bdrF2 of Lyme Disease Spirochetes Is Coexpressed with a Series of Cytoplasmic Proteins and Is Produced Specifically during Early Infection

ABSTRACT The Bdr proteins are polymorphic inner membrane proteins produced by most Borrelia species. In Borrelia burgdorferi B31MI, the18 bdr genes form three subfamilies, bdrD, bdrE, and bdrF. The production of at least one of the Bdr paralogs, BdrF2, is up-regulated in host-adapted spirochetes, suggesting a role for the protein in the mammalian environment. Here, we demonstrate using reverse transcriptase (RT) PCR that BBG29, BBG30, BBG31, and BBG32, which reside upstream of bdrF2, are cotranscribed with bdrF2 as a five-gene operon. While the functions of most of these proteins are unknown, BBG32 encodes a putative DNA helicase. Real-time RT-PCR analyses demonstrated higher levels of bdrF2 transcript relative to other genes of the operon, suggesting that bdrF2 may also be transcribed independently from an internal promoter. Internal promoters were detected using the 5′ rapid amplification of cDNA ends system. The putative promoter associated with bdrF2 was found to be highly similar in sequence to the multiple promoters associated with the ospC gene. Real-time RT-PCR analyses, performed to assess the expression of these genes in infected mice, revealed that genes of the bdrF2 locus are expressed only during early infection, suggesting a role in the establishment of infection. To further characterize the proteins encoded by the bdrF2 locus, which have unknown functions, the cellular localizations of these proteins were determined by Triton X-114 extraction and phase partitioning. BBG29 and BBG31 were found to be cytoplasmic. To determine if these proteins elicit an antibody (Ab) response during infection, immunoblot analyses were performed. Abs to these proteins were not detected. Based on the analyses presented here, we offer the hypothesis that BdrF2 and other proteins encoded by the operon form an inner-membrane-associated protein complex that may interact with DNA and which carries out its functional role during transmission or the early stages of infection.

[1]  Gregory A. Price,et al.  Demonstration of the Genetic Stability and Temporal Expression of Select Members of the Lyme Disease Spirochete OspF Protein Family during Infection in Mice , 2001, Infection and Immunity.

[2]  G. Baranton,et al.  Linear chromosome of Borrelia burgdorferi. , 1989, Research in microbiology.

[3]  D. Roberts,et al.  Evolutionary and molecular analyses of the Borrelia bdr super gene family: delineation of distinct sub-families and demonstration of the genus wide conservation of putative functional domains, structural properties and repeat motifs. , 2000, Microbial pathogenesis.

[4]  F. Liang,et al.  An Immune Evasion Mechanism for Spirochetal Persistence in Lyme Borreliosis , 2002, The Journal of experimental medicine.

[5]  D. Roberts,et al.  Environmental Regulation and Differential Production of Members of the Bdr Protein Family of Borrelia burgdorferi , 2002, Infection and Immunity.

[6]  R. Marconi,et al.  Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes , 1993, Journal of bacteriology.

[7]  M. Lovett,et al.  Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114 , 1988, Journal of bacteriology.

[8]  Ruth R. Montgomery,et al.  Temporal pattern of Borrelia burgdorferi p21 expression in ticks and the mammalian host. , 1997, The Journal of clinical investigation.

[9]  R. Marconi,et al.  Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange , 1994, Journal of bacteriology.

[10]  J. Radolf,et al.  Decorin-Binding Protein of Borrelia burgdorferi Is Encoded within a Two-Gene Operon and Is Protective in the Murine Model of Lyme Borreliosis , 1998, Infection and Immunity.

[11]  W. Zückert,et al.  Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element , 1996, Journal of bacteriology.

[12]  S. Salzberg,et al.  Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi , 1997, Nature.

[13]  D. Roberts,et al.  Molecular and Immunological Analyses of theBorrelia turicatae Bdr Protein Family , 2000, Infection and Immunity.

[14]  S. Casjens,et al.  Profiling of Temperature-Induced Changes in Borrelia burgdorferi Gene Expression by Using Whole Genome Arrays , 2003, Infection and Immunity.

[15]  Andrew T. Revel,et al.  DNA microarray analysis of differential gene expression in Borrelia burgdorferi, the Lyme disease spirochete , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  E. Fikrig,et al.  Borrelia burgdorferi transcriptome in the central nervous system of non-human primates , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Norgard,et al.  Regulation of Expression of the Paralogous Mlp Family in Borrelia burgdorferi , 2003, Infection and Immunity.

[18]  J. D. Young Underreporting of Lyme disease. , 1998, The New England journal of medicine.

[19]  A. Barbour,et al.  Biology of Borrelia species. , 1986, Microbiological reviews.

[20]  M. Li,et al.  Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family , 1996, Journal of bacteriology.

[21]  F. Cabello,et al.  Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN–RpoS regulatory pathway , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Barbour,et al.  Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends. , 1987, Science.

[23]  T. Schwan,et al.  Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Radolf,et al.  Regulation of OspE-Related, OspF-Related, and Elp Lipoproteins of Borrelia burgdorferi Strain 297 by Mammalian Host-Specific Signals , 2001, Infection and Immunity.

[25]  P. B. Donzis Corneal ulcers from contact lenses during travel to remote areas. , 1998, The New England journal of medicine.

[26]  S. Wikel,et al.  Changes in Temporal and Spatial Patterns of Outer Surface Lipoprotein Expression Generate Population Heterogeneity and Antigenic Diversity in the Lyme Disease Spirochete, Borrelia burgdorferi , 2002, Infection and Immunity.

[27]  R. Marconi,et al.  Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete Borrelia hermsii , 2004, Journal of bacteriology.

[28]  A. Barbour,et al.  Cross‐species hybridization of a Borrelia burgdorferi DNA array reveals infection‐ and culture‐associated genes of the unsequenced genome of the relapsing fever agent Borrelia hermsii , 2003, Molecular microbiology.

[29]  D. Roberts,et al.  Analysis of the Cellular Localization of Bdr Paralogs in Borrelia burgdorferi, a Causative Agent of Lyme Disease: Evidence for Functional Diversity , 2000, Journal of bacteriology.

[30]  J. Carlyon,et al.  Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from Borrelia turicatae, a Causative Agent of Relapsing Fever , 1998, Journal of bacteriology.

[31]  J. Radolf,et al.  Identification, Characterization, and Expression of Three New Members of the Borrelia burgdorferi Mlp (2.9) Lipoprotein Gene Family , 1999, Infection and Immunity.

[32]  P. S. Hefty,et al.  Global Analysis of Borrelia burgdorferi Genes Regulated by Mammalian Host-Specific Signals , 2003, Infection and Immunity.

[33]  D. Roberts,et al.  The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution. , 2000, Emerging infectious diseases.