Liggins Departmental Seminar: Fireflies & superbugs - when science & nature collide by Dr Siouxsie Wiles

Fireflies and superbugs:when science and nature collide

Dr Siouxsie Wiles

Dept. Molecular Medicine & PathologyUniversity of Auckland

ORCID: 0000-0002-0467-0015

@SiouxsieW

#BSGSW

http://youtu.be/kP_RaHo1Pmw

Meet the Lampyridae

Daniel Keogh AKA

Professor Funk

Tales of cannibals & orgies ….

Glow worms (Arachnocampa luminosa)

….and invisibility cloaks!

Hawaiian bobtail squid and Vibrio fischeri

BioluminescenceEnzyme catalysed reaction

D-Luciferin Light(560nm)

Luciferase

ATP AMP

CO2O2

Oxyluciferin +

FFluc

Firefly (Photinus pyralis)

lux operon

C D A B E

R T S

RT S

-

Fatty Acid(RCOOH)

Aldehyde(RHO)

? ?

? ?

Luciferase

FMNH FMN

NAD(P) NAD(P)HH+

Flavin oxidoreductase

Light (490nm)

O2 H2O

lux operon

C D A B E

R T S

RT S

-

Fatty Acid(RCOOH)

Aldehyde(RHO)

? ?

? ?

Luciferase

FMNH 2 FMN

NAD(P)H+


Light (490nm)

O2 H2O

lux operon

C D A B E

R T S

RT S

RT S

Fatty acid reductase complex-

Fatty Acid(RCOOH)

Aldehyde(RHO)

α β

? ?α β

Luciferase

FMNH FMN

NAD(P)H+


Light (490nm)

O2 H2O

Photorhabdus luminescens

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

Photonic imaging

A highly sensitive & non-toxic analytical

technique based on the detection of light by

specialised charge coupled device (CCD) cameras

Equipment

• Light-tight box

• Sensitive charge coupled device camera

• Number of machines commercially available

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


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




100000

90000

80000

70000

60000

50000

40000

30000

ImageMin = -44712



Max = 100000


Bioluminescence imaging



100000

90000

80000

70000

60000

50000

40000

30000

ImageMin = -44712



Max = 100000


Measured as photons/sec/cm2/sr

= number of photons leaving a square cm of tissue and radiating into a solid angle of 1 sr

sr = steradian = unit of solid angle for a sphereClick # SW20070413151554

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


100000

90000

80000

70000

60000

50000

40000

30000

ImageMin = -44712



Max = 100000


ROI 1=1.1398e+06

ROI 2=2.7412e+05

ROI 3=7.8168e+05

ROI 4=6.575e+05

ROI 5=2.2645e+05

ROI 6=6.9768e+05

ROI 7=7.4472e+05

ROI 8=2.7444e+05

ROI 9=3.6689e+05

Total: Area Flux = 5.16333e+06

http://en.wikipedia.org/wiki/Image:Steradian.svg

3Rs - Refinement

Incorrectly dosed animals can therefore be removed from study

Intraperitoneal administration Oral gavage

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

Massive reduction in animal use

1010

108

106

102

104

0 5 10 15 20 25

Time (days)

Num

ber

of bac

teria

(cfu

per

gra

m t

issu

e)

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

Massive reduction in animal use

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

Biophotonic imaging

Not just for mice….

Limits of detection

• Level of reporter expression

• Emission wavelength

• Availability of cofactors (e.g. O2 and ATP)

• Location of signal (depth and type of tissue)

• Signal impedance factors (melanin, etc)

Monitoring biological processes

• Where are cells of interest?

• Are they dead or alive?

• What genes are being expressed?

Mycobacterium tuberculosis:the captain of death

• 1/3 people latently infected

• 4,500 people die every day

• Resistant to common antibiotics

• Now have strains that are untreatable– Surgery– Incarceration

Desperate need for new antibiotics!

