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Bacterial Pathogenicity
in a Historical and Experimental
Perspective
Microbial Evolution and co-Adaptation
A Workshop in Honor of Joshua A Workshop in Honor of Joshua
LederbergLederberg
GENETIC RECOMBINATION IN
BACTERIA: A DISCOVERY
ACCOUNT
Joshua LederbergAnnual Review of Genetics, Vol:21, 1987
“The importance of bacteria as agents of infectious disease was clearly established by 1876, but this motivated little interest in their fundamental biology until about sixty-five years later.”
DNA as the "Stuff of Genes": The Discovery of the Transforming PDNA as the "Stuff of Genes": The Discovery of the Transforming Principlerinciple
19401940--19441944
Oswald Avery Colin McLeod Macyln McCarty
Beyond its details, the Beyond its details, the
revolutionary contribution of revolutionary contribution of
Avery, MacLeod and McCarty Avery, MacLeod and McCarty
was the refocusing on DNA was the refocusing on DNA
by a generation of scientists by a generation of scientists
that followed.that followed.
Lederberg noted that upon reading this paper he feltthat this research was “unlimited in its implications.
The Phage Group1945
Delbruck and Luria
“The principal encouragement to think about genes in bacteria had come
from Luria & Delbriick’s experiments on the statistics of mutation in
E. coli”
GENETIC RECOMBINATION IN
BACTERIA: A DISCOVERY
ACCOUNT
Joshua LederbergAnnual Review of Genetics, Vol:21, 1987
But bacteria were thought and taught to be
“Schizomycetes,” that is, asexual, primitive plants.”
and Microbiology was at the time the..
“last stronghold of Lamarckism”
Joshua Lederberg
1946
The Discovery of Bacterial Conjugation
“If bacteria could be crossed, a new repertoire of biological materials for experimental analysis would be available to physiological geneticsand biochemistry. This work might also have important practical applicationsfor vaccine improvement and the understanding of virulence-a latterdayextension of Pasteur’s primitive techniques.”
Any piece of bacterial DNA can be incorporated into the phage
This type of transduction is termed generalized transduction
Norton Zinder
Bruce Stocker
“the possibility of gene recombination in“the possibility of gene recombination in
the natural history of bacteria was presented by the natural history of bacteria was presented by
taxonomic tables of the species or serotypes of taxonomic tables of the species or serotypes of
Salmonella “Salmonella “
1952
19
19531953The Watson – Crick Model of DNA
Elie Wolman
1957
F. Jacob
‘Rough Sex’
1961
Sydney BrennerSydney Brenner
Discovery of mRNADiscovery of mRNA
With Meselson and JacobWith Meselson and Jacob
2002
Marshall NirenbergMarshall Nirenberg
19611961--19671967
Cracking the Genetic CodeCracking the Genetic Code
In 1958 I Wondered What Made Organisms Pathogenic
E. coli Shigella Salmonella
Baron had worked with Lederberg and told me that Josh
felt that if an experiment had more than 6 plates and 4 pipettes,
It was over-designed
Medical Microbiologists taughtA Pathogen is any organism that causes disease
Microbiologists taughtPathogens are degenerate microbes that grow at the expense of their host and cause damage by doing so .
A very famous scientist in the audience said“Falkow, no one give a @%&$ about Typhoid, Or pathogens. Why Don’t You Work on SomethingImportant. “
I said at a seminar at Cold Spring HarborIn 1964 that pathogens had evolved unique genetic traits that made them that way
What is a Pathogen?What is a Pathogen?
