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Selection of Borrelia burgdorferi Promoter Sequences Active During Mammalian Infection Using In Vivo Expression Technology

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1690)

Abstract

In vivo expression technology (IVET) has been applied to a variety of organisms to identify active promoters in specific environments or growth conditions of interest. Here, we describe modifications to employ this genome-wide screening method for Borrelia burgdorferi, the Lyme disease spirochete, during an active murine infection. Utilization of this technique provides valuable insights into the B. burgdorferi transcriptome during infection, despite the low bacterial numbers in the mammalian host environment.

Key words

Borrelia burgdorferi Lyme disease In vivo expression technology Promoter trap Genomic library Pathogenicity Gene expression Mammalian infection Transcriptome 

Notes

Acknowledgment

Thank you to Dr. Andrew Camilli, Dr. Patti Rosa and members of the Rosa lab for initial guidance on development of IVET for B. burgdorferi. Thank you to members of the Jewett lab for all of their hard work on this project. In particular, we recognize the invaluable contributions of Angelika Linowski and Dr. Sunny Jain. This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers K22AI081730 and R01AI099094 to M.W.J.

References

  1. 1.
    Mahan MJ, Slauch JM, Hanna PC et al (1993) Selection for bacterial genes that are specifically induced in host tissues: the hunt for virulence factors. Infect Agents Dis 2(4):263–268PubMedGoogle Scholar
  2. 2.
    Mahan MJ, Slauch JM, Mekalanos JJ (1993) Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259(5095):686–688CrossRefPubMedGoogle Scholar
  3. 3.
    Slauch JM, Camilli A (2000) IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues. Methods Enzymol 326:73–96CrossRefPubMedGoogle Scholar
  4. 4.
    Jackson RW, Giddens SR (2006) Development and application of in vivo expression technology (IVET) for analysing microbial gene expression in complex environments. Infect Disord Drug Targets 6(3):207–240CrossRefPubMedGoogle Scholar
  5. 5.
    Hanin A, Sava I, Bao Y et al (2010) Screening of in vivo activated genes in Enterococcus faecalis during insect and mouse infections and growth in urine. PLoS One 5(7):e11879. doi: 10.1371/journal.pone.0011879 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lee SW, Cooksey DA (2000) Genes expressed in Pseudomonas putida during colonization of a plant-pathogenic fungus. Appl Environ Microbiol 66(7):2764–2772CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Mendez J, Reimundo P, Perez-Pascual D et al (2011) A novel cdsAB operon is involved in the uptake of L-cysteine and participates in the pathogenesis of Yersinia ruckeri. J Bacteriol 193(4):944–951. doi: 10.1128/JB.01058-10. JB.01058-10 [pii]CrossRefPubMedGoogle Scholar
  8. 8.
    Ellis TC, Jain S, Linowski AK et al (2014) In Vivo expression technology identifies a novel virulence factor critical for Borrelia burgdorferi persistence in mice. Plos Pathog 10(6):e1004260. doi: 10.1371/journal.ppat.1004260. ARTN e1004260CrossRefPubMedGoogle Scholar
  9. 9.
    Adams PP, Flores Avile C, Popitsch N et al (2017) In vivo expression technology and 5′ end mapping of the Borrelia burgdorferi transcriptome identify novel RNAs expressed during mammalian infection. Nucleic Acids Res 45(2):775–792. doi: 10.1093/nar/gkw1180 CrossRefPubMedGoogle Scholar
  10. 10.
    Radolf JD, Caimano MJ, Stevenson B et al (2012) Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10(2):87–99. doi: 10.1038/nrmicro2714 PubMedPubMedCentralGoogle Scholar
  11. 11.
    Bestor A, Stewart PE, Jewett MW et al (2010) Use of the Cre-lox recombination system to investigate the lp54 gene requirement in the infectious cycle of Borrelia burgdorferi. Infect Immun 78(6):2397–2407. doi: 10.1128/IAI.01059-09. IAI.01059-09 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jewett MW, Jain S, Linowski AK et al (2011) Molecular characterization of the Borrelia burgdorferi in vivo-essential protein PncA. Microbiology 157(Pt 10):2831–2840. doi: 10.1099/mic.0.051706-0 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Purser JE, Lawrenz MB, Caimano MJ et al (2003) A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host. Mol Microbiol 48(3):753–764CrossRefPubMedGoogle Scholar
  14. 14.
    Ramamoorthy R, McClain NA, Gautam A et al (2005) Expression of the bmpB gene of Borrelia burgdorferi is modulated by two distinct transcription termination events. J Bacteriol 187(8):2592–2600. doi: 10.1128/JB.187.8.2592-2600.2005 187/8/2592. [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Rosa PA, Tilly K, Stewart PE (2005) The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3(2):129–143. doi: 10.1038/nrmicro1086. nrmicro1086 [pii]CrossRefPubMedGoogle Scholar
  16. 16.
    Rego RO, Bestor A, Rosa PA (2011) Defining the plasmid-borne restriction-modification systems of the Lyme disease spirochete Borrelia burgdorferi. J Bacteriol 193(5):1161–1171. doi: 10.1128/JB.01176-10. JB.01176-10 [pii]CrossRefPubMedGoogle Scholar
  17. 17.
    Casselli T, Bankhead T (2015) Use of in vivo expression Technology for the Identification of putative host adaptation factors of the Lyme disease spirochete. J Mol Microbiol Biotechnol 25(5):349–361. doi: 10.1159/000439305 CrossRefPubMedGoogle Scholar
  18. 18.
    Barbour AG (1984) Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57(4):521–525PubMedPubMedCentralGoogle Scholar
  19. 19.
    Rosa PA, Hogan D (1992) Colony formation by Borrelia burgdorferi in solid medium: clonal analysis of osp locus variants. In: Munderloh UG, Kurtti TJ (eds) Proceeding of the first international conference on tick borne pathogens at the host-vector Interface. University of Minnesota, St. Paul, Minnesota, pp 95–103Google Scholar
  20. 20.
    Elias AF, Stewart PE, Grimm D et al (2002) Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect Immun 70(4):2139–2150CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Grundemann D, Schomig E (1996) Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21(5):898–903PubMedGoogle Scholar
  22. 22.
    Samuels DS (1995) Electrotransformation of the spirochete Borrelia burgdorferi. In: Nickoloff JA (ed) Methods in molecular biology, Electroporation protocols for microorgansisms, vol 47. Humana Press, Inc., Totowa, N.J, pp 253–259Google Scholar
  23. 23.
    Rego RO, Bestor A, Stefka J et al (2014) Population bottlenecks during the infectious cycle of the Lyme disease spirochete Borrelia burgdorferi. PLoS One 9(6):e101009. doi: 10.1371/journal.pone.0101009 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Troy EB, Lin T, Gao L et al (2013) Understanding barriers to Borrelia burgdorferi dissemination during infection using massively parallel sequencing. Infect Immun 81:2347–2357. doi: 10.1128/IAI.00266-13 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Immunity and Pathogenesis, Burnett School of Biomedical SciencesUniversity of Central Florida College of MedicineOrlandoUSA

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