Mycobacterium tuberculosis:a very unusual bug

• Complex lipid-rich cell wall

• Acid fast bacilli (AFBs)

• Lives inside the immune system’s frontline troops

• Grows very slowly

Need something faster than waiting for them to grow!

Imaging TB Consortium

● BMGF-funded consortium (Drug Accelerator Programme)

● To develop in vivo imaging for Mycobacterium tuberculosis

● LSHTM (Schaible & Bancroft) – in vivo Barts and the London (Parish) – fluorescent reporters Imperial (Wiles & Robertson) – bioluminescent reporters

Bioluminescent mycobacteria

lux-expressingluc-expressing

Mycobacterial strain

Light

leve

ls

(rel

ativ

e lig

ht

units)

Andreu et al (2010). PLoS One. 5(5): e10777

Light reflects cell numbers

Light

leve

ls

Answer in minutes rather than weeks!Bacterial numbers Bacterial numbers

Light

leve

ls

lux-expressingluc-expressing

Drug screening in vitro

Bacterial culture+

Drug

Labour-intensive

Slow!

Drug testing in immune cells

Doubling dilutions from 160 ug/ml

Chloramphenicol

Answer in ~2-4 days!

Andreu et al (2012). J. Antimicrobial Chemotherapy, 67:404

No antibiotic

Highest antibiotic concentration

Light

leve

ls

GSK acute in vivo drug assay

UntreatedTreated

Before treatment

8 days treatment

• Mice intranasally infected with 105 cfu Mtb

• 24h post infection begin daily drug dosing by oral gavage

• Organs harvested at day 9 for cfu’s

Andreu et al . Submitted

UntreatedTreated

2 days treatment

8 days treatment

Non-invasive drug screening

Andreu et al . Submitted

Auckland - FMHS

Auckland - FMHS

First new class of drugs to be developed for TB in 50 years

Evolution in Action: C. rodentium

A/E

• The etiological agent of mouse transmissible colonic hyperplasia

• Colonises gastro-intestinal tract through attaching/effacing (A/E) lesion formation (in vivo)

Mundy et al (2005). Cellular Microbiology 7:1697-1706.

Via

ble

counts

(l

og c

fu g

-1 s

tool)

1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

0 5 10 15 20 25

wild-type

intimin-

Conventional model….

Time (days)

In vivo 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

3Ph

oto

ns sec

-1 cm -2 sr

-1

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

Transmissible….

Click # SC2004120810155508 Dec 2004 10:16:24Bin:1, FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34

Series: IACUC #: Experiment: cageLabel: 11Comment: Analysis Comment: 1x1

20

15

10

5

x106

ImageMin = -8.7851e+05Max = 4.2345e+07

p/sec/cm^2/sr

Color BarMin = 6.375e+05Max = 2.125e+07



Series: IACUC #: Experiment: cageLabel: 11Comment: Analysis Comment: 1x1

20

15

10

5

x106

ImageMin = -8.7851e+05Max = 4.2345e+07

p/sec/cm^2/sr

Color BarMin = 6.375e+05Max = 2.125e+07


~108 cfu mouse-1 24h-1

Lum

ines cen

ce(P

hoto

ns sec

-1 cm2

-1 sr-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



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

50000

40000

30000

20000

10000

ImageMin = -3083.3


Color BarMin = 5000

Max = 50000



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

50000

40000

30000

20000

10000

ImageMin = -3271.7


Color BarMin = 5000

Max = 50000


d2

Infection by natural transmission

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

Natural transmission changes tissue tropism and infectious dose


Series: IACUC #: Experiment: lux nat green d3Label: Comment: Analysis Comment: 1x1

500

400

300

200

100

ImageMin = -23.633Max = 4621.9

counts

Color BarMin = 100Max = 500



Series: IACUC #: Experiment: 106 LB grown inocLabel: gavaged mouse (U)Comment: Analysis Comment: 1x1

