Sara Garbom, Åke Forsberg, Hans Wolf-Watz, and Britt-Marie Kihlberg 2004, Infection and Immunity



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Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes

  • Sara Garbom, Åke Forsberg, Hans Wolf-Watz, and Britt-Marie Kihlberg

  • 2004, Infection and Immunity, v. 72

  • pp. 1333-1340


Hypothesis

  • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host}



Hypothesis

  • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host}

  • THEN{Genes expressed in vivo and shared by pathogens may be “amenable” targets for antibacterial agents}



Why target in vivo expressed virulence factors?



Why target in vivo expressed virulence factors?



Why target in vivo expressed virulence factors?



Method:



Method:

  • In silico: Find novel putative virulence genes through comparative analysis

  • In vitro: Assay genes for essentiality to survival



Method:

  • In silico: Find novel putative virulence genes through comparative analysis

  • In vitro: Assay genes for essentiality to survival

  • In vivo: Assay genes for virulence in an animal model



Goal:

  • “the rapid emergence of multiply [antibiotic] resistant bacterial strains…demands the development of new antibacterial agents by engaging strategies that specifically counteract the development of resistance”



In silico:



In silico:

  • Gathered genes of unknown function from a pathogenic organism

    • “Conserved hypotheticals” or “unknown”
  • Compared these genes to those of other pathogens



In silico:

  • Gathered genes of unknown function from a pathogenic organism

    • “Conserved hypotheticals” or “unknown”
  • Compared these genes to those of other pathogens

  • Considered all genes found in all pathogens “virulence-associated genes (vag)”











Control:



Control:

  • 99 in vivo expressed genes

    • STM (signature tagged mutagenesis) and “selected capture of transcribed sequences”
  • Compared to (same) 6 genomes



Control:

  • 99 in vivo expressed genes

    • STM (signature tagged mutagenesis) and “selected capture of transcribed sequences”
  • Compared to (same) 6 genomes

  • 5 conserved genes classified as vir genes

    • Also conserved among many bacteria
    • No human homologues


In vitro:



In vitro:

  • Mutagenized conserved genes

    • Insertion mutagenesis
  • Analyzed cytotoxicity with HeLa cells



In vitro:

  • Mutagenized conserved genes

    • Insertion mutagenesis
  • Analyzed cytotoxicity with HeLa cells

  • Measured Yop secretion

    • Yersinia outer proteins
    • Known virulence factors
    • Encoded on a plasmid
    • Belonging to a type III secretion system


Hypothesized:



Hypothesized:

  • 3 mutations were lethal

  • 14 remaining mutants

    • vagE - impaired growth / uncharacteristic morphology / delayed cytotoxic response*
    • vagH - lowered Yops secretion
    • vagI - lowered Yops secretion but no loss of cytotoxicity


Hypothesized:

  • 3 mutations were lethal

  • 14 remaining mutants

    • vagE - impaired growth / uncharacteristic morphology / delayed cytotoxic response*
    • vagH - lowered Yops secretion
    • vagI - lowered Yops secretion but no loss of cytotoxicity
  • 11 “indistinguishable from the wild type”



In vivo:

  • Infected model organisms with mutagenized strains

    • Oral infection of mice


In vivo:

  • Infected model organisms with mutagenized strains

    • Oral infection of mice
  • Lethal vs. non-lethal/delayed-lethal classification of virulence

    • WT killed 50% mice at 107 CFU/mL in 5-8 days
    • “Attenuated” strains were not lethal at same dose


Hypothesized:

  • 5 were virulent

  • Control:

  • 2 were virulent



Hypothesized:

  • 5 were virulent

  • 9 were attenuated

  • Control:

  • 2 were virulent

  • 3 were attenuated



In vivo:



In vivo:

  • In-frame deletion mutagenesis

    • Prevent downstream effects of insertion mutagenesis
  • Meant to verify results of insertion mutagenesis



Hypothesized:



Hypothesized:



Hypothesized:

  • 1 deletion mutant could not be made

  • 3 mutants regained virulence

    • Genes in virulence-associated operons
  • 5 mutants remained attenuated

    • 1 of these having exhibited non-WT like growth (in vitro)


Hypothesized:

  • 1 deletion mutant could not be made

  • 3 mutants regained virulence

    • Genes in virulence-associated operons
  • 5 mutants remained attenuated

    • 1 of these having exhibited non-WT like growth (in vitro)
  • 4~5 in vivo-only virulence genes were successfully discovered

  • Control:

  • 3 remain attenuated



Experimental Control

  • 211 genes initially considered



Experimental Control

  • 211 genes initially considered

  • 17 (8%) conserved across pathogens



Experimental Control



Experimental Control

  • 211 genes initially considered

  • 17 (8%) conserved across pathogens

  • 9 (4%) in or around virulence genes

  • 5 (2%) confirmed virulence genes



Hypothesis

  • IF{Genomes of pathogenic bacteria are reduced to smallest set needed for growth in an animal host}

  • THEN{Genes expressed in vivo and shared by pathogens may be “amenable” targets for antibacterial agents}



Amenable(…



Amenable(…



Amenable(…



Amenable(…

  • Traditional screening not possible

    • Microarrays?


Amenable(…

  • Traditional screening not possible

    • Microarrays?
  • Targeting gene products isn’t as easy as in-frame deletion mutagenesis

    • …especially when human homologues exist for 4 out of 5 of the genes IDed


Amenable(…

  • Traditional screening not possible

    • Microarrays?
  • Targeting gene products isn’t as easy as in-frame deletion mutagenesis

    • …especially when human homologues exist for 4 out of 5 of the genes IDed
  • Response of normal human microflora unknown



Amenable(…

  • Traditional screening not possible

    • Microarrays?
  • Targeting gene products isn’t as easy as in-frame deletion mutagenesis

    • …especially when human homologues exist for 4 out of 5 of the genes IDed
  • Response of normal human microflora unknown



Conclusion

  • Genes responsible for virulence were identified

  • I’m “amenable” to calling the method a success



Why start with T. pallidium when Y. pestis was the organism of interest and Y. pseudotuberculosis was used for testing?

  • Why start with T. pallidium when Y. pestis was the organism of interest and Y. pseudotuberculosis was used for testing?

  • How would deletion mutagenesis of homologous genes in non-pathogens alter their growth?

  • How target-able were the products of the genes knocked out?

    • What’s the best way to assay target-ability of an uncharacterized gene product?
  • Was there any overlap between the set of vag genes and the control (vivo + silico) set?




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