Consortium leader: Professor SVEN BERGSTRÖM,
Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden, Tel.: +46 (0)90 7856726, sven.bergstrom@molbiol.umu.se

Partner of the consortium:
Professor Matti Viljanen,
Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland, Tel.: +358-2-3337330, email: matti.viljanen@utu.fi , homepage: http://www.utu.fi/research/tic/viljanen/

Reserchers in Sweden:
- Christer Larsson, Department of Molecular Biology, Umeå University, christer.larsson@molbiol.umu.se
- Marie Andersson, Department of Molecular Biology, Umeå University, marie.andersson@molbiol.umu.se
- Pär Comstedt, Department of Molecular Biology, Umeå University, par.comstedt@molbiol.umu.se
- Betty Guo, Department of Molecular Biology, Umeå University, bettu.guo@molbiol.umu.se
- Annika Nordstrand, Department of Molecular Biology, Umeå University, annika.nordstrand@molbiol.umu.se

Researchers in Finland:
- Juha Suhonen, Department of Medical Microbiology, University of Turku
- Heta Yrjänäinen, Department of Medical Microbiology, University of Turku
- Pauliina Hartiala, Department of Medical Microbiology, University of Turku
- Jemiina Neuvonen, Department of Medical Microbiology, University of Turku
- Jukka Hytönen, Department of Medical Microbiology, University of Turku
- Jenni Pelkonen, Department of Medical Microbiology, University of Turku
- Markus Penttinen, Department of Medical Microbiology, University of Turku
- Jarmo Oksi, Department of Medicine, University of Turku
- Helena Tuominen-Gustafsson, Department of Medical Microbiology, University of Turku

Bergström's results:

The main aim of this project was to gain knowledge of the interactions between Borrelia spirochetes and the host during infection. We have been using both the Lyme borreliosis and the relapsing fever borreliosis spirochetes as model organisms. We have particularly investigated how the Borrelia spirochetes can circumvent the immunological defence, how they spread form the infectious focus, reach various sites in the mammalian body, and how the spirochetes can live in these tissues at a dormant state, reactivate, return to the circulatory system and cause acute disease again. We also aimed to characterize and define the components involved in the interactions between Borrelia and human cells, including the cells of the innate and adaptive immunity.

Adhesion of Borrelia to endothelium is mediated by specific integrin binding.
This project will further extended to identify outer membrane proteins, i.e. P66, P13, OspA-C etc in pore formation, adhesion to host cells, and tissue tropism. Structural characteristics of some of these proteins are known and will be used to design inhibitory compounds for the binding and interaction process. P66 (Oms66) is a chromosomally-encoded 66-kDa integral outer membrane protein of the Lyme disease agent Borrelia burgdorferi. To further characterize this protein we have investigated the oligomeric state and channel-forming activity of native P66. We purified P66 by FPLC to homogeneity and demonstrated that the native protein has channel-forming activity with a single-channel conductance of about 11.5 nS in 1 M KCl using the planar lipid bilayer assay. Further characterization of the channels formed by P66 suggested that it is a slightly anion-selective and voltage-dependent channel. That P66 is oligomeric was shown by cross-linking with glutaraldehyde. In addition, the channel-forming activity was blocked after incubation of P66 with polyclonal antibodies. P66 channel-forming activity was identified in outer membrane fractions of both non-infectious (B313, B31-A, and HB19) and infectious (N40) B. burgdorferi strains. Furthermore, we inactivated the p66 gene in the B31-A (WT) strain and two other channel-forming protein mutant strains, P13-18 (Dp13), and Dbba01, by allelic-exchange mutagenesis. Characterization of the p66 knock-out strains HB19/K02, HB19/K04, B31-A/Dp66,  Dp13/Dp66 and Dbba01/Dp66 revealed that they lacked 11.5-nS channel-forming activity. A construct with the p66 gene on a shuttle vector was used to complement the channel-forming activity, and the regained 11-nS channel-forming activity obtained from this complementation was abolished after pre-incubation with polyclonal antiserum. In conclusion, using genetic and biochemical methods we have confirmed that the integrin-binding, integral outer membrane protein P66 of Borrelia is also a porin.

