Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle

Authors Organisations
  • Tom A. Mendum(Author)
    University of Surrey
  • Aneesh Chandran(Author)
    University of Surrey
  • Kerstin Williams(Author)
    University of Surrey
  • Hans-Martin Vordermeier(Author)
    Animal and Plant Health Agency
  • Bernardo Villarreal-Ramos(Author)
    Animal and Plant Health Agency
  • H. Wu(Author)
    University of Surrey
  • Albel Singh(Author)
    University of Birmingham
  • Alex A. Smith(Author)
    University of Surrey
  • Rachel E. Butler(Author)
    University of Surrey
  • Aravind Prasad(Author)
    Centre for Development of Advanced Computing, India
  • Neeraj Bharti(Author)
    Centre for Development of Advanced Computing, India
  • Ruma Banerjee(Author)
    Centre for Development of Advanced Computing, India
  • Sunitha M. Kasibhatla(Author)
    Centre for Development of Advanced Computing, India
  • Apoorva Bhatt(Author)
    University of Birmingham
  • Graham R. Stewart(Author)
    University of Surrey
  • Johnjoe McFadden(Author)
    University of Surrey
Type Article
Original languageEnglish
Article number431
JournalBMC Genomics
Volume20
Issue number1
DOI
Publication statusPublished - 28 May 2019
Externally publishedYes
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Abstract

Background

BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria.

Results

To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host.

Conclusion

These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy.

Keywords

  • Cattle, Cyclopropane-fatty-acyl-phospholipid synthase, Mycobacterium bovis BCG, Mycobacterium tuberculosis, Pyruvate carboxylase, Rv1006, Transposon library

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