liggins departmental seminar: fireflies & superbugs - when science & nature collide by dr...
DESCRIPTION
Talk by Dr Siouxsie Wiles (ORCID: 0000-0002-0467-0015) at the Liggins Institute, University of Auckland on the 27 February 2013. Bioluminescence (literally ‘living light’) has evolved in a wide variety of fascinating organisms with many different purposes. It allows glow worms to glow worms to lure food, fireflies to find a mate and nocturnal squids to camouflage themselves from predators. The light is produced as a by-product of an enzyme (‘luciferase’) reaction, emitted when a substrate (‘lucferin’) is exposed to oxygen. Siouxsie will talk about her research using bioluminescence to better understand infectious diseases, from tracking infections in living animals to discovering new drugs for tuberculosis.TRANSCRIPT
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+
Flavin oxidoreductase
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+
Flavin oxidoreductase
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
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
Bioluminescence imaging
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
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
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 = 30090
Max = 100000
bkg subflat-fieldedcosmic
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
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
bkg subflat-fieldedcosmic
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
bkg subflat-fieldedcosmic
~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
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.
Natural transmission changes tissue tropism and infectious dose
Click # SC2004120315025803 Dec 2004 15:03:25Bin:1, FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
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
bkg subflat-fieldedcosmic
Click # SC2004121609522916 Dec 2004 09:52:56Bin:1, FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
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
bkg subflat-fieldedcosmic
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
bkg subflat-fieldedcosmic
Click # SC2004121013013210 Dec 2004 13:02:00Bin:1, FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
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
bkg subflat-fieldedcosmic
Click # SC2004120810475108 Dec 2004 10:48:20Bin:1, FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
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
bkg subflat-fieldedcosmic
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)
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)
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)
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)
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)
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)
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)
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)
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)
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)
‘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
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