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Bacterial Virulence Factors
Dongwoo Shin
Laboratory of Molecular BacteriologyDepartment of Molecular Cell Biology
Sungkyunkwan University School of Medicine
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
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
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
Bacteria and Disease
Virulence factors: Extracytoplasmic components
- Cell wall components: Teichoic (Lipoteichoic) acid, LPS
- Lipoproteins
- Capsule
- Pili
Bacteria and Disease
Virulence factors: Extracytoplasmic components
- Cell wall structure: Gram (+) bacteria
Bacteria and Disease
Virulence factors: Extracytoplasmic components
- Gram (+) bacteria cell wall: Teichoic acid (polymers of modified ribose or glycerol connected by phosphate
Bacteria and Disease
Virulence factors: Extracytoplasmic components
- Cell wall structure: Gram (-) bacteria
Lipopolysacchride (LPS)
-Somatic O polysacchride + Core polysaccharide + Lipid A
Bacteria and Disease
Bacteria and Disease
Virulence factors: Various proteins with different functions
Bacteria and Disease
Biology of Salmonella Infection
Bacteria and Disease
“Environmental Signals inside Host”
Expression of Necessary Proteins Expression of Necessary Proteins (Virulence Proteins) in the Correct Tissues(Virulence Proteins) in the Correct Tissues
Virulence Genes
Genetic screenings of bacterial virulence factors
1. Pre-genomic era: 1990’s
2. Post-genomic era: 21C
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)
Auxotrophic selection method
Step 1: Creating transcriptional fusions of random fragments of the Salmonella chromosome with a promoterless purA gene; introducti
on of this library into a purA mutant
a mutant that cannot synthesize purines (auxotroph)
Auxotrophic selection method
Step 2: Integration of a plasmid construct into chromosome of a purA
mutant via single crossover
Auxotrophic selection method
Step 3: Host infection with the pool of fusion strains and selection
X-gal plate
Differential fluorescence induction (DFI)
Rationale: trapping the gene promoters that are activated inside macrophages; using GFP
macrophage
Activation of mgtC transcription
Salmonella
PmgtC gfp
Differential fluorescence induction (DFI)
Step 1: Cloning of random fragments of Salmonella chromosome into a promoterless gfp plasmid; introduction of plasmids into Salmon
ella
Differential fluorescence induction (DFI)
Step 2: Infection of macrophages with Salmonella harboring gfp fusio
n plasmids; sorting GFP-active Salmonella with FACS
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 Salmonell
a’s ability to infect host
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 coun
t Salmonella
Validation of a screened candidate for virulence
- Gentamicin protection assay:
a mutant that cannot produce SPI-2 TTSS
Validation of a screened candidate for virulence
- Animal experiments: Mouse infection model
- Oral infection and Intraperitoneal infection
- Immunocompromised mouse and Immunocompetent mouse
Validation of a screened candidate for virulence
- Animal experiments:
Genetic screenings of bacterial virulence factors
1. Pre-genomic era: 1990’s
2. Post-genomic era: 21C
Announcement of Salmonella genome sequence: Nature (2000)
“Now, one can predict which genes are important for Salmonella virulence and experimentally test them.”
In this paper, the authors evaluated the role of every single transcription factor in Salmonella virulence
- 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