Additionally, the Borrelia species that causes relapsing fever binds and aggregates erythrocytes as a possible additional mechanism for evasion of the immune system. A potential adhesin involved in this aggregation has been identified. Pore forming assays and aggregation assays will be used to measure the inhibitory effects of test compounds chemical inhibitors that block the function of Borrelia species. Thus, we have demonstrated in earlier studies that some species of relapsing fever Borrelia adhere to erythrocytes, causing the formation of erythrocyte rosettes. This aggregate of Borrelia and red blood cells may protect the spirochetes and contribute to the delayed immune response seen in rosette-forming strains compared non-rosette-forming strains. Mice infected with a rosette-forming strain exhibited more severe pathology and reduced blood flow compared to mice infected with a non-rosette-forming strain.  Therefore, adhesins and receptors involved in this interaction would lead to possible therapeutic tools against relapsing fever. We have now identified a 27 kDa Borrelia protein that binds to a component of human erythrocyte membranes. We are in the process of purifying this protein in order to capture the erythrocyte receptor by affinity chromatography and subsequently identify it by mass spectrometry. A Borrelia library will be constructed and screened using the purified receptor. ¨

Borrelia use host proteases to spread from the infection focus to blood, and to invade distant organs. The initial studies concerning this part were published in 2001(Nordstrand A., et al. "Delayed kidney and brain invasion by Borrelia crocidurae in plasminogen knock-out mice". Infect Immun 2001, 69: 5832-5839). Further protease evaluation is described below, where B. crocidurae, B. hermsii and B. duttoni are investigated in relation to invasion capability and characteristics. A murine model has been established to test Borrelia-mediated activation of host proteases and modulation of the immune responses in the induction of abortion associated with relapsing fever borreliosis. Immune cell subsets and the role of proteases are being investigated. So far matrix metalloproteases and plasmin have been tested. However, no up-regulation has been documented so far, although other proteases will be tested.

Relapsing fever borreliosis caused by Borrelia duttonii is a common cause of pregnancy complications, miscarriage and neonatal death in sub-Saharan Africa. To study the pathological development of these complications, we established a murine model of gestational relapsing fever infection. We demonstrate that B. duttonii infection during pregnancy results in intrauterine growth retardation as well as placental damage and inflammation, impaired fetal circulation and decreased maternal hemoglobin level. We show that spirochetes frequently cross the maternal-fetal barrier, resulting in a congenital infection. Further, we compare infection severity in pregnant and non-pregnant animals, showing that pregnancy has a protective effect. This model closely parallels the consequences of human gestational infection and our results provide insight into the mechanisms behind the pregnancy complications reported in human relapsing fever infection (submitted manuscript).

Borrelia may, by crossing very tight barriers, such as the blood-testis and blood-brain barriers, invade organs and use these as reservoirs for an extensive length of time. Reactivation of infection can occur from these sites. Results indicate that B. duttoni are able to reside in the immune privileged organ brain an extensive time after their disappearance from blood, and can be reactivated to appear in blood by immunosuppresing conditions. This appears to be a unique feature of this species. Relapsing fever, is generally considered a transient, self-limiting disease in humans. However, our studies reveals that murine infection by B. duttonii can be reactivated after an extended time as a silent infection in the brain, with no bacteria appearing in the blood and spirochete load comparable to the numbers in an infected tick. The host cerebral gene expression pattern is indistinguishable from that of uninfected animals, indicating that persistent bacteria are not recognized by the immune system nor cause noticeable tissue damage. Silent infection can be reactivated by immunosuppression, inducing spirochetemia comparable to that of initial densities. B. duttonii has never been found in any host except man and the tick vector. We therefore propose the brain to be a possible natural reservoir of the spirochete. The view of relapsing fever as an acute disease should be extended to include in some cases prolonged persistence, a feature characteristic of the related spirochetal infections Lyme disease and syphilis. Interestingly, we have also found that the RF Borrelia species tested in this project has a great ability to pass the blood-placenta barrier indicating an effective mechanism to also pass this important physical barrier to an immune privileged site.

