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Structure and Function of the Borrelia burgdorferi Porins, P13 and P66
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Sven Bergström)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Borrelia burgdorferi is an elongated and helically shaped bacterium that is the causal agent of the tick-borne illness Lyme disease. The disease manifests with initial flu-like symptoms and, in many cases, the appearance of a skin rash called erythema migrans at the site of the tick bite. If left untreated the disease might cause impairment of various organs such as the skin, heart, joints and the nervous system. The bacteria have a parasitic lifestyle and are always present within a host. Hosts are usually ticks or different animals and birds that serve as reservoirs for infection. B. burgdorferi are unable to synthesize building blocks for many vital cellular processes and are therefore highly dependent on their surroundings to obtain nutrients. Because of this, porins situated in the outer membrane, involved in nutrient uptake, are believed to be very important for B. burgdorferi. Except for a role in nutrient acquisition, porins can also have a function in binding extracellular matrix proteins, such as integrins, and have also been implicated in bacterial adaptation to new environments with variations in osmotic pressure.

P13 and P66 are two integral outer membrane proteins in B. burgdorferi previously shown to have porin activities. In addition to its porin function, P66 also has integrin binding activity. In this thesis, oligomeric structures formed by the P13 and P66 protein complexes were studied using the Black lipid bilayer technique in combination with nonelectrolytes. Initial attempts were also made to study the structure of P13 in Nanodiscs, whereby membrane proteins can insert into artificial lipid bilayers in their native state and the structure can be analyzed by electron microscopy. In addition, the role of P13 and P66 in B. burgdorferi osmotic stress adaptation was examined and also the importance and role of the integrin-binding activity of P66 in B. burgdorferi infections in mice.

Using Black lipid bilayer studies, the pore forming activity of P13 was shown to be much smaller than previously thought, exhibiting activity at 0.6 nS. The complex formed by P13 was approximately 300 kDa and solely composed of P13 monomers. The channel size was calculated to be roughly 1.4 nm. Initial Nanodisc experiments showed a pore size of 1.3 nm, confirming the pore size determined by Black lipid bilayer experiments. P66 form pores with a single channel conductance of 11 nS and a channel size of 1.9 nm. The porin assembles in the outer membrane into a large protein complex of 420 kDa, 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.

Abstract [sv]

Borrelia burgdorferi är en bakterie med många unika egenskaper som orsakar sjukdomen Lyme borrelios. Borrelia kan idag lätt behandlas med antibiotika om sjukdomen upptäcks i ett tidigt stadium. Det är först om sjukdomen tillåts fortgå som symptom som nervsmärta och ansiktsförlamning kan uppstå och dessutom vara svåra att koppla till en Borrelia-infektion. Multiresistenta bakterier har blivit en stor del av vår vardag och även om Borrelia-bakterierna idag inte är resistenta mot flertalet antibiotika är det kanske speciellt viktigt, innan det är för sent, med forskning som kan leda till upptäckter av unika angreppsställen för nya läkemedel.

Målet med denna avhandling var att studera hur två Borrelia proteiner, P13 och P66, ser ut, är uppbyggda och även vilken funktion de har. Dessa proteiner är tänkbara vaccinkandidater eftersom de sitter i yttre membranet hos bakterierna och sticker ut på ytan mot våra värdceller, vilket gör att vi reagerar mot dem vid en infektion. P13 och P66 är också viktiga kanaler för bakterierna vid upptag av näringsämnen och byggstenar från omgivningen. Ämnen som bakterierna inte kan producera själva. Pga. denna funktion är P13 och P66 tänkbara proteiner för blockering med ett läkemedel som skulle förhindra bakterien från att föröka sig i och med att de förlorar möjligheten att tillgodogöra sig näring. Detta i sin tur skulle leda till att vårt eget immunförsvar hinner rensa undan bakterierna innan infektionen blivit för stor och vi blivit sjuka. P66 har förutom porin funktionen även en adhesions funktion när proteinet kan binda integriner som sitter på olika typer av celler i vår kropp, bl. a. immunceller och epitelceller i våra blodkärl och vävnader. Den integrin bindande funktionen är viktig för bakterierna vid en infektion eftersom det gör det möjligt för bakterierna att binda till våra celler. Ett steg som är viktigt för att de senare ska kunna ta sig ut från blodkärlen till våra vävnader.

