Umeå University Medical Dissertation, New series No 1719
Structure and function of the Borrelia burgdorferi porins, P13 and P66
Mari Bonde
Akademisk avhandling
som med vederbörligt tillstånd från Rektor vid Umeå universitet för avläggande av filosofie doktorsexamen framläggs till offentligt försvar i sal E04, byggnad 6E, NUS,
fredagen den 8 maj 2015, kl. 13.00
Avhandlingen kommer att försvaras på engelska
Fakultetsopponent: Associate Professor Melissa J. Caimano University of Connecticut Health Center Farmington, Connecticut, USA
Department of Molecular Biology Umeå Center for Microbial Research (UCMR)
Laboratory for Molecular Infection Medicine Sweden (MIMS) Umeå University
Umeå 2015
Organization Document type Date of publication Umeå University Doctoral thesis 17 April 2015 Department of Molecular Biology
Author Mari Bonde Title
Structure and function of the Borrelia burgdorferi porins, P13 and P66 Abstract
Borrelia burgdorferi is a helically shaped bacterium with a parasitic life style, always present within a host. The bacterium is transmitted by means of infected ticks to small rodents or birds that usually serve as reservoirs. Infected ticks can bite humans and there the bacterium cause Lyme disease. Early infection involves the skin and a in many cases a typical skin rash called erytema migrans, can be seen at the site of the tick bite.
If left untreated the disease can cause impairments of various organs such as joints, heart and nervous system.
Porins are water-filled channels situated in the outer membrane of bacteria. These proteins are important in the sieving mechanism of the outer membrane, allowing important compounds to pass into the bacterial cell, while excluding harmful substances. Borrelia spp. are limited in metabolic and synthetic pathways and are therefore highly dependent on their surrounding to supply them with important compounds. Because of this, porins are believed to be very important for Borrelia spp.
Except a role in nutrient acquisition, porins can function as ashesins and in bacterial adaptation to new environments with variations in osmotic pressure.
P13 and P66 are two integral outer membrane proteins in B. burgdorferi, previously seen to have porin activities. In addition to the porin activity, P66 is also an adhesin, binding integrins present on for instance epithelial cells in the human body. In this thesis structural characterisations and functional studies of P13 and P66 were assessed. In the Black lipid bilayer assay, the pore forming activity of P13 was shown to be much smaller than previously thought, exhibiting activity at 0.6 nS with a channel size of 1.3 nm. Initial Nanodisc experiments displayed a pore size of 1.3 nm, confirming the Black lipid bilayer results. The 300 kDa protein complex formed by P13 contained sole P13 monomeres. P66 forms pores with a single channel conductance of 11 nS and a channel size of 1.9 nm. The porin assembles into a large protein complex of 420 kDa in the outer membrane, containing exclusively P66 monomers. The integrin-binding function of P66 was found to be important for efficient bacterial dissemination in the murine host but was not essential for B. burgdorferi infectivity. Neither P13 nor P66 had an active role in osmotic stress adaptation. Instead, two p13 paralogs were up- regulated at the transcript level in B. burgdorferi cultured under glycerol-induced osmotic stress.
Keywords
Borrelia, P13, P66, porin, protein complex, dissemination, osmotic stress Language ISBN ISSN Number of pages English 978-91-7601-264-2 0346-6612 58+5 papers