500

400

300

200

100

ImageMin = -15.979Max = 35.716

counts

Color BarMin = 10

Max = 500


Click # SC2004121610054616 Dec 2004 10:06:01Bin:HR (2), FOV12, f1, 5mFilter: OpenCamera: IVIS 82, DW34

Series: IACUC #: Experiment: 109 LB grown from stool inocLabel: gavaged mouse (U)Comment: Analysis Comment: 5x2

500

400

300

200

100

ImageMin = -212.36Max = 2496.7

counts

Color BarMin = 10

Max = 500



Series: IACUC #: Experiment: natural transmission - without bolusLabel: contact mouse (G)Comment: Analysis Comment: 1x1

500

400

300

200

100

ImageMin = -24.169Max = 256.56

counts

Color BarMin = 10

Max = 500



Series: IACUC #: Experiment: natural transmission - no bolusLabel: contact mouse (U)Comment: Analysis Comment: 1x1

20

18

16

14

12

10

8

6

x106

ImageMin = -8.3287e+05Max = 1.5605e+08

p/sec/cm^2/sr

Color BarMin = 4.25e+06

Max = 2.125e+07


Lum

ine scen

ce(Ph

oto

ns sec

-1 cm2

-1 sr-1)

NaturalTransmission

Host Passaged (106)

Lab Passaged (106)

Lab Passaged (109)

Tissue distribution at 3 days post-infection

Wiles et al (2005). Cellular Microbiology 7:1163-1172.

Evolution in Action!

Lab-grown bacteria used to inoculate a seed mouse for each colony

Phase 1: Experimental evolution

d1

d2

1. Monitor transmission and infection dynamics by BPI.

2. Collect bacteria during infection and archive.

Phase 2: Measuring adaptationArchived samples mixed 1:1 and competed (i) growing in culture, and (ii)

in the environments the organisms were evolved in. Outcome of competition measured by monitoring colour of dominant strain

or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)



d1

d2





or(i)

(ii)

‘Environment’ I (n=5)

‘Environment’ II (n=5)

Evolution in Action!

‘Environment’ I

6-7 wk female C57Bl/6 mice

‘Environment’ II

6-7 wk female C57Bl/6 mice+ antibiotics

Bac

teria/

gra

m s

tool

Crowdfunding

• US$ 4,480 = NZ$ 5,750

• 79 contributors – 51% strangers

• Mean contribution = $55.31

• NZ based = 52%

• UK/Europe = 22%

• USA/Canada = 26%115

137

775

US$252,811 raised so far

Round 4 coming soon!

And finally…

corriecanuck.wordpress.com

Biostat Decision Tool

A maths-free decision making tool for statistical data analysis, particularly aimed at biologists

that have small n-number data sets

… an app for Android & iPhone!

Free web based version coming soon….

Nuria Andreu

Brian Robertson

Paul Elkington

Taryn Fletcher

Nitya Krishnan

Andrea Zelmer

Ulrich Schaible

Greg Bancroft

Anne Bishop

John Fraser

Fiona Clow

Simon Swift

Debbie Williamson

Fiona Radcliff

Bill Denny

Brian Palmer

Phil Crosier

David Jenkins

Shaun Lott

Steve Richie Greg Cook

Jimmy Dalton

Benedict Uy

Hannah Read

Sarah Johnson

Grant Mills

John Boikov

Joon Cho

Tim Yang

Vic Arcus

www.youtube.com/user/Skeptimoo

@SiouxsieW

www.sciblogs.co.nz/infectious-thoughts/

www.siouxsiewiles.blogspot.co.nz/

Get in touch!

Liggins Departmental Seminar: Fireflies & superbugs - when science & nature collide by Dr Siouxsie Wiles

Education

pre cage bin

analysis comment

1x4 comment

1x4 camera

dw34 comment

sr pseccm

bkg sub sr

open label