P13 och P66 visade sig kunna bilda stora proteinkomplex i ytter membranet hos bakterierna med en storlek på 300 kDa respektive 420 kDa. De är inga specifika poriner som bara kan transportera en viss typ av molekyl med t.ex. en viss laddning, utan kan ombesörja upptaget av många olika typer av ämnen. Eliminering av p66 orsakade att ett annat adhesionsprotein, uppreglerades. En omplacering av ett normalt cytoplasmatiskt lokaliserat chaperon-protein till ytter-membranet hos bakterierna kunde också ses i frånvaro av P66. Chaperonet GroEL har i andra bakterier, bl. a. Helicobacter pylori, bakterien som orsakar magsår, beskrivits som ett protein som kan förflytta sig till ytan av bakterierna och där ha en liknande funktion som P66, dvs. att binda extracellulära matrisprotein. Förändringen i uttryck av adhesionsproteinet och förflyttningen av chaperonet till membranet var en följd av p66-eliminering och mest troligt ett sätt för bakterierna att komplettera den förlorade integrinbindande funktionen av P66.

Det har tidigare visats att poriner är involverade i skyddet mot osmotisk stress i andra bakterier. Denna funktion hos P13 och P66 i Borrelia kunde inte ses när bakterier utsattes för osmotisk stress med glycerol, som orsakar en form av membranstress. Däremot kunde vi med hjälp av transkriptomanalys se att Borrelia-bakterier uppreglerade transkriptionen av två paraloger till P13 vid hyper-osmotisk stress. Borrelia bakteriens användning av dessa paraloga proteiner har tidigare trotts ske enbart i frånvaro av ett funktionellt P13 protein. Nu visade det sig att P13-paraloger har en egen funktion även i närvaro av P13, nämligen att vara involverade i regleringen av hyperosmotisk stress och därmed skydda bakterierna i denna stressituation. Andra gener som påverkades av osmotisk stress med glycerol var gener för stressfaktorer och pumpar i inre membranet hos bakterien.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2015.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1719
Keyword [en]
Borrelia, P13, P66, porin, protein complex, dissemination, osmotic stress
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-101801ISBN: 978-91-7601-264-2 (print)OAI: oai:DiVA.org:umu-101801DiVA: diva2:802842
Public defence
2015-06-13, Major Groove, NUS - Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-04-17 Created: 2015-04-13 Last updated: 2015-05-08Bibliographically approved
List of papers
1. Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin
Open this publication in new window or tab >>Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, e78272- p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library of Science, 2013
Keyword
nutrient uptake, outer membrane transport, Lyme disease, porin radius determination, black lipid bilayer
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-83891 (URN)10.1371/journal.pone.0078272 (DOI)000326656200037 ()
Funder
Swedish Research Council
Available from: 2013-12-11 Created: 2013-12-10 Last updated: 2017-12-06Bibliographically approved
2. Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin
Open this publication in new window or tab >>Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 27, 18614-18624 p.Article in journal (Refereed) Published
Abstract [en]

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 M KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to +/-100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2014
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-91837 (URN)10.1074/jbc.M113.539528 (DOI)000339062900001 ()
Available from: 2014-08-27 Created: 2014-08-18 Last updated: 2017-12-05Bibliographically approved
3. Structural analysis of the B. burgdorferi integral outer membrane protein, P13, in lipid bilayer Nanodiscs
Open this publication in new window or tab >>Structural analysis of the B. burgdorferi integral outer membrane protein, P13, in lipid bilayer Nanodiscs
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-101950 (URN)
Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2015-12-21Bibliographically approved
4. Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity
Open this publication in new window or tab >>Integrin binding by Borrelia burgdorferi P66 facilitates dissemination but is not required for infectivity
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2015 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 17, no 7, 1021-1036 p.Article in journal (Refereed) Published
Abstract [en]

P66, a Borrelia burgdorferi surface protein with porin and integrin-binding activities, is essential for murine infection. The role of P66 integrin-binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202–208). Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αvβ3. Integrin-binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post-subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202–208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post-inoculation, but bacterial burdens recovered to wild-type levels at 4 weeks post-inoculation. The delay in tissue colonization correlated with reduced migration of the Del202–208 strains across microvascular endothelial cells, similar to Δp66bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-101796 (URN)10.1111/cmi.12418 (DOI)000356605900008 ()
Available from: 2015-04-13 Created: 2015-04-13 Last updated: 2017-12-04Bibliographically approved
5. Effects of osmotic stress in P13 and P66 deficient Borrelia burgdorferi mutants
Open this publication in new window or tab >>Effects of osmotic stress in P13 and P66 deficient Borrelia burgdorferi mutants
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-101951 (URN)
Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2015-04-17Bibliographically approved

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