Transcript No Slide Title

MGH-PGA
Genomic Analysis of Stress and Inflammation:
Pseudomonas aeruginosa Infection
Nicole T. Liberati, Dan G. Lee, Jacinto M. Villanueva
and Frederick M. Ausubel
Department of Molecular Biology
Massachusetts General Hospital
Carolyn Cannon, Fadie Coleman, Mike Kowalski
Jeff Lyczak, Martin Lee, Gloria Meluleni, and Gerald Pier
Channing Laboratory
Brigham and Women’s Hospital
Ausubel/Pier PGA Project
Colonization of the lungs of cyst ic fibrosis (CF) pat ient s by the opportunist ic bacterial
pathogen Pseudom onas aeruginosa is the major cause of mortality in CF populations.
The overall goal of this project is to identify novel P. aeruginosa virulence factors
involved in lung infect ions in CF pat ients. The novel st rategic approach that we have
developed involves the use of alternat ive non-vertebrate hosts in which we can model P.
aeruginosa pathogenesis. We have identified a P. aeruginosa isolat e (st rain PA14) that
is not only infectious in several mouse models, including an oropharynx colonization
model in transgenic CF mice, but also causes disease in the insects Drosophila
m elanogaster and Galleria m ellonella (wax moth caterpillar) and in t he plant Arabidopsis
thaliana. P A14 also kills t he nematode Caenorhabditis elegans. Remarably, most of the
P. aeruginosa genes that have been tested that are required for killing in t he insect,
nematode or plants models are also required for pat hogenesis in mice. One advantage of
using non-vertebrate host s is that thousands of bacterial clones from a mutagenized
library can be individually screened for avirulent mutants in separate host organisms. In
this way, the ent ire set of P. aeruginosa non-essent ial genes can be readily surveyed for
ones that encode virulence-related factors. Our specific st rategy is to randomly
mutagenize P. aeruginosa st rain PA14 wit h transposon TnphoA, determine the sites of
insert ion of approximat ely 30,000 mutants, and from this collect ion, choose a subs et of
mutants corresponding to a single t ransposon insert ion in each annotated gene. This socalled “ uni-gene” library consist ing of approximately 4,800 members will be screened for
avirulent mutant s using the nematode and wax moth killing models. The mutant s
ident ified in this screen will subsequent ly be screened in the CF mouse model.
Virulence-related genes identified in this screen may encode suitable targets for novel P.
aeruginosa anti-infect ive compounds.
Contents of Slide Show:
Section I: Background Information on Multi Host Pathogenesis
System
Section II: Background Information on Screening Methodology
and Rationale for Constructing the Uni-Gene Library
Section III: Progress Report on Uni-Gene Library Construction
and Detailed Methodology
Section IV: Development of a Linux MySQL Uni-Gene Library
Relational Database
Section V: CF Mouse Oropharynx Colonization Model
Section I
Background Information on Multi-Host
Pathogenesis System
Pseudomonas aeruginosa
• Gram-negative rod
• Found throughout the environment in soil, water and plants
• Opportunistic human pathogen:
- Nosocomial pulmonary infections
- Immune compromised patients (chemotherapy/burns)
- 85% of adult CF patients suffer from chronic pulmonary
P. aeruginosa infections
P. aeruginosa Multi-Host
Pathogenesis System
Humans
Mice
P. aeruginosa
Strain PA14
Plants
Nematodes
Insects
P. aeruginosa Kills C. elegans
and Colonizes the C. elegans Intestine
100
P. aeruginosa
E. coli
% Nematodes Killed
80
60
40
20
0
0
20
40
60
Hours of Feeding on
P. aeruginosa strain PA14
80
P. aeruginosa Kills Galleria mellonella
(Wax Moth Caterpillar) Larvae; LD50 = 1
Section II
Background Information on
Screening Methodology and
Rationale for Constructing the
Uni-Gene Library
Identification of P. aeruginosa Virulence Factors
by Screening “UniGene” Library for Mutants that
do not Kill Wax Moth Caterpillars or Nematodes
PA14
Random Transposon Mutagenesis
Sequence Insertion Sites and Identify a Non-Redundant “Unigene” Set
Screen Unigene Set for Mutants that Do Not Kill C. elegans or Wax Moths
Test Mutants that Do Not Kill C. elegans in CF Mouse Model
Generation of Transposon Insertion Mutations
Transposase
E. Coli
Transposon: Kanr
6 Mb
PA14
Select for insertions with Kanamycin
Unigene Library:
A collection of P. aeruginosa strains
containing a disruption in each
non-essential open reading frame (ORF)
in the P. aeruginosa genome
Wild type
Mutant #1
Mutant #2
Unigene Library Size
6 Mb genome
(S. cerevisiae)
4800 non-essential genes
~6 Mb
5 fold saturation
24,000 insertions
Recovery failure
30,400 insertions
Selection of Unigene Library Mutants
30,400 insertions
Approximately 5 hits per ORF:
~6 Mb
Choose the most 5’ disruption
within the actual coding sequence
~4800 catalogued Unigene mutants
Advantages of Unigene Library Screening
1) Mutation previously identified
2) Limited number of mutants to screen (4800)
3) Non-redundant mutations
4) Built-in confirmation of the involvement of
known pathways.
