelia species

Abstract

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

“…Among the Borrelia genospecies, sequence variation in the predicted surfaceexposed loop is greater than that found throughout the rest of the P66 sequence, indicating that the loop may be under immune selection pressure during mammalian infection (28,29). Furthermore, Skare et al demonstrated that native P66 forms pores in lipid bilayer assays (30), and similar properties have been described for P66 from B. garinii and B. afzelii (31). In vitro analyses have implicated P66 as an adhesin that binds specifically to ␤ 3 -chain integrins (32)(33)(34).…”

The physical properties of lipid monolayers and bilayers containing calixarenes sensitive to cytochrome c

“…DPhPC contain branched hydrocarbon chains and are important component of the membranes of halobacteria (Kates et al 1965;Barcena-Uribarri et al 2010). We showed that CX forms stable monolayers at an air-water interface, as well as stable mixed DPhPC/calixarene monolayers.…”

DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates

“…The Lyme disease strains used in this study were B. afzelii [76], B. garinii LU185 [77] and B. burgdorferi strains B31 (ATCC 35210) and B. burgdorferi Δp66, a p66 knock-out strain of B. burgdorferi B31-A [49] and the Osp-deficient B. burgdorferi B313 [78]. The relapsing fever bacterial isolates used were B. crocidurae CR2 (from the strain collection of Alan G Barbour UC Irvine), B. duttonii 1120 [79], B. hermsii (ATCC35209), B. hispanica CR1 [79] and B. recurrentis A1 [59].…”