biliary signature of a bacterial pathogen

Biliary Signature of aBacterial Pathogen

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Biliary Signature of aBacterial PathogenListeria monocytogenes

The Deadly Deli

The Deadly Deli

Spectrum of Listeriosis• Mild fever and malaise• Mild to severe gastritis

• Severe meningitis in newborns• Bacteremia and sepsis in newborns

• Osteomyelitis and arthritis• Endocarditis

• Asymptomatic carriage• Age-dependent (young and old)

• More frequent in pregnancy• Immune status is critical

L. monocytogenes Intracellular Life CycleTilney and Portnoy

entry

Escape(LLO)

ActA

comet tail

actin cloud

cell-cell spread

LLO+PLCs

Organs Infected by L. monocytogenes

• Intestine• Lymph nodes

• Liver • Spleen

• Brain• CNS

• Bones • Heart

How can one hope to monitor all of these in every animal?

In Vivo Bioluminescence Imaging (BLI)

1000

200

Xenogen Corp.Alameda CA, USA

Photorhabdus luminescens

Day 1

Day 3

Day 2

Day 4

Day 5

2000

200

Survived

1 LD50 (4x104 CFU)

Hardy et al., Science 303:851-3

Localization of Focal Signal to the Gall Bladder Lumen

1. BLI Signal3. Colonies fromlumen on a petri

dish

2. Excised gall bladder4. Bacteria in the

lumen by histology

Hardy et al., Science 303:851-3

Oral Infection

Hardy et al., Science 303:851-3

2000

200

Frequency of Gall Bladder Signals

20 CD1 femalemice given

5x105 CFU of Listeria, imaged

on Day 2

Hardy et al., Infect. Immun. 74(3): 1819-27

Unusual Morphologyof Intralumenal

L. monocytogenes

Pauline Chu, Comparative Medicine

Uninfected

Intralumenal L. monocytogenes

The Gall Bladder

Liver

Gall Bladder

Intestine

Bile duct

The Gall Bladder

Liver

Gall Bladder

Intestine

Bile duct

Questions at this Point

•What is the food source of the bacteria?•Do they adhere to mucus, and if so, how?•What genes are expressed?•Does this process contribute to disease?•What effects are seen on the tissue and contentsof the organ?

•How do the bacteria mitigate the toxicity of the bile salts and other components of bile?

Can they ever GET OUT?

Liver Liver

Gall BladderGall Bladder

3.5 mm4 mm

2.5 mm3 mm

2500

15,000

Jeff Margolis

Hardy et al., Infect. Immun. 74(3): 1819-27

Alan Hofmann

A

B

C

(+)

Daughters of 542.3

147.3

Daughters of 542.3

147.3

m:z 542.3=Monosodium 18:2 Lysophosphatidylcholine

R' = 18-Carbon chain with two double bonds

18:2 Lysophosphatidylcholine

Choline group

Uninfected Animals

Uninfected

Infected

2000

200

Infected

T=0

30 min

60 min

90 min

120 min

20 hours

Human Bile1:3 in H2O

+106 CFU of Listeria

L. monocytogenes Grows in Pure Bile

Bile Culture Dilutions

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

4.00E+06

4.50E+06

T=

0 m

in

T=

5 m

in

T=

10 m

in

T=

15 m

in

T=

20 m

in

T=

25 m

in

T=

30 m

in

T=

35 m

in

T=

40 m

in

T=

45 m

in

T=

50 m

in

T=

55 m

in

T=

60 m

in

T=

65 m

in

T=

70 m

in

T=

75 m

in

T=

80 m

in

T=

85 m

in

T=

90 m

in

time (min)

undiluted

1 to 2

1 to 4

1 to 16

1 to 64

1 to 256

1 to 1024

1 to 4096

Water

Proteins in Mouse Bile

•Pancreatic amylase•Immunoglobulins (antibodies)

•Pancreatic lipases•Sterol esterases

•Carboxypeptidases•Collagen

•α Andrenergic receptor?•Transcription initiation factor TFIID?

The Future• More in vitro cultures with mouse bile, which is

cleaner and has less variation• More mice with the attenuated strain which grows

for a long time without making the mice sick• More protein data from both in vitro and mouse

samples to look for Listeria proteins (!) • Use pure substances and look for changes upon

culture, which will be tricky• Use bacterial mutants such as phospholipase

mutants

People to Thank

Jeff MargolisPauline Chu

Alan HofmannLee Hagey

Janet Bueno

Cormac Gahan

Allis ChienKarolina KrasinskaTheresa McLaughlinLindsay Comeaux

Chris Adams

Christopher H. Contag