Reprogramming biological functionalities for autonomous …€¦ · • Metabolic...
Transcript of Reprogramming biological functionalities for autonomous …€¦ · • Metabolic...
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Reprogramming biological functionalities for autonomous therapeutic cells
Matthew Chang
Department of Biochemistry, Yong Loo Lin School of MedicineNUS Synthetic Biology for Clinical and Technological Innovation (SynCTI)
National University of Singapore
http://SynCTI.org/
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MICROBIOMEDENIZENS OF THE HUMAN BODY AND AFFECTS OUR HEALTH
25SPECIESIn the stomach include
500-1,000SPECIESIn the intestines include:
600+SPECIESIn the mouth, pharynx and respiratory system include:
1,000SPECIESIn the skin include:
60SPECIESIn the
urogenital tract include:
Reprogramming commensal microbes to function as
therapeutic units?
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Functional commensal microbesResponding to disease conditions in an autonomous manner
Anti-infection
Prevention
Drug delivery
Anti-inflammatory
Anti-cancer
Infections
Inflammation
Cancers
Metabolic disorders
Disease conditions
Input Output
Therapeutic functionalities
Commensal microbes that autonomously respond to
disease/health states
Reprogramming
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Most major classes of antibiotics developed in 1940�s to 1960�sSince 1980�s, the pharmaceutical industry focusing on chronic diseases that require life-long daily treatmentFewer new classes of antibiotics available
ANTIBIOTIC RESISTANCE: PROBLEM?
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POTENTIAL SOLUTIONS?
Develop new classes of antibiotics
Diversify our antimicrobial strategies– Exploiting synthetic biology approaches?
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RESEARCH OBJECTIVE
• Intentional design– Programmable behaviors
• Sensing, seeking and killing a target pathogen
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DESIGN: INPUT & OUTPUTQUORUM SENSING (QS) & BACTERIOCINS
QS molecules– Intercellular
communication– Transition between
cellular lifestyle
BacteriocinsCell-specific lysis
FEBS Lett (2010); BBRC (2010); Chem Eng Sci (2013); ChemBioChem (2013)
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Pathogen-killing microbesPrototype
Our Engineered E. coli
P. aeruginosa
PluxRlasR Lysis E7 Pyocin S5PluxRPtetR
3OC12HSL
Pyocin S5 protein
Lysis E7 protein
lasR Protein
PluxR Activator
3OC12HSL
Pyocin S5 protein
Lysis E7 protein
lasR Protein
PluxR Activator Quorum Sensing
Pathogen Inhibition
Lysis ActivationPyocin Accumulation
Hwang et al. ACS Syn Biol, 2014Rasouliha et. ChemBioChem, 2013
Saeidi et al. Mol Sys Biol, 2011Ling et al. FEBS Lett, 2010
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How our synthetic microbes work..Courtesy of West German Broadcasting (WDR)
http://www.wdr.de/tv/quarks/sendungsbeitraege/2011/1213/008_bakterien_2.jspHwang et al. ACS Syn Biol, 2014
Rasouliha et. ChemBioChem, 2013Saeidi et al. Mol Sys Biol, 2011
Ling et al. FEBS Lett, 2010
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PATHOGEN-KILLING MICROBESPROTOTYPES - STILL EVOLVING
Our Engineered E. coli
P. aeruginosa
PluxRlasR Lysis E7 Pyocin S5PluxRPtetR
3OC12HSL
Pyocin S5 protein
Lysis E7 protein
lasR Protein
PluxR Activator
3OC12HSL
Pyocin S5 protein
Lysis E7 protein
lasR Protein
PluxR Activator Quorum Sensing
Pathogen Inhibition
Lysis ActivationPyocin Accumulation
• Molecular Systems Biology (2011), Featured in Reuters, Nature, Science, etc.