5) Easy to confirm the importance of the mutated
gene using other mutant alleles.
Section III
Progress Report on Uni-Gene Library
Construction and Detailed Methodology
Generation of Unigene Library of Transposon
Insertions in Non-Essential Genes
Pick ~30,000 colonies with Qbot into bar-coded 96- well plates
containing media + selective antibiotics
Grow overnight
25 ml for arbitrary
(ARB) PCR reactions
Add glycerol to 15%
Divide into 3 plates:
384-well (Master copy)
384-well (Duplicate copy)
96-well (Working copy)
Current Status of the Unigene Library
1) 48 x 96 (4608) mutants created.
60% of the insertion sites identified.
2) Insertion site identification protocol optimized.
(1152 mutants created and identified in 2.5 weeks)
3) Accompanying database is operational.
Quality assurance testing is in progress.
Library Construction: Mutagenesis/Plating
TnPhoA: Kanr/Neor
E. Coli
PA14
LB + Irgasan + Neomycin
(3,000-5,000 colonies)
Library Construction: Colony Picking/Culture
• Inoculate 250 µL
LB + Irgasan [50 µg/mL]
Kanamycin [200 µg/mL]*
• Grow 40 hrs at 37°C
(no shaking)
Library Construction: Biomek-Automated Liquid Manipulation
-80°C Storage
Working (wor)
Master (mas)
Duplicate (dup)
Culture (wor)
(280 µL)
70 µL
Add glycerol
Mix and Seal
-20°C Storage
Supernatant (sup)
Library Construction: Bar Coding
B Side: Human Readable
A Side: Unique ID#
PA14_PhoA_100_xxx
wor
sup
mas
dup
ar1
ar2
seq
Library Construction: Arbitrary PCR to Amplify
Sequence Adjacent to Transposon Insertion
1
2
3
LEGEND
Genomic DNA
1st PCR Reaction
1
2
Transposon
Transposon-specific Primer
2nd PCR Reaction
PCR Cleanup and Sequencing
Arbitrary PCR Primers
Library Construction: Details of ARB1 PCR
Supernatant (sup)
Thaw, 99°C/6 min.,
pellet 3K/5 min.