The Episome ConceptThe Episome Concept
(later to be called Plasmid, a term first coined by (later to be called Plasmid, a term first coined by
Joshua Lederberg)Joshua Lederberg)
1955-1961
Wollman andJacob
Alan Campbell 1960
Infectious
Multiple
Drug
Resistance
Tn9
Tn
21
Tn 10
Tn8
RTF
R determinant
CsCl Density GradientCsCl Density Gradient
CentrifugationCentrifugation
Julian Davies
‘Life is too short
To drink bad wine’
H. Williams (Willie) Smith
Not only could multiple resistance be transmitted by plasmids but also Toxins, Adhesins and, to some extent, Host Specificity
1970
Wally Gilbert
DNA Sequencing1975
Fred Sanger
1995
Hemophilus InfluenzaeChromosome Sequence
Hamilton Smith,Claire FraserCraig Venter
Redefining Bacterial
Pathogenicity Using the Tools
of Molecular Genetics
All in All Pathogens are Impressive Cell Biologists
Pathogenic bacteria Interfere or Manipulate for Their Own Benefit Normal Function(s) of
the Host Cell
Horizontal Gene Transfer and
Bacterial Pathogenicity
Transposons:
ST enterotoxin genes in E. coli
Prophages:
Shiga-like toxins in EHEC
Diptheria toxin gene, Cholera toxin
Botulinum toxins
Plasmids:
Shigella, Salmonella, Yersinia
Pathogenicity Islands:
Uro/Entero-pathogenic E.
coli
Salmonella typhimurium
Yersinia spp.
Helicobacter pylori
Vibrio cholerae
Genomics has reGenomics has re--emphasized the importance of Mobile Genetic emphasized the importance of Mobile Genetic
Elements in Bacterial EvolutionElements in Bacterial Evolution
How Do We Define Virulence Genes How Do We Define Virulence Genes
and Their Function?and Their Function?
What is different about pathogens?
Salmonella infection
Monack et al. 2004 Nature Reviews Microbiology
selC
epithelial cell invasion/ apoptosis
SPI-5
enteropathogenesis
valVserT
intramacrophage survival
intramacrophage survival
SPI-2 SPI-1 SPI-3 SPI-4
0' 20' 31' 63' 82' 92' 100'
PATHOGENICITY ISLANDS OF SALMONELLA ENTERICA
Microarray-based negative selection strategy
Mutantlibrary
Infect 50 129Sv mice IP8x104
A B C D
Mix probes hyb to array
A
C
Dlabel redC
AB
B AB
C
D label greenA
CD
Regrowon plates
in vitro pool
Plate spleen& liver 1, 2, 3, 4, & 7 wks
in vivo pool
Time-dependent.Selection ofPersistencegenes
Fimbrialgenes
Yellow= absentBlue/Blk= present
PathogenicityIslands and LPS
Virulence PlasmidVirulence Plasmid
host cell
Bacterial effectormolecules
OM
IM
PM
SPI-1 of Salmonella enterica
sptPinvH F G E A B C I J sicA sipB C D A prgH I J KspaOP Q R S hilA DiagBiacP orgA hilC sprB avrAsicP
Salmonella Pathogenicity Island 1
Mutant absentMutant Present
Lawley et al. Plos Pathogens (2006)
Microarray-based negative selection screen
PooledMutantlibrary
Infect mice IP
A B C D
Mix probes hyb to array
Regrowon plates
in vitro pool
in vivo pool, from spleens taken each week after infection
SPI 2 (30.5 centisomes)
secretion apparatus
OMPeriplasm
CM
ssa sse ssc sse ssc sse ssa ssr
similarto: yscU T S R Q O N lcrD yscL J yscF espB espD espA yscE D C B barA glnG
U T S R Q P O N V M L K J I H G G F B E D C A B A E D C B A B
26 kb
ippI sycD
The Salmonella Pathogenicity Island 2 Type III
secretion apparatus in the Salmonella cell envelope
Bacterial Burden after Oral infection (34 days)
WT P
Pss
eI- P
P
WT M
LNss
eI- M
LNW
T Sple
en
sseI
- Sple
enW
T Liv
er
sseI
- Liv
erW
T cec
um
sseI
- cec
um
100
101
102
103
104
105
CF
U/o
rgan
sseI mutant is attenuated for establishing infection at systemic sites, liver and spleen
PP MLN Spleen Liver Cecum
G. Govoni
= WT SseI
= ∆∆∆∆sseI
• Position specific iterative BLAST found homology to acetyltransferases including murine NAT2, human NAT1, and Salmonella NhoA.
