1. Bacterial Virulence Factors Dongwoo Shin Laboratory of Molecular Bacteriology Department of Molecular Cell Biology Sungkyunkwan University School of Medicine

2. What is a pathogen? -An organism capable of colonizing a host organism where the interaction results in disease Bacteria and Disease -opportunistic pathogens -strict pathogens

3. Bacteria and Disease Establishing connection: Koch’s Postulates Proving cause and effect in infectious disease research -first raised in the 1800s by Robert Koch Koch’s postulates -association of the bacteria with the lesions of the disease -isolating the bacterium in pure culture -showing that the isolated bacterium causes disease in humans or animals -reisolating the bacterium from the intentionally infected animal

4. Bacteria and Disease What is virulence (pathogenicity)? -The capacity of a pathogen to cause damage or disease in the host -Virulence factors: Cell wall components, DNA, Proteins

5. Bacteria and Disease Virulence factors: Extracytoplasmic components - Cell wall components: Teichoic (Lipoteichoic) acid, LPS - Lipoproteins - Capsule - Pili

6. Bacteria and Disease Virulence factors: Extracytoplasmic components - Cell wall structure: Gram (+) bacteria

7. Bacteria and Disease Virulence factors: Extracytoplasmic components - Gram (+) bacteria cell wall: Teichoic acid (polymers of modified ribose or glycerol connected by phosphate

8. Bacteria and Disease Virulence factors: Extracytoplasmic components - Cell wall structure: Gram (-) bacteria

9. Lipopolysacchride (LPS) -Somatic O polysacchride + Core polysaccharide + Lipid A Bacteria and Disease

10. Virulence factors: Various proteins with different functions

11. Bacteria and Disease Biology of Salmonella Infection

12. Bacteria and Disease “Environmental Signals inside Host” Expression of Necessary Proteins (Virulence Proteins) in the Correct Tissues Virulence Genes

13. Genetic screenings of bacterial virulence factors 1.Pre-genomic era: 1990’s 2.Post-genomic era: 21C

14. In Vivo Expression Technology (IVET) 1.Isolation of Salmonella genes whose expression is induced inside the host (i.e. genes whose products are necessary for host infection) 2.Auxotrophic selection method (Science, 1993) and Differential fluorescence induction method (Science, 1997)

15. Auxotrophic selection method Step 1: Creating transcriptional fusions of random fragments of the Salmonella chromosome with a promoterless purA gene; introduction of this library into a purA mutant a mutant that cannot synthesize purines (auxotroph)

16. Auxotrophic selection method Step 2: Integration of a plasmid construct into chromosome of a purA mutant via single crossover

17. Auxotrophic selection method Step 3: Host infection with the pool of fusion strains and selection X-gal plate

18. Differential fluorescence induction (DFI) Rationale: trapping the gene promoters that are activated inside macrophages; using GFP macrophage Activation of mgtC transcription Salmonella PmgtC gfp

19. Differential fluorescence induction (DFI) Step 1: Cloning of random fragments of Salmonella chromosome into a promoterless gfp plasmid; introduction of plasmids into Salmonella

20. Differential fluorescence induction (DFI) Step 2: Infection of macrophages with Salmonella harboring gfp fusion plasmids; sorting GFP-active Salmonella with FACS

21. Validation of a screened candidate for virulence 1.In vitro method: Gentamicin protection assay for evaluations of Salmonella invasion into and survival within host cells 2.In vivo method: Animal experiments for evaluations of Salmonella’s ability to infect host

22. Validation of a screened candidate for virulence - Gentamicin protection assay: Infection of epithelial cells (e.g. Hep-2) or macrophages (e.g. J774.A) with Salmonella Incubation allowing for Salmonella to invade into epithelial cells or for macrophages to engulf Salmonella (i.e. phagocytosis) Gentamicin treatment to kill bacteria outside host cells Detergent treatment to lyse host cells; plating onto agar plate to count Salmonella

23. Validation of a screened candidate for virulence - Gentamicin protection assay: a mutant that cannot produce SPI-2 TTSS

24. Validation of a screened candidate for virulence - Animal experiments: Mouse infection model - Oral infection and Intraperitoneal infection - Immunocompromised mouse and Immunocompetent mouse

25. Validation of a screened candidate for virulence - Animal experiments:

26. Genetic screenings of bacterial virulence factors 1.Pre-genomic era: 1990’s 2.Post-genomic era: 21C

27. Announcement of Salmonella genome sequence: Nature (2000) “Now, one can predict which genes are important for Salmonella virulence and experimentally test them.”

28. In this paper, the authors evaluated the role of every single transcription factor in Salmonella virulence

31. -A revolutionary method for construction gene deletion mutants in E. coli: PNAS (2000) -Applicable to other enteric bacteria: Salmonella, Klebsiella, Yersinia, Enterobacter etc. -Accurate, fast, and cheap method