3µL supnt
Arb PCR 1 (ar1)
Arb PCR 1
Temporary
Storage
-20°C
Library Construction: Details of ARB2 PCR
ar1
5µL
Arb2 PCR (ar2)
ARB2 PCR
Temporary
Storage
-20°C
Library Construction: PCR Cleanup: EXOSAP-IT
Library Construction: PCR Cleanup
ar2
7µL
Sequencing plate (seq)
+ ExoSAP-IT
15’ at 37°C
15’ at 80°C
Temporary
Storage
-20°C
Library Construction: Sequencing
seq
Add Sequencing primer
to a [final] of 5 ng/µL and Seal
Send to DNA Core for Sequencing
(Store at 4°C)
Example of High Quality Sequence
TnPhoA
Sequence Length + Mixed + TnPhoA = Sequence Success Index
Example of Low Quality Sequence
TnPhoA
Optimization of [Taq] in Sequencing Reactions
300
Sequencing Success Index
2.5U
1.25U
250
0.6125U
200
150
100
50
0
Mutant 1
Mutant 2
Mutant 3
Mutant 4
Mutant 5
Mutant 6
Mutant 7
Mutant 8
Unigene Library Mutant Identification Optimized for:
• Taq Manufacturer
Roche vs. Promega vs. Prepared Master Mixes
• Final Taq Concentration
1.25 U sufficient
• PCR Master Mix Preparations
Fresh Master Mixes vs Stored (4°C) Master Mix
• Hybaid vs. MJ Research PCR Machines
• PCR Cleanup Protocol
ExoSAP-IT vs. Clontech NucleoSpin
Relevant Background Sequence:
Template Independent Genomic Sequence
1
2
3
Template-Specific
Tn/Genomic Sequence
1
2
3
No Sequence
2
3
A Template-Independent
Genomic Sequence
2
High Quality Sequence (cont’d)
NNNNNNNNN
ARB PRIMER Sequence
Trouble Shooting: Buried ARB Sequence
NNNNNNNNN
ARB PRIMER Sequence
High Quality
Sequence
Relevant Background Sequence: Buried ARB Primer Sequence
1
3
2
1
1
2
2
3
1
2
1
2
+
1
2
Library Construction:Time Line for 4608 colonies (48 sup plates)
Time
Mutagenesis/growth on Qbot plate
3 days
Qbot picking/growth in 96 well culture plate
2 days
Biomek
1 day
ARB1/ARB2 Reactions/PCR Cleanup/Seq prep
10 days
Sequencing
?
Total
For 7 sets of 48 plates:
16+ days
114 days
P. aeruginosa PA14 Virulence-Related Factors
Involved in Mammalian Pathogenesis Identified in
Non-Vertebrate Hosts
Category
#
Genes
Regulators
6
gacA, gacS, algU, plcR, ptsP, lasR
Membrane Protein
1
aefA
Biosynthetic Enzymes
3
phzB, hrpM, fabF
Modifying Enzyme
1
dsbA
Multi-Drug Transport
2
mexA, mtrR
Type III Secretion
1
pscD
Helicases
2
phoL, lhr
Unknown Proteins
16
?
Section IV
Development of a Linux MySQL
Uni-Gene Library Relational Database
Unigene Library:
Overview of Bioinformatics
Catalog each sample in relational database
Retrieve DNA sequence for each sample
Process DNA sequence to remove low-quality and contaminant
sequences (i.e. - vector)
BLAST searches to distinguish:
a. Pseudomonas sequence vs.
contaminants.
b. PA01 vs. PA14 sequences.
BLAST searches to identify:
a. Disrupted ORF.
b. Coding sequence vs. putative
promoter disruption.
What will the MySQL Database Do?
1) Store/catalog all of the data.
2) Process DNA sequences and perform BLAST
searches.
3) Display the results and allow for user queries.
How will the Database Store the Data?
• The data will be stored in a relational database.
• Individual tables can be thought of as separate
Excel spreadsheets with rows and columns.
• The tables are connected to each other via specified
relationships.
How will the Database Store the Data?
• Tables will be populated (i.e. - individual cells in the
table will be filled with entries) as plates, samples,
and/or data are generated.
• Data entry into the Database will be “restricted” to
parallel the creation of the physical library.
– Order of different types of inputs is restricted.
– Prevent duplicate entries.
How will the Database Store the Data?
• The Database will store organizational information:
–
–
–
–
–
Date created.
Created by.
Storage locations.
Bacterial strain.
Mutagen/Transposon used.
• The Database will store experimental data:
–
–
–
–
DNA sequences obtained by PCR.
Location of insertion with respect to PAO1 genome.
Identity of PAO1 ORF disrupted.
Phenotypic data?
ProcessPlateLink
ProcessExecution
Plate
ExecutionID
ExecutionID
PlateID
PlateType
PlateID
InOrOut
ProcessType
PlateID
PlateType
ExecutionID
PlateID
InOrOut
ExecutionID
Process
Type
5
PCR1
12
5
In
12
ARB2
PCR
6
PCR2
12
6
Out
ProcessPlateLink
ProcessExecution
Plate
ExecutionID
ExecutionID
PlateID
PlateType
PlateID
InOrOut
ProcessType
Protocol
PlateSample
SampleID
PlateID
RawSequence
Well Position
Mutant
RawSeqID
MutantID
MutID
MutantID
ChromatPath
Library
Sequence
How will the Database Analyze the Data?
