Erythrocyte-aggregating relapsing fever spirochete Borrelia crocidurae induces formation of microemboli.

The African relapsing fever spirochete Borrelia crocidurae forms aggregates with erythrocytes, resulting in a delayed immune response. Mice were infected with B. crocidurae and monitored during 50 days after infection. Spirochetes were observed extravascularly at day 2 after infection. Two days later, inflammatory responses, cell death, and tissue damage were evident. The pathologic responses in lungs and kidneys were similar, whereas the symptoms in the brains were delayed, with a less pronounced inflammatory response. Microemboli were found in the blood vessels, possibly a result of the erythrocyte aggregation. The B. crocidurae invasion emerged more rapidly than has been described for Lyme disease-causing Borrelia species. In addition to erythrocyte rosetting, the presence of extravascular B. crocidurae indicates a novel route for these bacteria to propagate and cause damage in the mammalian host. The histopathologic findings in this study may explain the clinical manifestations of human relapsing fever.

Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes.

A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before.

The spirochete Borrelia crocidurae causes erythrocyte rosetting during relapsing fever.

Several species of the genus Borrelia exhibit antigenic variation of variable major proteins on their surface during relapsing fever. We have investigated the African relapsing fever species Borrelia crocidurae during infections in mice and compared it with the thoroughly studied North American species Borrelia hermsii. A major difference between the two species is that B. crocidurae can bind and become completely covered with erythrocytes. In addition, B. crocidurae causes a prolonged spirochetemia which coincides with a delayed appearance of antiborrelial antibodies. We show that the antibody response against an unrelated antigen is not delayed and that antibiotic treatment, which dissociates rosettes and inhibits the spirochetes, also leads to an early antibody response. Taken together, the erythrocyte aggregation and prolonged spirochetemia hint at a new mode of immune evasion where erythrocyte-covered spirochetes may avoid contact with the phagocytic cells and B cells of the immune system, thereby delaying the onset of a specific immune response.

Expression of the flagellin gene in Borrelia is controlled by an alternative sigma factor.

The flagellin genes from six Borrelia species were cloned, sequenced and characterized at the molecular level. The flagellin genes of two relapsing fever Borrelia species, B. hermsii and B. crocidurae, three Lyme disease genomic species, B. burgdorferi, B. afzelii and B. garinii, and the avian borreliosis agent, B. anserina, were compared and showed an 85-93% sequence identity to each other. Comparison of the fla genes from the different Lyme borreliosis spirochaetes revealed that they were 94-99% identical. Nucleotide sequencing of the fla gene and primer extension on isolated mRNA from both B. hermsii (as transcribed in Escherichia coli) and B. burgdorferi (as transcribed in the natural host) identified the putative transcriptional start points, the ribosomebinding sites and the promoter regions of these genes. The deduced promoter of the Borrelia flagellin gene resembled neither the sigma 70 promoter of prokaryotes, as seen for the genes for the outer-surface proteins A and B in Lyme disease Borrelia and the genes for the variable major proteins 7 and 21 of B. hermsii, nor the sigma 28 consensus promoter region of motility genes from other bacteria. Instead, the promoter of the fla gene in Borrelia has most similarity to the bacteriophage SP01 sigma gp33-34 promoter sequence of Bacillus subtilis.

Antigenic variation and strain heterogeneity in Borrelia spp.

Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i.e. B. burgdorferi and the relapsing fever borreliae. In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i.e. from inner sites to telomeric sites of the plasmid). As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon. In B. burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease. Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC). There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon. With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B. burgdorferi is immunologically and genetically more heterogeneous, as previously believed. The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level. The same degree of genetic non-identity was observed for the pC protein. Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity. Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E. coli and these proteins are immunologically reactive. This allows further research for development of vaccines and diagnostic tools. B. burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors. Comparison of the different methods has shown good agreement when the same strains have been investigated. No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice. A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed. Serotyping a large number of B. burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular characterization of Borrelia burgdorferi isolated from Ixodes ricinus in northern Sweden.

Ixodes ricinus ticks, harbouring Borrelia burgdorferi, were found in an area in northern Sweden, not thought to be endemic for Lyme borreliosis. This investigation took place at Norrbyskär, an island situated in the Bothnian Gulf, 63 degrees 33'N/19 degrees 52'E. One of 42 nymphal and 8/43 adult I. ricinus ticks collected carried spirochetes as seen by phase contrast microscopy. Pure bacterial cultures were obtained from 2 of the ticks. Western blot analysis using species-specific monoclonal antibodies showed that the isolated spirochetes were B. burgdorferi. The identity of the isolated spirochetes was confirmed by DNA amplification using B. burgdorferi OspA and flagellin gene specific oligonucleotides as well as partial DNA sequencing of the respective OspA and flagellin genes. The 2 isolated spirochaete populations were different as shown by their protein profiles in sodium dodecyl sulphate polyacrylamide gels. Moreover, the demonstration of Lyme borreliosis in a patient from the island of Norrbyskär indicates the need for clinical consideration of this disease in northern Sweden.

Variable antigen genes of the relapsing fever agent Borrelia hermsii are activated by promoter addition.

Borrelia hermsii, an agent of relapsing fever, avoids the host's immune response by means of multiphasic antigenic variation. Serotype specificity is determined by variable antigens called the Vmp lipoproteins. Through recombination between linear plasmids a formerly silent vmp gene replaces another vmp gene at a telomeric expression locus. We examined strain HS1 borreliae before and after a switch from serotype 7 to serotype 21. The nucleotide sequences of 5' regions of silent and expressed vmp7 and vmp21 were determined. Silent and active vmp7 and vmp21 genes shared a block of homologous sequences surrounding their 5' ends. Sequences upstream of silent vmp7 and vmp21 genes lacked the promoter and substantially differed from each other. In this antigenic switch a vmp gene was activated by a recombination that placed it downstream of a promoter.

Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome.

The relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer-membrane proteins, known as Vmp proteins. The genes for the variant-specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2 kD) and 364 amino acids (37.1 kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome.