Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi.

The gene of the immunodominant major protein pC of Borrelia burgdorferi was previously cloned and sequenced (R. Fuchs, S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, and E. Soutschek, Mol. Microbiol. 6:503-509, 1992). pC is abundantly expressed on the outer surface of B. burgdorferi, as demonstrated by immunoelectron microscopy with monoclonal antibody L22 1F8. Accordingly, pC is renamed OspC, by analogy to the outer surface proteins OspA and OspB. Western immunoblot analysis of 45 B. burgdorferi isolates with monoclonal antibodies revealed that OspC is immunologically heterogeneous. Partial sequence analysis of the ospC gene confirmed the protein heterogeneity at the genetic level. We found that the degree of identity between the ospC partial sequences of five strains representing different OspA serotypes was only 63.3 to 85.4%. Immunological heterogeneity was also observed among representatives of the three newly designated genospecies of B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, and group VS461. Heterogeneity was confirmed for B. garinii at the genetic level. The ospC gene was also cloned from strains that did not express OspC, and antibody-reactive OspC was expressed in Escherichia coli. In addition, OspC-expressing variants were obtained from a nonexpressing strain by plating single colonies on solid medium. These findings confirm that the ospC gene is also present in nonexpressing strains. Because OspC is an immunodominant protein for the early immune response in Lyme borreliosis and was effective as a vaccine in an animal model, the immunological and molecular polymorphisms of ospC and OspC have important implications for the development of diagnostic reagents and vaccines.

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.

Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection.

Serious infection due to Borrelia burgdorferi and the disseminated infection characteristic of the disease possess unique treatment problems. The wide and still increasing incidence of Lyme borreliosis as well as the problems in treatment call for effective prevention strategies by active immunization. Vaccination experiments were done to determine if active immunization of gerbils with recombinant OspA and pC protects against infection with strains of B. burgdorferi. Gerbils were vaccinated with recombinant OspA and pC (20 kDa protein) and challenged four weeks later with a clone (derived from B. burgdorferi strain PKo) which expresses an abundant amount of pC but only little OspA. Non-immunized gerbils challenged with the same B. burgdorferi strain were used as controls. Both groups of immunized gerbils developed antibodies against the recombinant vaccines. The pC vaccinated group was protected against infection, whereas the OspA vaccinated group showed signs of infection. The non-vaccinated group developed generalised infection. These results show that pC should be considered as a further vaccine candidate and probably needs to be combined with OspA for an efficient vaccine against B. burgdorferi.

Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa 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.

Molecular analysis of the outer surface protein A (OspA) of Borrelia burgdorferi for conserved and variable antibody binding domains.

The outer surface protein A (OspA) of Borrelia burgdorferi is a major candidate for development of a borrelia vaccine. However, vaccine development may be aggravated by the immunological heterogeneity of OspA. In this respect the knowledge about conserved and variable epitopes is of major interest. In this study truncated proteins derived from two different OspA serotypes of B. burgdorferi were mapped for conserved and specific antibody-binding domains. The OspA fragments were reacted in the Western blot with eight different OspA-specific monoclonal antibodies recognizing between one and seven of the seven OspA serotypes previously described. The two broadly reacting antibodies (recognizing all serotypes) react with N-terminal fragments of 93 and 214 amino acids, respectively, whereas antibodies recognizing only one and two to four of the seven serotypes are reactive with C-terminal fragments of amino acid 143-273 and 109-273, respectively. Thus, conserved antibody-binding domains are located nearer to the N terminus than serotype-specific ones. Comparison of the results from western blot mapping with OspA sequence data suggested certain conserved or variable regions as probable candidates for antigenic sites involved in linear or conformationally dependent epitopes. This, however, needs to be confirmed by epitope mapping using the respective synthetic peptides.

Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): identification of catalytic and cellulose-binding domains.

The nucleotide sequence of the celZ gene coding for a thermostable endo-beta-1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 bp which encodes a preprotein of 986 amino acids with an Mr of 109,000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fluorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.

Xylan degradation by the thermophile Clostridium stercorarium: cloning and expression of xylanase, beta-D-xylosidase, and alpha-L-arabinofuranosidase genes in Escherichia coli.

Seven genes related to arabinoxylan degradation were isolated from a genomic library of the thermophilic bacterium Clostridium stercorarium. The cloned genes include a xylanase gene (xynA), two beta-D-xylosidase genes (bx1A and bx1B), two alpha-L-arabinofuranosidase genes (arfA and arfB), and two genes (celW and celX) encoding enzymes termed celloxylanases, which hydrolyze both xylans and beta-D-cellobiosides. The genes xynA, celX, and bxlB were found to encode the major xylanolytic enzyme activities induced by growth of C. stercorarium on xylan.