Chromatogram
Phred
Raw Sequence
Trimmed Sequence
Quality Scores
How will the Database Analyze the Data?
Trimmed Sequence
Remove transposon,
vector, and/or other
contaminant sequences.
Processed Sequence
BLAST PAO1
genome
BLAST PAO1
annotated ORFs
How will the Database Analyze the Data?
• Other BLAST searches that can be performed in the
future:
– Internal BLAST against the contents of the database to
identify siblings vs. adjacent independent insertions.
– BLAST against other public databases to determine gene
identity of ORFs not found in PAO1.
How will we Retrieve/View the Contents of
the Database?
• Current status:
– A web-accessible table viewer can allow us to examine
the contents of each table in the database.
– To organize and search the contents, the html file can be
opened in Excel and then sorted.
• Future goals:
– A web-accessible browser with multiple query and view
options.
How will we Retrieve/View the Contents of
the Database?
Types of queries:
–
–
–
–
–
–
Insertions in a given gene.
Insertions upstream of a given gene.
Insertions near a given gene.
Insertions near a given physical location.
Insertions in PAO1 non-coding sequences.
Insertions in sequences NOT found in PAO1.
–
–
–
–
Insertions in genes of a particular pathway/family.
Insertions in PAO1 ORFs of known function.
Insertions in putative PAO1 ORFs of unknown function.
Multiple queries.
How will we Retrieve/View the Contents of
the Database?
Options for Viewing Database Contents:
– Table view in alphabetical order.
– Table view in linear order.
– Graphical view with ORF orientation and transposon
orientation (zoom in/out/, click on ORF or transposon,
etc).
Future Steps
• Select members for unigene (non-redundant)
library.
• Physically pick members for unigene library.
• Store, duplicate and disseminate unigene library.
• Incorporate non-PAO1 sequences into unigene set.
• “Completing” the unigene set (targeted deletions,
inducible antisense?).
Trouble Shooting
• Test cases - designed to test either the entire
system (i.e. - start to finish) or a particular module.
• Can be designed to test “ideal” inputs or
“incorrect” inputs.
• Example:
– Input 96 (unknown) chromatogram files that contain a few known
sequences at defined well positions.
– Determine if the expected BLAST hits are associated with the
appropriate well position.
– This tests:
• Ability to process the chromatogram files.
• Ability to correctly perform BLAST searches.
• Ability to correctly map the resulting BLAST search onto the correct
well-position.
Trouble Shooting:
Example Test Case
Chromatogram
After chromatograms are
retrieved, is each wellposition mapped correctly
as the sequences are
processed and BLASTed?
Phred
Raw Sequence
Quality Scores
Trimmed Sequence
Remove transposon, vector, and/or
other contaminant sequences.
Processed Sequence
BLAST PAO1 genome
BLAST PAO1 ORFs
Trouble Shooting:
Example Test Case
A: aroE
B: braB
C: coxA
D: dnaA
E: exoT
F: fabF
G: galE
H: hmgA
Trouble Shooting:
Example Test Case
A: aroE
B: braB
C: coxA
D: dnaA
E: exoT
F: fabF
G: galE
H: hmgA
Plate 1
Plate 2
Plate 3
Example Test Case:
Expected BLAST Results
1A1 : aroE
1A2 : --1A3 : --1A4 : --1A5 : --1A6 : --1A7 : --1A8 : --1A9 : --1A10 : --1A11 : --1A12 : ---
1B1 : braB
1B2 : --1B3 : --1B4 : --1B5 : --1B6 : --1B7 : --1B8 : --1B9 : --1B10 : --1B11 : --1B12 : ---
1C1 : coxA
1C2 : --1C3 : --1C4 : --1C5 : --1C6 : --1C7 : --1C8 : --1C9 : --1C10 : --1C11 : --1C12 : ---
1D1 : dnaA
1D2 : --1D3 : --1D4 : --1D5 : --1D6 : --1D7 : --1D8 : --1D9 : --1D10 : --1D11 : --1D12 : ---
“System Requirements”
• Programming language / database platform.
– Microsoft Access vs. MySQL.
• Backups and database restore.
• Storage issues.
