Genetic Screenings for Studying
Bacterial Pathogenesis
Dongwoo Shin, Ph.D.
Associate Professor, Department of Molecular
Cell Biology, Sungkyunkwan University School
of Medicine
Structure of a Bacterial Cell
What is a pathogen?
-An organism capable of colonizing a host
organism where the interaction results in disease
-opportunistic pathogens
-strict pathogens
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)
[NOTE] Colonization vs. Infection
 Exposure of an individual to an organism
1. Transient (hours or days) colonization
2. Permanent colonization
3. Disease production (Infection)
[NOTE] Normal microbiota (microflora)
 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
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
 Lipopolysacchride (LPS)
-Somatic O polysacchride + Core polysaccharide + Lipid A
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
 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
 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
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
Salmonella
Enterobacteriaceae
Escherichia coli
Shigella
Serovars
Salmonella enterica
Salmonella bongori
Typhimurium
Enteritidis
Typhi
Paratyphi
Gastroenteritis
Systemic disease
Complex Lifestyle of Salmonella
Soil, water, food
Host: Humans and animals
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
Serves
(Human) and Typhoid fever (Mouse)
as a model system for other intracellular
pathogens
Biology of Salmonella Infection
“Environmental Signals inside Host”
Virulence Genes
Expression of Necessary Proteins
(Virulence Proteins) in the Correct Tissues
Stomach
Invasion into epithelial cells
of small intestine
Type III Secretion Systems (TTSSs)
Survival inside macrophages
Systemic disease
Expression of SPI-1 Genes Mediates
Salmonella-Invasion into Host Cells
Expression of ~30 genes in Salmonella Pathogenecity Island 1 (SPI-1)
Type III Secretion System
SPI-1 TTSS-Induced Changes in Host Cells
Stomach
Invasion into epithelial cells
of small intestine
Type III Secretion Systems (TTSSs)
Survival inside macrophages
Systemic disease
Bacterial killing processes inside phagosome
Antimicrobial peptides
Phagosome
Bacterial
Killing
ROS & RNS production
Phagosome-lysosome fusion
Induction of the SPI-2 T3SS within Macrophage
Host cells
effector proteins
secretion system
Salmonella
The SPI-2 T3SS prevents bacterial killing by macrophages
Antimicrobial peptides
LPS modification
Phagosome
SPI-2 TTSS
Preventing recruitment
of NADH oxidase
ROS & RNS production
Alteration of vesicle
trafficking
Phagosome-lysosome fusion
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 promoterless purA and lacZ genes;
introduction 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
PmgtC
gfp
Activation of mgtC
transcription
Salmonella
macrophage
Differential fluorescence induction (DFI)
Step 1: Cloning of random fragments of Salmonella chromosome into
a promoterless gfp plasmid; introduction of plasmids into
Salmonella
Differential fluorescence induction (DFI)
Step 2: Infection of macrophages with Salmonella harboring gfp
fusion 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
Salmonella’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
count 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 (83 regulators) 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
Identification of 14 regulators required for Salmonella virulence