• Contains catalytic triad: cysteine-histidine-aspartate
SseI
N C
1 32251% GC 33% GC142
•No major homology to any knownProtein
Identification of SseI function: Protein binding partners in the macrophage
Coomassie stain
mark
er
mar
ker
10% mini-gel 4-20% gradient-gel
20
30
40
506070
Puri
fied
SseI-G
ST
20
30
14
66
220
97
45
SseI-G
ST
GS
H-r
esin
only
SseI-G
ST
GS
T
• Mass Spec.
G Govoni, LM McLaughlin
IQGAP1
CHD
1 1657
IQ GRD
• Cytoskeleton and cell motility regulator:• binds actin and induces actin cross-linking through interaction
with Arp2/3 complex and N-WASP and Cdc42.
• binds CLIP-170, microtubule binding protein
• localizes to the lamellipodia and filopodia and required for protrusion
• Activated Cdc42 and Rac1 binds IQGAP1, and IQGAP1-inturn prolongs their activation by inhibiting slowing GTPase activity
• Cdc42 and Rac1 are required for IQGAP1-mediated cell migration
-5
0
5
10
15
20
25
30
35
non-infected WT
sseI::WT SseI
sseI::empty vector
sseI::sseIC
178A
ST strain used for infection
% B
MD
M m
igra
tio
n s
pecif
ically
tow
ard
heat-
kil
led
ST
SseI inhibits migration toward heat-killed S. typhimurium (ST) in infected BMDM
*
LM McLaughlin
The Host as a Reporter The Host as a Reporter
of Response to of Response to
Infection Infection
Isolate RNA
amplification
label hybridize to mousecDNA array
Gene expression in the peripheral blood from infected mice during acute and persistent infection
Lucy Thompson
Response to Salmonella Infection
Naïve animals prior to challenge
Naïve animals after WT infection (d4-9 pc)
SAM: naïve animals pre vs. post-challenge
with WT
FDR: 0.93%, 2-fold change
334 induced
105 repressed
Network 1
JAK/STAT
signaling
Leukocyte extravasation
signaling
Infiltration
of
neutrophils
Interferon
signaling
Early infection in 129sv mice,
•• Primary and secondary Primary and secondary
responses are distinctresponses are distinct
•• Response to attenuated Response to attenuated
strain is less dramatic strain is less dramatic
than WTthan WT
•• Similar genes Similar genes
categories induced in categories induced in
this early infection as this early infection as
was seen in susceptible was seen in susceptible
micemice
•• Immunity ‘SignatureImmunity ‘Signature’
WTWT aroAaroA--
aroAaroA--
immunizedimmunized
WTWT
challengedchallenged
Induced in persistently infected
mice (129sv): 336/3190 genes
Ten Months Postinfection
Major GO categories induced (DAVID):
• Antigen Presentation: MHCI plus Fc R IgG and IgE
• Defense response: beclin 1, IFN induced genes, G-CSFR, lysozyme, Phospholipase A2, MCP-1 receptor etc.
• Regulation of Cellular Processes: caspase 4, annexinA1, nudix, SOCS3, Bcl-2 like 1, fibrinogen-like protein 2 etc
WT infected
Uninfected controls
Most 6
M s
am
ple
s m
ovin
g to
ward
s “n
orm
al”-
4 h
ave a
stro
ng s
ignatu
re re
main
ing
BJ183-T28BJ180-T28BJ181-T28BJ166-T28BJ173-T28BJ171-T28BJ176-T28BJ177-T28BJ179-T28BJ178-T6MBJ170-T28BJ178-T28BJ182-T28BJ175-T28BJ167-T28BJ181-T6MBJ63-T6MBJ176-T6MBJ184-T28BJ169-T28BJ168-T6MBJ177-T6MBJ175-T6MBJ179-T6MBJ171-T6MBJ172-T6MBJ60-T6MBJ180-T6MBJ174-T6MBJ182-T6MBJ63-T28MC4MC2BJ167-T6MBJ172-T28BJ168-T28MC4MC4MC3MC9MC7MC1BJ57-T6MBJ67-T6MBJ67-T28BJ66-T6MBJ60-T28MC16MC15MC8MC3MC6MC11MC14MC12MC13BJ59-T28MC6MC5MC10BJ64-T6MBJ169-T6MBJ184-T6MBJ64-T28BJ66-T28MC5BJ173-T6MBJ166-T6MMC1BJ174-T28
Typh
oid
T28
Typh
oid
T6M
Health
y C
.
