Renate Fuchs

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22kDa protein (pC) in Escherichia coli

We describe the cloning and expression of the pc gene which encodes a major immunodominant protein of Borrelia burgdorferi, the causative agent of Lyme borreliosis. The pC protein was purified from lysates of B. burgdorferi strain PKo. After tryptic digestion of the pC protein the resulting oligopeptides were applied to a gas‐phase sequenator. Thus partial amino acid sequences were obtained. The deduced oligonucleotides were used as hybridization probes. After Southern blotting a reactive band in the 3 kb range of PstI‐digested genomic DNA was detected. The insertion of these fragments into pUC vectors finally resulted in pc‐positive Escherichia coli clones. The gene (encoding a protein with 212 amino acids) was expressed in E. coli with varying deletions at the 5′ end. A sequence comparison with other outer membrane proteins of B. burgdorferi indicates a processing of pC that is similar to that of lipoproteins.

Cross-reactive proteins ofBorrelia burgdorferi

The specificity of serological tests for Lyme borreliosis is impaired by cross-reacting antibodies. In order to select antigens for more specific tests, specific and cross-reactive proteins ofBorrelia burgdorferi must be identified. Therefore, to analyze cross reactions ofBorrelia burgdorferi with other bacteria, rabbit immune sera against heterologous bacteria (Borrelia hermsii, Treponema pallidum, Treponema phagedenis, Leptospira interrogans (serogroupgrippotyphosa),Neisseria meningitidis, Haemophilus influenzae, Yersinia enterocolitica (serotypes O3 and O9),Campylobacter jejuni, Listeria monocytogenes O1,Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Shigella flexneri andLegionella micdadei) were examined by Western blot usingBorrelia burgdorferi as antigen. Broad cross reactivity was shown forBorrelia proteins of the 60–75 kDa range. Other broadly cross-reacting proteins were at the level of p40, p33 and two proteins in the range of 20 kDa. Some of the cross reactions were eliminated by absorption of the sera withTreponema phagedenis. The absorbed antibodies were directed mainly against bands at the level of p33 and bands of the 60 to 75 kDa range. Showing the lowest potential for cross reactivity, p100, p41, OspA and pC seem to be the most suitable antigens for serodiagnosis. In contrast to p100 and OspA, however, p41 and pC showed cross reactivity with immune sera against bacteria not belonging to the genusBorrelia.

Purification of the Borrelia burgdorferi flagellum by use of a monoclonal antibody.

The flagellum associated polypeptide p41 is an immunodominant antigen of Borrelia burgdorferi in the early and late stages of Lyme borreliosis. p41 was prepared by affinity chromatography using monoclonal antibody specific for p41. An immunoglobulin class specific ELISA (IgM-, IgG-ELISA) was established with purified p41 as antigen and compared to the conventional ELISA with whole cell ultrasonic antigen. Whereas the sensitivity of IgM- and IgG-ELISA was comparable in both antigen preparations, crossreactivity of sera from syphilitic patients was reduced in the p41 IgG-ELISA. Discrepant results obtained by use of ultrasonic antigen or p41 antigen, were controlled by Western blots. A correlation between the results of p41-ELISA and Western blot was shown.