Selected publications
Widhe M, Jarefors S, Ekerfelt C, Vrethem M, Bergström S, Forsberg P and Ernerudh J. Borrelia specific IFN-g and IL-4 secretion in CSF and blood during the course of Human Lyme borreliosis: relation to clinical outcome. J Inf Dis 2004, 189: 1881-1891

Östberg Y., Carrol JM, Pinne M, Rosa P and Bergström S. Pleiotropic effects of inactivating a carboxyl-terminal protease, CtpA, in Borrelia burgdorferi. J. Bacteriol. 2004 186: 2074-2084

Pinne M, Östberg Y, Comstedt P, and Bergström S. Molecular analysis of the channel-forming protein P13 and its paralog family 48 from different Lyme disease Borrelia species. Microbiology. 2004, 150: 549-559

Östberg Y, Bunikis I, Bergström S and Johansson J  The etiological agent of Lyme disease, Borrelia burgdorferi, appears to contain only a few small RNA molecules. J Bacteriol. 2004 186:8472-8477

Alitalo A, Meri T, Comstedt P, Jeffrey LA, Tornberg J, Strandin T, Lankinen H, Bergström S, Cinco M, Vuppala S, Akins D, and Meri S (2005 ) Expression of complement factor H binding proteins in Borrelia garinii isolated from patients with neuroborreliosis. Eur J Immunol., 35:3043-53

M. Pinne, K. Denker, E. Nilsson, R. Benz, and S. Bergström. 2006. The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13. Accepted in J Bacteriol

Viljanen's results

We have previously shown that contact with B. burgdorferi induces maturation and IL-8 production of immature dendritic cells (DCs) in a similar manner as contact with LPS, a known maturation inducer. However, gene expression studies are essential to investigate in more detail the possibility that borreliae somehow manipulate DCs to their benefit. We have now completed the microarray experiments where we determined the global gene expression profiles of borrelia-stimulated and -unstimulated DCs, and compared the borrelia-induced changes in DC gene expression to the effects of LPS. Changes in gene expression were analysed in four time points, and each time point was done in triplicate resulting in 36 microarray hybridizations with two technical repeats. We have identified the differential expression of up to several hundred genes, many of which are important regulators of immune response. Confirmatory experiments with quantitative PCR and protein arrays are underway. From our point of view, the most interesting observation was that borrelia does not induce upregulation of the gene of CD38 on DCs as LPS does. CD38 plays a central role in the chemotaxis of DCs into inflamed tissues and in the migration of DCs into local lymph nodes after antigen contact. By evading CD38 upregulation, borrelia could spread in the body without encountering efficient defense. This would be a completely novel mechanism for microbes to interfere with crucial stages of immune response. Our further studies will focus on role of CD38 regulation on DCs in immune evasion by borrelia and microbes in general.

We have succeeded in establishing animal models for the studies on Lyme arthritis and borrelial latency by infecting SJL or C3H/He mice with B. garinii Å218 or B. burgdorferi s.s. N40 bacteria and by treating the animals with ceftriaxone. Our results suggest that the presence of vegetative spirochetes is no prerequisite for the persisting symptoms. However, we have also found out that when the mice are treated with anti-TNFa after a latent period of up to four weeks, borreliae are activated from a dormant state and the animals develop spirochetemia. The tissues where borreliae reside will be identified by culture or PCR, fluorescence imaging, and by position emission tomography (PET) scanning. The genomes of borreliae recovered from the dormant state are analysed through hybridization on micro-array slides covering the whole borrelia genome to find out the genetic alterations taking place during dormancy. These experiments will provide essential information concerning the pathogenesis of and possible therapeutic approaches in antibiotic treatment resistant Lyme arthritis and in borreliosis in general. The results may also have an impact on the understanding of other persistent infections like tuberculosis, chlamydia infections and certain viral diseases.

Selected publications:

Tuominen-Gustafsson H, Penttinen M, Hytönen J, Viljanen MK. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) staining of Borrelia burgdorferi sensu lato offers benefits in studies on interaction with human neutrophils. Submitted.

Yrjänäinen H, Hytönen J, Söderström K-O, Oksi J, Hartiala K, Viljanen MK. Persistent joint swelling and borrelia-specific antibodies in Borrelia garinii infected mice after eradication of vegetative spirochetes with antibiotic treatment. Microbes and Infection, accepted.

Yrjänäinen H, Hytönen J, Song XR, Oksi J, Hartiala K, Viljanen MK. Anti-TNF-a-treatment activates Borrelia burgdorferi infection in ceftriaxone treated C3H/He mice. Submitted.

An abstract of the research plan (January 2003)