T28 C
luste
rC
ontro
l Clu
ste
rIn
term
. Clu
ste
r
Exit the Host
One of the least studied aspects of pathogenicity
Exit the Host
Because transmission is the ultimate key to success
Mouse Models of Salmonella
Infection80days80days
80+ days80+ days
• “Susceptible” Acute Model
– Resembles Enteric Fever
– Higher proliferation of bacteria in RES organs
– Death 5-7 days post infection
– Limited to Innate Immune Response
SpleenSpleen
• “Resistant” Model
– Bacteria can persist for up to a
year
– MLN act as a reservoir
– Immune Response involves:
Innate
Adaptive
Ag specific
Denise Monack
Experimental design to study horizontal transmission of serovar Typhimurium among mice
orogastric infection of donor micewith 108 cfus
(wait 2-8 days for non-colonizing bacteria to pass through GI tract)
naïve age matched mice
Co-mingle (5 mice/cage) for 1-60 days
Monitor fecal shedding of Typhimurium
Trevor Lawley
Infection and Immunity, Infection and Immunity, 7676 . . 403403--416 2008416 2008
Wild type S. typhimurium
-5 0 5 10 15 20 25 30 35 40 45 50 55 601.0×1000
1.0×1001
1.0×1002
1.0×1003
1.0×1004
1.0×1005
1.0×1006
1.0×1007
1.0×1008
1.0×1009
1.0×1010
days post-mingling
cfu
/g F
eces
S. typhimurium-specific IgA from feces
0 5 10 15 20 25 30 35 40 45 500
1
2
3
days post-mingling
OD
405
Shedding IgA
naïve mouse
wild type bacteria
Cecum PP MLN Spleen Liver1.0×100
1.0×101
1.0×102
1.0×103
1.0×104
1.0×105
1.0×106
1.0×107
1.0×108
cfu
/g t
issu
e
Colonization, 58 daysS. typhimurium-specific Ig from serum
1.0×100
1.0×101
1.0×102
1.0×103
1.0×104
1.0×105
an
ti-S
tm a
nti
bo
dy t
iter
seroconversion
naive uninf.
Fecel Shedding of Salmonella typhimuriumfrom Experimentally Infected Laboratory Mice
0 5 10 15 20 251.0×1000
1.0×1002
1.0×1004
1.0×1006
1.0×1008
1.0×1010
mouse 1
mouse 3
mouse 5mouse 4
mouse 2
days post infection
cfu
of
Salm
on
ella/
gra
m f
eces
““SupersheddersSupershedders””
Remember that these mice are inbredRemember that these mice are inbred
-3 0 3 6 9 12 15 181.0×10 00
1.0×10 02
1.0×10 04
1.0×10 06
1.0×10 08
1.0×10 10
moderate shedder
1 naive 129X1
3 naive C57BL/62 naive 129X1
super shedder
2 Donor Transmission Experiment
days of co-mingling
cfu
/g
-3 0 3 6 9 12 15 181.0×10 00
1.0×10 02
1.0×10 04
1.0×10 06
1.0×10 08
1.0×10 10
1 naive 129X1
2 naive 129X1
4 naive C57BL/63 naive C57BL/6
super shedder
Super Shedder Donor Transmission Experiment
days of co-mingling
cfu
/g
-3 0 3 6 9 12 15 181.0×10 00
1.0×10 02
1.0×10 04
1.0×10 06
1.0×10 08
1.0×10 10
moderate shedder
2 naive 129X1
4 naive C57BL/63 naive C57BL/6
1 naive 129X1
Moderate Shedder Donor Tranmission Experiment
days of co-mingling
cfu
/g
co-minglesuper
shedderwith 4 naive
mice
co-minglemoderateshedder
with 4 naivemice
But …Bacteria from But …Bacteria from SupersheddersSupershedders
Are No More Virulent or TransmissibleAre No More Virulent or Transmissible
Than Are WildThan Are Wild--Type BacteriaType Bacteria
NonNon--Genetic Factors at Play?Genetic Factors at Play?
