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