Quorum Sensing Disruption through pvdQ...
Transcript of Quorum Sensing Disruption through pvdQ...
Quorum Sensing Disruption
through pvdQ Production
Overview
1. Introduction 2. Project Design 3. Results 4. Human Practice
Input
Chemical
Signals
Processing Output
Chemical
Signals
MICROBIAL INTELLIGENCE
ANTIBIOTICS
HUMAN INTELLIGENCE
A GLOBAL
CRISIS:
ANTIBIOTIC
RESISTANCE
… but bacteria were more intelligent
A SINGLE
BACTERIUM
A COLONY OF BACTERIA
BIOFILMS
1. Attach to the Surface
2. Secrete Extra Polymeric Substance
3. Signal the Synthesis of Effector Molecules
4. Regulate Pathways -> Hyper Inflammation
BIOFILM FORMATION MECHANISM
http://botserv1.uzh.ch/microbio/site/research/introduction/Biofilm.php
EFFECTS OF BIOFILMS ON BACTERIA
• Slow penetration of toxic substances
• Accelerated horizontal gene transfer
• Slow growth rate
• Metabolically inert state
Antibiotic
Treatment
Not Effective
QUORUM SENSING MECHANISM
1. Reach a threshold cell density
2. AHL production
3. AHL concentration reaches threshold
4. AHL binds to regulatory protein
5. Transcription activation
http://www.lifesci.dundee.ac.uk/groups/sarah_coulthurst/research-areas
Solution
Signal molecule does not reach critical concentration
Virulence protein is not transcribed
AH
L C
on
cen
trat
ion
->
Threshold
LACTONASE vs ACYLASE
Lactonase: Ring opening of AHL -> AHL reforms [under acidic conditions] Acylase: 1. Fatty Acyl Chain: subsequently metabolized 2. Homoserine Lactone: growth inhibitor of bacteria
Pseudomonas. Volume 5: A Model System in Biology, Chapter 15 (2007)
pvdQ Isolation
Pseudomonas
aeruginosa
pvdQ Acylase
http://www.esrf.eu/UsersAndScience/Publications/Highlights/2010/sb/sb19
1. Constitutive pvdQ expression
EXPRESSION SYSTEMS
2. AHL inducible pvdQ expression (BBa_K855001)
EXPRESSION SYSTEMS
lacI+pL luxR pvdQ Lux pR
LuxR pvdQ
AHL
1. Constitutive pvdQ expression
2. AHL inducible pvdQ expression (BBa_K855001)
Cloning pvdQ Expression pvdQ Activity
Assay
Project Plan
Cloning pvdQ Expression pvdQ Activity
Assay
• A 18 bp his tag is attached to the C – terminal of the beta chain
Alpha Beta Signal peptide Spacer peptide
PAO1 pvdQ gene
His
tag
•A 18bp his tag is attached to the C – terminal of the beta chain • Remove the internal PstI site at (1,489-1,494 bp) by silent mutation.
• K855005: CTGCAG > CTCCAG Leucine codon • K855006: CTGCAG > CTGCAA glycin codon
Alpha Beta
Signal peptide Spacer peptide
PAO1 pvdQ gene
His
tag PstI
Cloning pvdQ Expression pvdQ Activity
Assay
1. BBa_ K855000:
2. BBa_ K855001:
3. BBa_ K855002:
pvdQ
pvdQ
Cloning pvdQ Expression pvdQ Activity
Assay
In PROGRESS: 1. BBa_K855005: PstI site removed version of K855000 by site
directed mutagenesis at leucine codon
2. BBa_K855006: PstI site removed version of K855000 by site directed mutagenesis at glycine condon.
3. Bba_K855007: Structural intermediate of AHL inducible system
4. BBa_K855008: AHL inducible production of pvdQ (BBa_K855005)
5. Bba_K855009: AHL inducible production of pvdQ (Bba_K855006)
Cloning pvdQ Expression pvdQ Activity
Assay
BL21 BL21
Cloning pvdQ Expression pvdQ Activity
Assay
Whole Cell Sonication Osmotic Shock
Cloning pvdQ Expression pvdQ Activity
Assay
1. Whole Cell Sonication Osmotic Shock
2. Supernatant
3. Pellet
4. Osmotic Shock Fluid
Cloning pvdQ Expression pvdQ Activity
Assay
1. Whole Cell Sonication Osmotic Shock
2. Supernatant
3. Pellet
4. Osmotic Shock Fluid
SDS-Page
Western Blot using Anti-His tag
Cloning pvdQ Expression pvdQ Activity
Assay
Alpha Beta
Signal peptide Spacer peptide
PAO1 pvdQ gene
His
tag
18 kDa 60 kDa
Cloning pvdQ Expression pvdQ Activity
Assay
Cloning pvdQ Expression pvdQ Activity
Assay
Sonication Whole Cell
Sonication 1. Whole Cell
2. Supernatant
3. Pellet
Cloning pvdQ Expression pvdQ Activity
Assay
Sonication 1. Whole Cell
2. Supernatant
3. Pellet
Controls: -Boiled Fractions :
4. Whole Cell
5. Sonication Supernant
6. Sonication Pellet
7. PBS
-Indication of natural
degradation of AHL
Cloning pvdQ Expression pvdQ Activity
Assay
15 ug AHL 0 ug AHL
100 μl sample
30 ℃ , 4 hours, 70 r.p.m shaker
AHL Assay
Cloning pvdQ Expression pvdQ Activity
Assay
Colorimetric
Determination of intact
AHL concentration
• Quick & Easy • Reliable linearity between the Absorbance (520nm) and AHL concentration
y = 0.0035x + 0.0022 R² = 0.9761
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
Ab
sorb
ance
(5
20
nm
)
Amount of C12 – AHL (ug)
C12-AHL Assay Standard Curve
Cloning pvdQ Expression pvdQ Activity
Assay
• Expressed pvdQ protein is functional. • It displays acylase activity that degrades C12-AHL
Cloning pvdQ Expression pvdQ Activity
Assay
Future Directions 1. Characterize the AHL inducible pvdQ production
system (BBa_K855001) . Construct the constitutive design. Compare the efficiency.
2. Extracellular secretion system of pvdQ.
3. Encapsulation of PseudoKill using alginate beads.
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References:
Bokhove, M., Jimenez, P. N., Quax, W. J., Quax, W. J., & Dijkstra, B. W. (2010). The quorum-quenching n-acyl homoserine lactone acylase pvdq is an ntn-hydrolase with an unusual substrate-binding pocket. PNAS, 107(2), 686–691. Yang, Y. H., Lee, T. H., Kim, J. H., Kim, E. J., Joo, H. S., Lee, C. S., & Kim, B. G. (2006). High-throughput detection method of quorum-sensing molecules by colorimetry and its applications. Analytical Biochemistry, (356), 297-299. Kievit, T. R. (2009). Quorum sensing in pseudomonas aeruginosa biofilms. Environmental Microbiology, 11(2), 279–288. doi: 10.1111/j.1462-2920.2008.01792.x Choi, J. H., & Lee, S. Y. (2004). Secretory and extracellular production of recombinant proteins using escherichia coli. Appl Microbiol Biotechnol, (64), 625–635. doi: 10.1007/s00253-004-1559-9 Wahjudi, M., Mariana, E., Hendrawati, O., Assen, A. H. G. V., Merkerk, R. V., Cool, R. H., & Poelarends, G. J. (2011). Pa0305 of pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the ntn hydrolase superfamily.Microbiology, (157), 2042-2055. doi: 10.1099/mic.0.043935-0