Supershedders are responsible for most transmission from animal toanimal…
Perturbation of the
Commensal Flora
Increases the Incidence of
‘Super Shedders”
What is a Pathogen?What is a Pathogen?****
What is the Difference Between a What is the Difference Between a
Pathogen and A Commensal?Pathogen and A Commensal?
Is Disease a Distraction?Is Disease a Distraction?
##
The term pathogen is derived from the Greek παθογένεια,"birth of pain.“
Steps in successful infection orHow to Become a Successful Pathogen
• Sex comes before disease– acquire virulence genes
• Sense environment
– and Switch virulence genes on and off
• Swim to site of infection
• Stick to site of infection
• Scavenge nutrients– especially iron
• Survive stress
• Stealth– avoid immune system
• Strike-back – Neutralize innate immune
elements
• Subvert– host cell cytoskeletal and
signalling pathways
• Set up– In specific host niche
• Scatter– If necessary
After Mark Pallen
OR, is it a Successful Commensal?OR BOTH?
What is the Difference Between a Pathogen What is the Difference Between a Pathogen and a Commensal?and a Commensal?
•• Pathogens possess the Pathogens possess the inherentinherent ability to cross ability to cross anatomic barriers or breach other host defenses that anatomic barriers or breach other host defenses that limit the survival or replication of other microbes and limit the survival or replication of other microbes and commensals.commensals.
•• Therefore, most pathogens often establish Therefore, most pathogens often establish themselves in a niche usually devoid of other stable themselves in a niche usually devoid of other stable microbial populations. microbial populations.
•• These invasive properties are These invasive properties are essentialessential for their for their survival in Nature. And, are often host specific.survival in Nature. And, are often host specific.
The Commensal PathogensThe Commensal Pathogens
•• Some Some micoroorganismsmicoroorganisms are commonly found in are commonly found in cultures of the normal flora but also regularly cultures of the normal flora but also regularly cause human disease.cause human disease.
•• These microbes have virulence determinants These microbes have virulence determinants that suggest they regularly come in intimate that suggest they regularly come in intimate contact with elements of the innate and contact with elements of the innate and adaptive immune system.adaptive immune system.
•• Immunization against such microbes not only Immunization against such microbes not only prevents human disease but also eliminates prevents human disease but also eliminates their ability to colonize the human host their ability to colonize the human host efficientlyefficiently
For example,
Streptococcus pneumoniaeStreptococcus pneumoniae
NeisseriaNeisseria meningitidismeningitidis
HaemophilusHaemophilus influenzaeinfluenzae bb
Streptococcus Streptococcus pyogenespyogenes
For example,
CapsuleCapsule
IgAIgA ProteaseProtease
PiliPili
AntiAnti--phagocytic proteinsphagocytic proteins
For example
HibHib ConjugateConjugate
PrevnarPrevnar
Meningococcal immunizationMeningococcal immunization
Group A Streptococcus Group A Streptococcus ---- 3.2 cases per 100,0003.2 cases per 100,000
(0(0--10% Asymptomatic Carriage Rate)10% Asymptomatic Carriage Rate)
Group B Streptococcus Group B Streptococcus ---- 6.9 cases per 100,0006.9 cases per 100,000
(4(4--18% Asymptomatic Genital Carriage Rate)18% Asymptomatic Genital Carriage Rate)
HaemophilusHaemophilus influenzaeinfluenzae ---- 1.3 cases per 100,0001.3 cases per 100,000
(1.3(1.3--3.0% Asymptomatic Carriage Rate) 3.0% Asymptomatic Carriage Rate)
NeisseriaNeisseria meningitidismeningitidis ---- 0.8 cases per 100,000 0.8 cases per 100,000
(10(10--15% Asymptomatic Carriage Rate)15% Asymptomatic Carriage Rate)
Streptococcus pneumoniaeStreptococcus pneumoniae ---- 21.9 cases per 100,00021.9 cases per 100,000
(10(10--55% Asymptomatic Carriage Rate)55% Asymptomatic Carriage Rate)
Prevalence of Endemic Carriage and Clinical DiseasePrevalence of Endemic Carriage and Clinical Disease
Caused by ‘Commensal’ PathogensCaused by ‘Commensal’ Pathogens
Are Are ““Virulence Factors,Virulence Factors,”” or or ““Pathogenic Pathogenic
DeterminantsDeterminants”” really a subset of really a subset of ““Adaptive Adaptive
FactorsFactors””, the claws, fangs, fur, and scales that , the claws, fangs, fur, and scales that
allow microbes to adapt to a particular niche. allow microbes to adapt to a particular niche.
