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/


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?


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


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?


POTENTIAL SOLUTIONS?

Develop new classes of antibiotics

Diversify our antimicrobial strategies– Exploiting synthetic biology approaches?


RESEARCH OBJECTIVE

• Intentional design– Programmable behaviors

• Sensing, seeking and killing a target pathogen


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)


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


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


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


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


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


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


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)


DELIVERY STRATEGIESTargeted killing by protein secretion

Protein secretion

ACS Syn Biol (2014)


Pseudomonas aeruginosa

CELLULAR DRUG DELIVERY STRATEGIESMotility via chemotaxis pathway


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


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)


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

reprogramming biological functionalities for autonomous …€¦ · • metabolic...

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