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

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:

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

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:

• Gathered genes of unknown function from a pathogenic organism

• “Conserved hypotheticals” or “unknown”

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:

• 99 in vivo expressed genes

• STM (signature tagged mutagenesis) and “selected capture of transcribed sequences”

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:

• Mutagenized conserved genes

• Insertion mutagenesis

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:

• 3 mutations were lethal

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

• All 3 non-WT like (in vitro) mutants were attenuated

• Control:

• 2 were virulent

• 3 were attenuated

In vivo:

• In-frame deletion mutagenesis

• Prevent downstream effects of insertion mutagenesis

In vivo:

• In-frame deletion mutagenesis

• Prevent downstream effects of insertion mutagenesis

• Meant to verify results of insertion mutagenesis

Hypothesized:

• 1 deletion mutant could not be made

Hypothesized:

• 1 deletion mutant could not be made

• 3 mutants regained virulence

• Genes in virulence-associated operons

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

• 211 genes initially considered

• 17 (8%) conserved across pathogens

• 9 (4%) in or around virulence genes

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(…

• Traditional screening not possible

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?