– Each plate generates ~30MB of chromatograms (I.e. - 3 X 96
chromatograms on a zip disk).
– Each chromatogram spawns several types of data: a raw sequence,
a quality score for each nucleotide of the raw score (~2.67 MB for an
average 96-well plate), a processed sequence, and blast results.
• Documentation of database development and test
cases.
Database Summary
• Data storage is mostly complete. Needs some
testing.
• Sequence analysis is currently being tested.
– Once it’s operational, sequence analysis will have to be
updated to include more complex scenarios (i.e. sequences not found in PAO1).
• Data retrieval/viewing is currently undeveloped.
• Non-redundant (unigene) library is undeveloped.
Section V
CF Mouse Oropharynx Colonization Model
Utility of transgenic CF mice for identifying
novel P. aeruginosa virulence factors
• To date, no apparent phenotype relevant to
acquisition and establishment of chronic P.
aeruginosa infection has been found in a variety of
transgenic CF mouse lines
• CF mice given acute P. aeruginosa infections
manifest increased inflammation and pathology but
do not get chronic infections
Our approach: try to recapitulate method of natural
acquisition of P. aeruginosa by CF patients
Aspects of chronic oropharyngeal colonization in mice
• Maintain mice on water with antibiotic to prevent P. aeruginosa
growth in water-0.1 mg gentamicin/ml
• Treat mice for 5-7 days with 250 ug levofloxacin/ml in drinking
water
– Eliminates oropharyngeal colonization by a mucoid Enterobacter that
grows on Pseudomonas isolation media and interferes with P. aeruginosa
colonization
– Remove 48 hrs prior to introduction of P. aeruginosa
Aspects of chronic oropharyngeal
colonization in mice
• Colonize mice by placing 107 CFU P. aeruginosa/ml
drinking water for 5 days
• Remove contaminated water, culture mouse throats,
give sterile water for 1 week followed by water
containing 0.1 mg gentamicin/ml to keep bacteria
from growing in it
• Monitor mice by throat culture every 1-2 weeks.
P. aeruginosa strain PA14 chronically colonizes
oropharynx of CF, but not wild-type, C57Bl/6 mice
Mouse Oropharyngeal Colonization Model
CWP
Percent
Positive
Throat
Cultures
100
80
CWP
60
C57sBl/6
CF mice
CWP
40
20
0
1
3
6
8
10 12
15
17
19
21
23
26
29
Time (Weeks) After Infection
CWP= contaminated water/Pseudomonas
A contribution of algD to pathogenesis is shown in a mouse
thermal injury model--the double mucD/algD mutant is more
attenuated for virulence than the mucD mutant alone
100
Percent
that
develop
sepsis
80
60
P=.06
40
20
0
P < .001
Wild-type
PA14
mucD
mutant
mucD mutant
complemented
in trans
algD
mutant
mucDalgD
double mutant
P. aeruginosa strain
From: Yorgey P, Rahme LG, Tan MW, Ausubel FM. The roles of mucD and alginate in the virulence of
Pseudomonas aeruginosa in plants, nematodes and mice. Mol Microbiol. 2001 Sep;41(5):1063-1076.
The algD mutant of P. aeruginosa PA14 fails to
chronically colonize the oropharynx of CF mice
C57Bl/6 WT-mice
CF mice
WT-PA14 in transgenic CF mice
CWP
Percentage
colonized
100
80
CWP
60
CWP
40
20
0
1
3
6
8 10 12 15 17 19 21 23 26 29
PA14 DalgD in transgenic CF mice
100
80
60
40
20
0
1
3
7
11
Time (Weeks) After Infection
CWP= contaminated water/Pseudomonas
15
17
19
21
23
Another mutant of P. aeruginosa PA14, with an interruption in
the gacA (global accessory regulator) gene, previously
shown to have reduced virulence in the multi-host pathogen
system, also has a reduced ability to chronically colonize the
oropharynx of CF mice
100
C57Bl/6 WT-mice
Percentage
colonized
80
CF mice
60
40
20
0
1
3
5
7
11
15
19
21
23
25
Time (Weeks) After Infection
27
Summary of CF Mouse Model
A model of chronic P. aeruginosa oropharyngeal colonization
in CF mice has been developed and tested for applicability for
confirming the role of P. aeruginosa multi-host virulence
factors.