Borreliose-Gesellschaft e.V.


Porins of Lyme Disease and Relapsing Fever Spirochetes – an Overview
(This talk will be in German)

Dr. Marcus Thein
Max Planck Institute for Developmental Biology
Department of Proteinevolution

The genus Borrelia belongs to the spirochete phylum, an ancient evolutionary branch of the domain bacteria that is only afar related to Gram-negative bacteria. Borreliae can be subdivided into the agents of the two borrelian-caused human diseases, Lyme disease and relapsing fever. Both disease patterns are closely related to the peculiar biology of Borrelia species and exhibit a wide spectrum of diverse clinical manifestations.    

Due to the small 0.91 Mb chromosome, borreliae have a lack of biosynthetic capacity and limited metabolic pathways. Thus, all Borrelia species are highly dependent on nutrients provided by their hosts. The transport of nutrients and other molecules across the outer membrane of Gram-negative bacteria, such as Borrelia species, is enabled by pore-forming proteins, so-called porins.

Porins are water-filled channels and can be subdivided into two different classes, general diffusion pores and substrate-specific porins. General diffusion pores sort mainly according to the molecular mass of the solutes and show a linear relation between translocation rate and solute concentration gradient. Specific porins with a substrate-binding site inside the channel exhibit Michaelis-Menten kinetics for the transport of certain solutes and are responsible for the rapid uptake of different classes of solutes. Among Gram-negative bacteria, the architecture and functions of porins are highly conserved. But, interestingly, the structure and function of Borrelia species seem to be completely different compared to other Gram-negative bacteria and underlines the outstanding properties and abilities of borreliae.  

In terms of the Lyme disease agent Borrelia burgdorferi, three putative porins were found in previous studies: P13, Oms28 and P66. In contrast to Lyme disease species, the porin knowledge of relapsing fever Borrelia is low, which means that not any porin has actually been described for representatives of these agents. Thus, the general aim of this work was to provide insight into the porin content of both, Lyme disease and relapsing fever spirochetes. This aim could be achieved by isolating and identifying porins from Borrelia outer membranes and by biophysically characterizing their properties in artificial lipid membranes.

Here, the first identification and characterization of a relapsing fever porin is presented. The pore-forming protein was isolated from outer membranes of Borrelia duttonii, Borrelia hermsii and Borrelia recurrentis and designated Oms38, for “outer membrane-spanning protein of 38 kDa”. Biophysical characterization of Oms38 was achieved by using the black lipid bilayer method and demonstrated that Oms38 forms small, water-filled channels with a single-channel conductance of 80 pS in 1 M KCl. The Oms38 channel did not exhibit voltage-dependent closure and is slightly selective for anions with a permeability ratio of cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequence demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions.

Subsequently, a protein homologous to Oms38 was identified in the Lyme disease agents Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii. The pore-forming protein of these species exhibits high sequence homology to Oms38 and similar biophysical properties, i.e. it forms pores of 50 pS in 1 M KCl. Interestingly, titration experiments revealed that this pore could be partly blocked by dicarboxylic anions, which means that this protein does not form a general diffusion pore but a channel with a binding-site specific for those compounds. Consequently, this porin was termed DipA, for “dicarboxylate-specific porin A”.  

In another set of experiments, the channel diameter and the constitution of Borrelia burgdorferi P66 were investigated in detail. Therefor, the P66 single-channel conductance in the presence of different nonelectrolytes with known hydrodynamic radii was analyzed in black lipid bilayers. The effective diameter of the P66 channel lumen was determined to be ~1.9 nm. Furthermore, as derived from multi-channel experiments the P66-induced membrane conductance could be blocked by certain nonelectrolytes, such as PEG 400, PEG 600 and maltohexaose. Additional blocking experiments on the single-channel level revealed seven subconducting states and indicated a heptameric constitution of the P66 channel. This indication could be confirmed by Blue native PAGE and immunoblot analysis which demonstrated that P66 units form a complex with a corresponding mass of approximately 440 kDa.

Taking together, this work describes detailed biochemical and biophysical investigations of both Lyme disease and relapsing fever Borrelia porins and represents an important step forward in understanding the outer membrane pathways for nutrient uptake of these strictly host-dependent, pathogenic spirochetes. Furthermore, it provides some knowledge of the outer-membrane protein composition of Borrelia spirochetes. Anyway, further studies, including genetic manipulations, growth experiments and X-ray analyses of protein crystals, remain to be done to understand the structure and functionality of Borrelia porins in detail. A profound knowledge of surface-exposed proteins, such as porins, is one precondition for the production of a successful vaccine and the drug design against the two borrelian-caused diseases.