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?

VirulentWT

DeadWT

Traditional Antibiotic

VirulentMutant

Why target in vivo expressed virulence factors?

VirulentWT

DeadWT

Traditional Antibiotic

VirulentMutant

Why target in vivo expressed virulence factors?

VirulentWT

Virulence-specific Antibiotic

AvirulentMutant

VirulentWT

DeadWT

Traditional Antibiotic

VirulentMutant

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”

Finding novel putative virulence genes through comparative analysis

In silico:

Gathered genes of unknown function from a pathogenic organism “Conserved hypotheticals” or “unknown”

Compared these genes to those of other pathogens

Finding novel putative virulence genes through comparative analysis

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)”

Finding novel putative virulence genes through comparative analysis

Organism Disease

Treponema pallidum Syphilis

Yersinia pestis Black death

Neisseria gonorrhoeae Gonorrhea

Heliobacter pylori Peptic ulcer disease

Borrelia bugdoreferi Lyme disease

Streptococcus pneumoniae

Pneumococcal meningitis

Pneumonia

“With the the exception of Y. pestis, all are causitive agents of chronic disease in humans.”

Organism Genes remaining

Treponema pallidum 211

Yersinia pestis

Neisseria gonorrhoeae

Heliobacter pylori

Borrelia bugdoreferi

Streptococcus pneumoniae

Organism Genes remaining

Treponema pallidum 211

Yersinia pestis 73

Neisseria gonorrhoeae

Heliobacter pylori

Borrelia bugdoreferi

Streptococcus pneumoniae

Organism Genes remaining

Treponema pallidum 211

Yersinia pestis 73

Neisseria gonorrhoeae

17Heliobacter pylori

Borrelia bugdoreferi

Streptococcus pneumoniae

Classified vagA – vagQ“[NCBI nr] database indicated that all of the vag genes exhibited homologous sequences in at least 35 other microorganisms… nine had products that also exhibited similarity [to human proteins].”

99 in vivo expressed genes STM (signature tagged mutagenesis) and

“selected capture of transcribed sequences”

In vivo analysis & in silico comparison

Control:

99 in vivo expressed genes STM (signature tagged mutagenesis) and

“selected capture of transcribed sequences” Compared to (same) 6 genomes

In vivo analysis & in silico comparison

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 vivo analysis & in silico comparison

Control:

In vitro:

Mutagenized conserved genes Insertion mutagenesis

Assaying genes for essentiality to survival and virulence

In vitro:

Mutagenized conserved genes Insertion mutagenesis

Analyzed cytotoxicity with HeLa cells

Assaying genes for essentiality to survival and virulence

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

Assaying genes for essentiality to survival and virulence

3 mutations were lethal

Hypothesized:Unchanged in vitro growth patterns

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:Unchanged in vitro growth patterns

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”

Hypothesized:Unchanged in vitro growth patterns

In vivo:

Infected model organisms with mutagenized strains Oral infection of mice

Assaying genes for virulence in an animal model

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

Assaying genes for virulence in an animal model

5 were virulent

Control: 2 were virulent

Hypothesized:Viable targets would be attenuated for virulence

5 were virulent 9 were attenuated

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

Control: 2 were virulent 3 were attenuated

Hypothesized:Viable targets would be attenuated for virulence

In vivo:

In-frame deletion mutagenesis Prevent downstream effects of insertion

mutagenesis

Assaying genes for virulence in an animal model (continued)

In vivo:

In-frame deletion mutagenesis Prevent downstream effects of insertion

mutagenesis Meant to verify results of insertion

mutagenesis

Assaying genes for virulence in an animal model (continued)

1 deletion mutant could not be made

Hypothesized:Viable targets would still be attenuated for virulence

1 deletion mutant could not be made 3 mutants regained virulence

Genes in virulence-associated operons

Hypothesized:Viable targets would still be attenuated for virulence

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:Viable targets would still be attenuated for virulence

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

Hypothesized:Viable targets would still be attenuated for virulence

Experimental Control

211 genes initially considered

99 genes initially considered

Experimental Control

211 genes initially considered

17 (8%) conserved across pathogens

99 genes initially considered

5 (5%) conserved across pathogens

Experimental Control

211 genes initially considered

17 (8%) conserved across pathogens

9 (4%) in or around virulence genes

99 genes initially considered

5 (5%) conserved across pathogens

3 (3%) 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

99 genes initially considered

5 (5%) conserved across pathogens

3 (3%) in or around virulence genes

3 (3%) 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(…

VirulentWT

DeadWT

Traditional Antibiotic

VirulentMutant

Amenable(…

VirulentWT

Virulence-specific Antibiotic

AvirulentMutant

VirulentWT

DeadWT

Traditional Antibiotic

VirulentMutant

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?

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?