1.E+07
1.E+08
1.E+09
1.E+10
0 5 10 15
Cel
l Vi
abili
ty
(cfu
/ml)
Time (hours)
ln7 & pTetR-LasR-pLux R-S 5-pLux R-E 7
ln7 & pTetR-LasR-pLux R-S 5
ln7 & pTetR-LasR-pLux R-E 7
PAO1 & pTetR-LasR-pLuxR- S5- pLuxR-E7
Killing efficiency: 99%
0
10
20
30
40
50
60
70
80
90
100
S5L S5 E7 PAO1
Perc
enta
ge B
iofil
m C
ell S
urvi
val (
%)
ln7 & pTetR-LasR-pLuxR-S5-pLuxR-E7 (S5L)ln7 & pTetR-LasR-pLuxR-S5 (S5)ln7 & pTetR-LasR-pLuxR-E7 (E7)PAO1 & pTetR-LasR-pLuxR-S5-pLuxR-E7 (PAO1)
A
B ln7 biofilm cultured with E. colipTetR-LasR-pLuxR-S5-pLuxR-E7
ln7 biofilm cultured with E. colipTetR-LasR-pLuxR-S5
ln7 biofilm cultured with E. colipTetR-LasR-pLuxR-E7
ln7 biofilm
Biofilm inhibition: 90%
• Targeted killing, minimizing negative effects on the microbiome
• Preventive effects
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System optimizationAdvancing the system towards more effective killing
Input(Signaling molecules)
Output (Killing program)
Sensor Killing device Release mechanism
Increased Sensitivity
Robust Expression
Enhanced E.coli per pathogen killing ratio
Disrupt Biofilm
Lysis timing for optimal cell killing
Secretion-tag
Pathogenic progress
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100 μm
PATHOGEN-KILLING MICROBESPROTOTYPES - STILL EVOLVING
Quorum Sensing!
Motility!
Killing!
Pathogenic target!
Biofilm degradation and cell killing by secreted DnaseI ( ) an Microcin S ( )!
DnaseI! mcsS!
cheZ!
lasR!
Sensing!
Pathogen seeking E. coli
• ACS Synthetic Biology (2014), Featured in The Economist, Nature News, etc.
[3O
-C12
HL]
M
Version 1 Improved QS
1. Sensitivity and robust expression2. Target localization (motility)3. Improved killing efficiency
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Advanced therapeutic functionalitiesProgrammed motility towards a target pathogen
ACS Synthetic Biology (2014), Featured in The Economist, Nature News, etc.
Quorum Sensing!
Motility!
Killing!
Pathogenic target!
Biofilm degradation and cell killing by secreted DnaseI ( ) an Microcin S ( )!
DnaseI! mcsS!
cheZ!
lasR!
Sensing!
Pathogen seeking E. coli
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PREVENTION OF INFECTION STRATEGIES Enzymatic dispersal of mature biofilm
Untreated P.aeruginosa
P. aeruginosa with engineered E. coli
0
20
40
60
80
100
120
140
PA alone EcN+E7 V2.0
Perc
enta
ge b
iofil
m c
ell (
%)
Engineered E.coli
ACS Syn Biol (2014)
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DELIVERY STRATEGIESTargeted killing by protein secretion
Protein secretion
ACS Syn Biol (2014)
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Pseudomonas aeruginosa
CELLULAR DRUG DELIVERY STRATEGIESMotility via chemotaxis pathway
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FULLY FUNCTIONAL MODELPROGRAMMED MOTILITY-DRIVEN KILLING
ACS Syn Biol (2014)
ΔcheZ
CheZ-YbaQ 0
50
100
150
200
250
∆cheZ CheZ WT (RP437)
CheZ-YbaQ
RFP
fluo
resc
ence
(RFU
)
Tryptone
PAO1 supernatant
Control YebF-DnaseI & YebF-McsS
50 µm
0
20
40
60
80
100
120
140
Control CheZ-YbaQ YebF-MccS & YebF-DnaseI
CheZ-YbaQ & YebF-MccS & YebF-DnaseI
Per
cent
age
rela
tive
Con
trol
(%)
Viable Biofilm cell Biofilm
0
20
40
60
80
100
120
140
Control CheZ-YbaQ YebF-MccS & YebF-DnaseI
CheZ-YbaQ & YebF-MccS & YebF-DnaseI
Perc
enta
ge re
lativ
e to
Con
trol
(%)
Viable PAO1 cells (CFU)
Pathogen-specific Localization Localized Cell Killing
Biofilm degradation Concerted Biofilm degradation and
Biofilm-encapsulated cells
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Antibiotic treatment
gut colonization
0
1
2
3
4
5
0 2 4 6 8 10 P. a
erug
inos
a (lo
g C
FU/g
)
Time (days)
Fecal
0
1
2
3
4
5
0 2 4 6
P. a
erug
inos
a (lo
g C
FU/g
)
Time (day)
Intestine
Programmed therapeutic cells
P.aeruginosa infection Treatment
infection
TEST MODELSKilling effects in eukaryotic models (Mus musculus)
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ALL-IN-A-PAGEWhat we have learned so far and what will come
INPUT• Communication
molecules• Metabolic produce• Cellular byproducts• Environmental cues
OUTPUT• Antibiotics• Metabolic
boosters/inhibitors• Immune boosters• Probiotic cell
maintenance
Metabolic disease
Infection
Cancer