fireflies & superbugs: when science & nature collide

Fireflies and superbugs: when science and nature collide

Dr Siouxsie Wiles

Dept. Molecular Medicine & Pathology

University of Auckland

Nuria Andreu

Brian Robertson

Paul Elkington

Taryn Fletcher

Nitya Krishnan

Andrea Zelmer

Ulrich Schaible

Greg Bancroft

John Fraser

Bill Wilson

Fiona Clow

Simon Swift

Debbie Williamson

Fiona Radcliff

Bill Denny

Brian Palmer

Phil Crosier

David Jenkins

Shaun Lott

Jimmy Dalton

Benedict Uy

Hannah Read

Sarah Johnson

Elin Rojdeby

Grant Mills

Christina Fehlmann

John Boikov

Greg Cook

Joanna Kirman

Vic Arcus

The detection of light by specialised

charge coupled device (CCD) cameras

Biophotonic imaging (BPI)

NZMS

Generating light: optical reporter genes

Bioluminescence

Enzyme –catalysed reaction

Fluorescence

Excitation of fluorophore

GFP ‘mFruits’

Shaner et al (2004) Nature Biotechnology 22:1567-1572

NZMS

Bioluminescence Enzyme catalysed reaction

D-Luciferin Light

(560nm)

Luciferase

ATP AMP

CO2 O2

Oxyluciferin +

FFluc

Firefly (Photinus pyralis)

lux operon

C D A B E

R T S

R T S

-

Fatty Acid

(RCOOH)

Aldehyde

(RHO)

? ?

? ?

Luciferase

FMNH FMN

NAD(P) NAD(P)H H +

Flavin oxidoreductase

Light (490nm)

O 2 H

2 O

lux operon

C D A B E

R T S

R T S

-

Fatty Acid

(RCOOH)

Aldehyde

(RHO)

? ?

? ?

Luciferase

FMNH 2

FMN

NAD(P) H +

Flavin oxidoreductase

Light (490nm)

O 2 H

2 O

lux operon

C D A B E

R T S

R T S

R T S

Fatty acid reductase complex -

Fatty Acid

(RCOOH)

Aldehyde

(RHO)

? ?

Luciferase

FMNH FMN

NAD(P) H +

Flavin oxidoreductase

Light (490nm)

O 2 H

2 O

Photorhabdus luminescens

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Why is bioluminescence so useful?

• No light unless luciferase genes present

• Dead cells don’t glow!

• The more bacteria there are, the more light there is

• Light travels through things – like flesh & skin!

• Potential for non-destructive real-time measurements

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BPI Equipment

• Light-tight box

• Sensitive charge coupled device camera

• Number of machines commercially available

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Procedure

• Animal anaesthetised for restraint purposes

• Add substrate if required

• Reference (grey-scale) photographic image taken under low illumination

• Image taken with/without illumination

Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34

Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:

Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34

Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:

100000

90000

80000

70000

60000

50000

40000

30000

ImageMin = -44712

Max = 5.9625e+05p/sec/cm 2̂/sr

Color BarMin = 30000

Max = 100000

bkg subflat-fieldedcosmic

Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34

Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:

Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34

Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:

100000

90000

80000

70000

60000

50000

40000

30000

ImageMin = -44712

Max = 5.9625e+05p/sec/cm 2̂/sr

Color BarMin = 30000

Max = 100000

bkg subflat-fieldedcosmic

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Multi-parameter imaging: spectral unmixing

GT

RT

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Biophotonic imaging

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Monitoring biological processes

• Where are cells of interest?

• Are they dead or alive?

• What genes are being expressed?

MWC Symposium

Real-time infection dynamics

Day 1 (10 min exp)

Day 3 (5 min exp)

Day 6 (1 min exp)

Day 8 (1 min exp)

Day 10 (1 min exp)

Day 14 (1 min exp)

10

3

10

6

10

3

Photo

ns s

ec

-1 cm

-2 s

r-1

Wiles et al (2006). Infection and Immunity 74:5391-5396.

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Real-time infection dynamics

Click # SC20041208101555

08 Dec 2004 10:16:24

Bin:1, FOV12, f1, 1m

Filter: Open

Camera: IVIS 82, DW34

Series:

IACUC #:

Experiment: cage

Label: 11

Comment:

Analysis Comment: 1x1

20

15

10

5

x10

6

Image

Min = -8.7851e+05

Max = 4.2345e+07

p/sec/cm^2/sr

Color Bar

Min = 6.375e+05

Max = 2.125e+07

bkg sub

flat-fielded

cosmic

Click # SC20041208101555

08 Dec 2004 10:16:24

Bin:1, FOV12, f1, 1m

Filter: Open

Camera: IVIS 82, DW34

Series:

IACUC #:

Experiment: cage

Label: 11

Comment:

Analysis Comment: 1x1

20

15

10

5

x10

6

Image

Min = -8.7851e+05

Max = 4.2345e+07

p/sec/cm^2/sr

Color Bar

Min = 6.375e+05

Max = 2.125e+07

bkg sub

flat-fielded

cosmic

~108 cfu mouse-1 24h-1

Lum

inescence

(P

hoto

ns s

ec

-1 cm

2 -1 s

r-1)

d1

Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34

Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:

50000

40000

30000

20000

10000

ImageMin = -3083.3

Max = 3.4125e+05p/sec/cm^2/sr

Color BarMin = 5000

Max = 50000

bkg subflat-fieldedcosmic

Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34

Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:

50000

40000

30000

20000

10000

ImageMin = -3083.3

Max = 3.4125e+05p/sec/cm^2/sr

Color BarMin = 5000

Max = 50000

bkg subflat-fieldedcosmic

Click # SW2005112313204623 Nov 2005 13:21:00Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34

Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: BG litle BR GIAnalysis Comment:

50000

40000

30000

20000

10000

ImageMin = -3271.7

Max = 1.212e+05p/sec/cm^2/sr

Color BarMin = 5000

Max = 50000

bkg subflat-fieldedcosmic

d2

Infection by natural transmission

Bishop et al (2007). Microb. Infect. 9:1316-1324.

NZMS

Mycobacterium tuberculosis: the captain of death

• 1/3 people latently infected

• 4,500 people die every day

• Resistant to common antibiotics

• Grows very slowly…

Desperate need for new antibiotics and faster ways to test them!

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Light reflects cell numbers

Lig

ht

levels

Answer in minutes rather than weeks!

Bacterial numbers Bacterial numbers

Lig

ht

levels

lux-expressing luc-expressing

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Untreated Treated

2 days treatment

8 days treatment

Non-invasive drug screening

Andreu et al . Submitted

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Auckland - FMHS

NZMS

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Massive reduction in animal use

1010

108

106

102

104

0 5 10 15 20 25

Time (days)

Num

ber

of

bacte

ria

(cfu

per

gra

m t

issue)

Wiles et al (2004). Cellular Microbiology 6:963-972.

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Massive reduction in animal use

Dennis et al (2008). Infection and Immunity. 76:4978-4988

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3Rs - Refinement

Incorrectly dosed animals can be removed from study

Intraperitoneal administration Oral gavage

Andreu et al (2011). FEMS Microbiology Reviews. 35(2):360-394

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Not just for mice….

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s.wiles@auckland.ac.nz

Get in touch!

@SiouxsieW

www.youtube.com/user/Skeptimoo