MGHPGA

1
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 Womens
Hospital
2
Ausubel/Pier PGA Project
3
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
4

• Section I
• Background Information on Multi-Host
• Pathogenesis System

5
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
6
P. aeruginosa Multi-Host Pathogenesis System
Humans
Mice
Plants
Nematodes
Insects
7
P. aeruginosa Kills C. elegans and Colonizes the
C. elegans Intestine
100
P. aeruginosa
E. coli
80
60
Nematodes Killed
40
20
0
0
20
40
60
80
Hours of Feeding on P. aeruginosa strain PA14
8
P. aeruginosa Kills Galleria mellonella (Wax Moth
Caterpillar) Larvae LD50 1
9
Section II Background Information on Screening
Methodology and Rationale for Constructing
the Uni-Gene Library
10
Identification of P. aeruginosa Virulence
Factors by Screening UniGene Library for
Mutants that do not Kill Wax Moth Caterpillars
or Nematodes
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
11
Generation of Transposon Insertion Mutations
Transposase
E. Coli
Transposon Kanr
PA14
Select for insertions with Kanamycin
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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
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Unigene Library Size
6 Mb genome
(S. cerevisiae)
4800 non-essential genes
5 fold saturation
24,000 insertions
Recovery failure
30,400 insertions
14
Selection of Unigene Library Mutants
30,400 insertions
Approximately 5 hits per ORF Choose the most 5
disruption within the actual coding sequence
4800 catalogued Unigene mutants
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Advantages of Unigene Library Screening

• Mutation previously identified
• Limited number of mutants to screen (4800)
• Non-redundant mutations
• Built-in confirmation of the involvement of known
  pathways.
• Easy to confirm the importance of the mutated
  gene using other mutant alleles.

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Section III Progress Report on Uni-Gene
Library Construction and Detailed Methodology
17
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)
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Current Status of the Unigene Library

• 48 x 96 (4608) mutants created.
• 60 of the insertion sites identified.
• 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.

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Library Construction Mutagenesis/Plating
TnPhoA Kanr/Neor
E. Coli
PA14
LB Irgasan Neomycin
(3,000-5,000 colonies)
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Library Construction Colony Picking/Culture

• Inoculate 250 µL
• LB Irgasan 50 µg/mL
• Kanamycin 200 µg/mL
• Grow 40 hrs at 37C
• (no shaking)

21
Library Construction Biomek-Automated Liquid
Manipulation
-80C Storage
-20C Storage
Culture (wor) (280 µL)
70 µL
Working (wor)
Add glycerol Mix and Seal
Supernatant (sup)
Master (mas)
Duplicate (dup)
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Library Construction Bar Coding
B Side Human Readable
A Side Unique ID
PA14_PhoA_100_xxx
wor sup mas dup ar1 ar2 seq
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Library Construction Arbitrary PCR to
Amplify Sequence Adjacent to Transposon Insertion
3
2
1
1
2
1st PCR Reaction
2nd PCR Reaction
PCR Cleanup and Sequencing
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Library Construction Details of ARB1 PCR
Supernatant (sup)
Thaw, 99C/6 min., pellet 3K/5 min.
3µL supnt
Temporary Storage -20C
Arb PCR 1 (ar1)
Arb PCR 1
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Library Construction Details of ARB2 PCR
ar1
5µL
Temporary Storage -20C
Arb2 PCR (ar2)
ARB2 PCR
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Library Construction PCR Cleanup EXOSAP-IT
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Library Construction PCR Cleanup
ar2
7µL
Temporary Storage -20C
Sequencing plate (seq) ExoSAP-IT
15 at 37C
15 at 80C
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Library Construction Sequencing
seq
Add Sequencing primer to a final of 5 ng/µL
and Seal
Send to DNA Core for Sequencing (Store at 4C)
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Example of High Quality Sequence
TnPhoA
Sequence Length Mixed TnPhoA Sequence
Success Index
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Example of Low Quality Sequence
TnPhoA
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Optimization of Taq in Sequencing Reactions
Sequencing Success Index
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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 (4C) Master Mix
• Hybaid vs. MJ Research PCR Machines
• PCR Cleanup Protocol
• ExoSAP-IT vs. Clontech NucleoSpin

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Relevant Background Sequence Template
Independent Genomic Sequence
3
2
1
Template-Specific Tn/Genomic Sequence
1
2
3
No Sequence
3
2
A Template-Independent Genomic Sequence
2
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High Quality Sequence (contd)
NNNNNNNNN
ARB PRIMER Sequence
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Trouble Shooting Buried ARB Sequence
NNNNNNNNN
ARB PRIMER Sequence
High Quality Sequence
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Relevant Background Sequence Buried ARB Primer
Sequence
1
3
2
1
1
2
2
3
2
1
1
1
2
2

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Library ConstructionTime Line for 4608 colonies
(48 sup plates)
Time 3 days 2 days 1 day 10 days ? 16
days
Mutagenesis/growth on Qbot plate Qbot
picking/growth in 96 well culture plate Biomek
ARB1/ARB2 Reactions/PCR Cleanup/Seq
prep Sequencing Total
For 7 sets of 48 plates
114 days
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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 ?
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Section IV Development of a Linux MySQL Uni-Gene
Library Relational Database
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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
• Pseudomonas sequence vs. contaminants.
• PA01 vs. PA14 sequences.

• BLAST searches to identify
• Disrupted ORF.
• Coding sequence vs. putative promoter disruption.

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What will the MySQL Database Do?

• Store/catalog all of the data.
• Process DNA sequences and perform BLAST searches.
• Display the results and allow for user queries.

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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.

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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.

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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?

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How will the Database Analyze the Data?
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How will the Database Analyze the Data?
Remove transposon, vector, and/or other
contaminant sequences.
BLAST PAO1 genome
BLAST PAO1 annotated ORFs
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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.

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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.

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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.

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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).

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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?).

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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.

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Trouble ShootingExample Test Case

• After chromatograms are retrieved, is each
  well-position mapped correctly as the sequences
  are processed and BLASTed?

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Trouble ShootingExample Test Case
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Trouble ShootingExample Test Case
Plate 1
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Example Test CaseExpected 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 ---
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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.

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Database Summary

• Data storage is mostly complete. Needs some
  testing.
• Sequence analysis is currently being tested.
• Once its 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.

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Section V CF Mouse Oropharynx Colonization Model
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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

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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

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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.

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P. aeruginosa strain PA14 chronically colonizes
oropharynx of CF, but not wild-type, C57Bl/6 mice
Mouse Oropharyngeal Colonization Model
CWP
100
Percent Positive Throat Cultures
80
CWP
C57sBl/6
60
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
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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
80
Percent that develop sepsis
60
P.06
40
20
P lt .001
0
mucD mutant complemented in trans
Wild-type PA14
mucD mutant
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 Sep41(5)1063-1076
.
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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
PA14 DalgD in transgenic CF mice
CWP
100
100
80
80
CWP
Percentage colonized
60
60
CWP
40
40
20
20
0
0
1
3
7
11
15
17
19
21
23
1
3
6
8
10
12
15
17
19
21
23
26
29
Time (Weeks) After Infection
CWP contaminated water/Pseudomonas
69
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
80
CF mice
60
Percentage colonized
40
20
0
1
3
5
7
11
15
19
21
23
25
27
Time (Weeks) After Infection
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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.