[show abstract][hide abstract] ABSTRACT: Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the sigma(N)-sigma(S) regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.
Infection and immunity 07/2008; 76(6):2498-511. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Members of the Borrelia burgdorferi paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from re-isolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in Borrelia residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for B. burgdorferi dissemination, tissue tropism, or evasion of the adaptive immune response.
Infection and Immunity 07/2007; 75(6):2753-64. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Results of previous immunological studies suggested that Borrelia burgdorferi regulates synthesis of the IpLA7 lipoprotein during mammalian infection. Through combined use of quantitative reverse transcription PCR, immunofluorescence analyses, ELISA and immunoblotting, it is now demonstrated that IpLA7 is actually expressed throughout mammalian infection, as well as during transmission both from feeding ticks to naïve mice and from infected mice to naïve, feeding ticks. However, proportions of IpLA7-expressing B. burgdorferi within tick midguts declined significantly with time following completion of blood feeding. Cultured bacteria differentially expressed IpLA7 in response to changes in temperature, pH and concentration of 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2, indicative of mechanisms governing IpLA7 expression. Previous studies also reported mixed results as to the cellular localization of IpLA7. It is now demonstrated that IpLA7 localizes primarily to the borrelial inner membrane and is not surface-exposed, consistent with the ability of these bacteria to produce IpLA7 throughout mammalian infection despite being the target of a robust immune response.
[show abstract][hide abstract] ABSTRACT: When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT-PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian-like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb-infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out-compete for binding. bba66 promoter mapping revealed putative sigma70 and sigmaS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the -35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either sigmaN or sigmaS resulted in loss of BBA66. Promoter-GFP fusion analysis indicated that the sigma70 and/or sigmaS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.