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Genetic Screenings for Studying Bacterial Pathogenesis
Dongwoo Shin, Ph.D.
Associate Professor, Department of Molecular Cell Biology, Sungkyunkwan University School
of Medicine
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Structure of a Bacterial Cell
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What is a pathogen?
-An organism capable of colonizing a host organism where the interaction results in disease
-opportunistic pathogens
-strict pathogens
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Opportunistic pathogens
- Most of infections
- Normal microbiota for causing disease
- No disease in normal setting but disease when introduced into unprotected sites (e.g. blood, tissues)
- Immunocompromised patients are more susceptible Staphylococcus aureus
Escherichia coli
Strict pathogens
- A few infections Mycobacterim tuberculosis (tuberculosis)
Neisseria gonorrhoeae (gonorrhea)
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Exposure of an individual to an organism
1.Transient (hours or days) colonization
2.Permanent colonization
3.Disease production (Infection)
[NOTE] Colonization vs. Infection
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In a healthy human,
- The internal tissues (e.g. brain, blood, muscles): normally free of microorganisms
- Conversely, the surface tissues (e.g. skin and mucous membrane): constantly in contact with environmental microorganisms and become colonized by certain microbial species
- Normal microbiota (= microflora, normal flora): mixture of microorganisms regularly found at any anatomical site
- Bacteria make up most of the normal microbiota over the fungi and protozoa
[NOTE] Normal microbiota (microflora)
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What is virulence (pathogenicity)?
-The capacity of a pathogen to cause damage or disease in the host
-Virulence factors: Cell wall components (LPS, LTA), DNA, Proteins
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Lipopolysacchride (LPS)
-Somatic O polysacchride + Core polysaccharide + Lipid A
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What is virulence (pathogenicity)?
-The capacity of a pathogen to cause damage or disease in the host
-Virulence factors: Cell wall components, DNA, Proteins
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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
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Establishing connection: Molecular Koch’s Postulates
• Raised in the 1988 by Stanley Falkow
• Molecular Koch’s postulates
-gene (or its product) should be found only in strains of bacteria that cause the disease
-gene should be “isolated” by cloning
-disruption of gene in virulent strain should reduce virulence
-gene is expressed by bacterium during infectious process in animal or human
Bacteria and Disease
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Identification of bacterial virulence factors
1. Understanding the molecular strategies used by a pathogen during host infection
2. Providing the targets in development of novel therapeutics for bacterial infection
-Currently used antibiotics → Targeting bacterial viability → Selective pressure
-Antivirulence therapy
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Salmonella
Escherichia coliEscherichia coli
ShigellaShigella
Salmonella entericaSalmonella enterica
Salmonella bongoriSalmonella bongori
EnterobacteriaceaeEnterobacteriaceae
Typhimurium Typhimurium EnteritidisEnteritidisTyphiTyphiParatyphiParatyphi
SerovarsSerovars
GastroenteritisGastroenteritis
Systemic diseaseSystemic disease
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Complex Lifestyle of Salmonella
Soil, water, food Host: Humans and animals
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Salmonella enterica serovar Typhimurium
A facultative intracellular pathogen
Infects millions of people worldwide every year resulting in ~500,000 deaths
Transmission via contaminated food or water
Gastroenteritis (Human) and Typhoid fever (Mouse)
Serves as a model system for other intracellular pathogens
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Biology of Salmonella Infection
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“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
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Invasion into epithelial cells Invasion into epithelial cells of small intestineof small intestine
Survival inside macrophagesSurvival inside macrophages
Systemic diseaseSystemic disease
StomachStomach
Type III Secretion Systems (TTSSs)
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Expression of SPI-1 Genes Mediates Salmonella-Invasion into Host Cells
Expression of ~30 genes in Expression of ~30 genes in SalmonellaSalmonella Pathogenecity Island 1 (SPI-1) Pathogenecity Island 1 (SPI-1)
Type III Secretion System Type III Secretion System
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SPI-1 TTSS-Induced Changes in Host Cells
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Invasion into epithelial cells Invasion into epithelial cells of small intestineof small intestine
Survival inside macrophagesSurvival inside macrophages
Systemic diseaseSystemic disease
StomachStomach
Type III Secretion Systems (TTSSs)
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Phagosome-lysosome fusionPhagosome-lysosome fusionROS & RNS productionROS & RNS production
Antimicrobial peptidesAntimicrobial peptides
Bacterial Bacterial KillingKilling
PhagosomePhagosome
Bacterial killing processes inside phagosome
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Salmonella
Host cellseffector proteins
secretion system
Induction of the SPI-2 T3SS within Macrophage
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Alteration of vesicle Alteration of vesicle traffickingtrafficking
LPS modificationLPS modification
Preventing recruitment Preventing recruitment of NADH oxidaseof NADH oxidase
SPI-2 TTSSSPI-2 TTSS
PhagosomePhagosome
Phagosome-lysosome fusionPhagosome-lysosome fusionROS & RNS productionROS & RNS production
Antimicrobial peptidesAntimicrobial peptides
The SPI-2 T3SS prevents bacterial killing by macrophages
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Genetic screenings of bacterial virulence factors
1. Pre-genomic era: 1990’s
2. Post-genomic era: 21C
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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)
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Auxotrophic selection method
Step 1: Creating transcriptional fusions of random fragments of the Salmonella chromosome with promoterless purA and lacZ genes;
introduction of this library into a purA mutant
a mutant that cannot synthesize purines (auxotroph)
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Auxotrophic selection method
Step 2: Integration of a plasmid construct into chromosome of a purA
mutant via single crossover
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Auxotrophic selection method
Step 3: Host infection with the pool of fusion strains and selection
X-gal plate
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Differential fluorescence induction (DFI)
Rationale: trapping the gene promoters that are activated inside macrophages; using GFP
macrophage
Activation of mgtC transcription
Salmonella
PmgtC gfp
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Differential fluorescence induction (DFI)
Step 1: Cloning of random fragments of Salmonella chromosome into a promoterless gfp plasmid; introduction of plasmids into
Salmonella
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Differential fluorescence induction (DFI)
Step 2: Infection of macrophages with Salmonella harboring gfp
fusion plasmids; sorting GFP-active Salmonella with FACS
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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
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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
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Validation of a screened candidate for virulence
- Gentamicin protection assay:
a mutant that cannot produce SPI-2 TTSS
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Validation of a screened candidate for virulence
- Animal experiments: Mouse infection model
- Oral infection and Intraperitoneal infection
- Immunocompromised mouse and Immunocompetent mouse
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Validation of a screened candidate for virulence
- Animal experiments:
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Genetic screenings of bacterial virulence factors
1. Pre-genomic era: 1990’s
2. Post-genomic era: 21C
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Announcement of Salmonella genome sequence: Nature (2000)
“Now, one can predict which genes are important for Salmonella virulence and experimentally test them.”
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In this paper, the authors evaluated the role of every single transcription factor (83 regulators) in Salmonella virulence
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- 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
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Identification of 14 regulators required for Salmonella virulence