(But not necessarily designed to cause (But not necessarily designed to cause
disease)?disease)?
Is Disease a Distraction?Is Disease a Distraction?
•• Human Medicine (and granting Human Medicine (and granting
agencies) demand that we focus on agencies) demand that we focus on
disease and its cure or amelioration.disease and its cure or amelioration.
•• But does this focus sometimes distract But does this focus sometimes distract
us from understanding the biology of us from understanding the biology of
the pathogen and the evolution of the the pathogen and the evolution of the
host parasite relationship?host parasite relationship?
Many HumanMany Human--Adapted Pathogens Cause Adapted Pathogens Cause
Persistent InfectionPersistent Infection
•• Mycobacterium tuberculosis Mycobacterium tuberculosis and otherand other
Mycobacterium species Mycobacterium species
•• TreponemaTreponema pallidumpallidum
•• Chlamydia Chlamydia trachomatistrachomatis
•• Salmonella typhiSalmonella typhi
•• Helicobacter pylori Helicobacter pylori
Which are AsymptomaticWhich are Asymptomatic
•• Mycobacterium tuberculosis Mycobacterium tuberculosis -- 90%90%
•• Salmonella typhi Salmonella typhi –– 80%80%
•• BartonellaBartonella henselaehenselae (in preferred Cat (in preferred Cat
host) host) -->90% >90%
•• Helicobacter pyloriHelicobacter pylori -- 80%80%
Infectious diseases
USA 1950-2000
MeaslesMumps
‘Immune disorders’
Multiple
sclerosis
Type 1
diabetes
Asthma
From: Bach JF, N Engl J Med. 2002; 347:911-20.
schizophrenia |�skitsə�frēnēə; -�frenēə|noun
a long-term mental disorder of a type involving a breakdown in the relation
between thought, emotion, and behavior, leading to faulty perception,
inappropriate actions and feelings, withdrawal from reality and personal
relationships into fantasy and delusion, and a sense of mental
fragmentation.
• (in general use) a mentality or approach characterized by inconsistent or contradictory elements
.DERIVATIVES
schizophrenic |-ˈfrenik| |ˈˈskˈtsəˈfrˈnˈk| |ˈˈskˈtsəˈfrinˈk| |-
ˈfrˈnˈk|
adjective & nounORIGIN early 20th cent.: modern Latin, from Greek skhizein ‘to split’ +
phrēn ‘mind.’
schizophrenia |�skitsə�frēnēə; -�frenēə|noun
a long-term mental disorder of a type involving a breakdown in the relation
between thought, emotion, and behavior, leading to faulty perception,
inappropriate actions and feelings, withdrawal from reality and personal
relationships into fantasy and delusion, and a sense of mental
fragmentation.
• (in general use) a mentality or approach characterized by inconsistent or contradictory elements This is especially applicable to discussions of Microbial Pathogenicity.DERIVATIVES
schizophrenic |-ˈfrenik| |ˈˈskˈtsəˈfrˈnˈk| |ˈˈskˈtsəˈfrinˈk| |-
ˈfrˈnˈk|
adjective & nounORIGIN early 20th cent.: modern Latin, from Greek skhizein ‘to split’ +
phrēn ‘mind.’
Sit down before fact as a little child, be prepared to give up every preconceived notion, follow humbly wherever and to whatever abysses nature leads,... Thomas Huxley