Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

Abstract

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.

Characterization of Stress and Innate Immunity Resistance of Wild-Type and Δp66 Borrelia burgdorferi

“…One known in vitro function of P66 is pore formation in the bacterial outer membrane (39). While the diameter of the pore has been estimated experimentally, 1.9 nm with a central constriction of 0.8 nm, it is still unclear what physiologically relevant substrates pass through the pore (45). Gram-negative bacteria use porins to allow diffusion of nutrients across the outer membrane.…”

“…The integrin binding function of P66 facilitates both transmigration out of the vasculature and bacterial dissemination but is not critical to establish infection (43,44). Although the size of the pore has been determined (opening diameter of 1.9 nm with a central constriction of 0.8 nm) (45), the in vivo importance of the pore formed by P66 remains unknown. These data, taken together, suggest that P66 functions to help B. burgdorferi evade the host innate immune system.…”

Borrelia burgdorferi HtrA: evidence for twofold proteolysis of outer membrane protein p66

“…P66, a chromosomally encoded 66 kDa integral membrane protein of B. burgdorferi, has homologs in the related Lyme borreliosis (LB)-causing Borrelia species B. afzelii and B. garinii in Europe and other LB-causing Borrelia species from Europe and South America (Bunikis et al, 1995;1998;Madden, 2002), as well as a number of relapsing fever Borrelia worldwide (Madden, 2002;Barcena-Uribarri et al, 2010). The outer membrane organization of p66 is oligomeric (Barcena-Uribarri et al, 2013;Kenedy et al, 2014), where individual monomers form outer membrane β-barrel structures (Kenedy et al, 2014). Predicted β-sheet regions in p66 suggestive of A. HtrA cleavage sites in p66 at 30 min (arrows).…”

Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

“…To check the integrity of the eluted P13 complex, the protein was loaded on a second and even a third BN-PAGE that displayed the same molecular mass and, therefore, retained its native state. It is noteworthy that the P66 complex previously eluted from BN-PAGE also retained its pore-forming activity after elution from BN-PAGE (52).…”