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

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? Virulent WT Dead WT Traditional Antibiotic Virulent Mutant

Why target in vivo expressed virulence factors? Virulent WT Dead WT Traditional Antibiotic Virulent Mutant

Why target in vivo expressed virulence factors? Virulent WT Virulence-specific Antibiotic Avirulent Mutant Virulent WT Dead WT Traditional Antibiotic Virulent Mutant

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

OrganismDisease Treponema pallidumSyphilis Yersinia pestisBlack death Neisseria gonorrhoeaeGonorrhea Heliobacter pyloriPeptic ulcer disease Borrelia bugdoreferiLyme disease Streptococcus pneumoniae Pneumococcal meningitis Pneumonia “With the the exception of Y. pestis, all are causitive agents of chronic disease in humans.”

OrganismGenes remaining Treponema pallidum 211 Yersinia pestis Neisseria gonorrhoeae Heliobacter pylori Borrelia bugdoreferi Streptococcus pneumoniae

OrganismGenes remaining Treponema pallidum 211 Yersinia pestis 73 Neisseria gonorrhoeae Heliobacter pylori Borrelia bugdoreferi Streptococcus pneumoniae

OrganismGenes remaining Treponema pallidum 211 Yersinia pestis 73 Neisseria gonorrhoeae 17 Heliobacter 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 10 7 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

ExperimentalControl  211 genes initially considered  99 genes initially considered

ExperimentalControl  211 genes initially considered  17 (8%) conserved across pathogens  99 genes initially considered  5 (5%) conserved across pathogens

ExperimentalControl  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

ExperimentalControl  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(… Virulent WT Dead WT Traditional Antibiotic Virulent Mutant

Amenable(… Virulent WT Virulence-specific Antibiotic Avirulent Mutant Virulent WT Dead WT Traditional Antibiotic Virulent Mutant

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?