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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

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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



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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


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Section I

Background Information on Multi-Host

Pathogenesis System


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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


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P. aeruginosa

Strain PA14

P. aeruginosa Multi-Host Pathogenesis System

Humans

Mice

Plants

Nematodes

Insects


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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


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P. aeruginosa Kills Galleria mellonella (Wax Moth Caterpillar) Larvae; LD50 = 1


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Section II

Background Information on

Screening Methodology and

Rationale for Constructing the

Uni-Gene Library


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PA14

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


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6 Mb

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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~6 Mb

Unigene Library Size

6 Mb genome

(S. cerevisiae)

4800 non-essential genes

5 fold saturation

24,000 insertions

Recovery failure

30,400 insertions


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~6 Mb

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


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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 Non-Essential Genes

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 Non-Essential Genes

TnPhoA: Kanr/Neor

E. Coli

PA14

LB + Irgasan + Neomycin

(3,000-5,000 colonies)


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Library Construction: Colony Picking/Culture Non-Essential Genes

  • Inoculate 250 µL

  • LB + Irgasan [50 µg/mL]

  • Kanamycin [200 µg/mL]*

  • Grow 40 hrs at 37°C

  • (no shaking)


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Library Construction: Biomek-Automated Liquid Manipulation Non-Essential Genes

-80°C Storage

-20°C 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 Non-Essential Genes

B Side: Human Readable

A Side: Unique ID#

PA14_PhoA_100_xxx

wor

sup

mas

dup

ar1

ar2

seq


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LEGEND Non-Essential Genes

Genomic DNA

Transposon

Transposon-specific Primer

Arbitrary PCR Primers

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 Non-Essential Genes

Supernatant (sup)

Thaw, 99°C/6 min.,

pellet 3K/5 min.

3µL supnt

Temporary

Storage

-20°C

Arb PCR 1 (ar1)

Arb PCR 1


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Library Construction: Details of ARB2 PCR Non-Essential Genes

ar1

5µL

Temporary

Storage

-20°C

Arb2 PCR (ar2)

ARB2 PCR



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Library Construction: PCR Cleanup Non-Essential Genes

ar2

7µL

Temporary

Storage

-20°C

Sequencing plate (seq)

+ ExoSAP-IT

15’ at 37°C

15’ at 80°C


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Library Construction: Sequencing Non-Essential Genes

seq

Add Sequencing primer

to a [final] of 5 ng/µL and Seal

Send to DNA Core for Sequencing

(Store at 4°C)


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Example of High Quality Sequence Non-Essential Genes

TnPhoA

Sequence Length + Mixed + TnPhoA = Sequence Success Index


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Example of Low Quality Sequence Non-Essential Genes

TnPhoA


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Optimization of [Taq] in Sequencing Reactions Non-Essential Genes

Sequencing Success Index


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Unigene Library Mutant Identification Optimized for: Non-Essential Genes

  • 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


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Relevant Background Sequence: Non-Essential Genes

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 (cont’d) Non-Essential Genes

NNNNNNNNN

ARB PRIMER Sequence


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Trouble Shooting: Buried ARB Sequence Non-Essential Genes

NNNNNNNNN

ARB PRIMER Sequence

High Quality

Sequence


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Relevant Background Sequence: Buried ARB Primer Sequence Non-Essential Genes

1

3

2

1

1

2

2

3

2

1

1

1

2

2

+


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Library Construction:Time 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 plates) 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 plates)

Development of a Linux MySQL

Uni-Gene Library Relational Database


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Unigene Library: plates)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? plates)

  • 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? plates)

  • 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? plates)

  • 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? plates)

  • 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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Plate plates)

ProcessPlateLink

PlateID

ExecutionID

PlateType

PlateID

InOrOut

ProcessExecution

ExecutionID

ProcessType


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Plate plates)

ProcessPlateLink

ProcessExecution

PlateID

ExecutionID

ExecutionID

PlateType

PlateID

ProcessType

InOrOut

Protocol

PlateSample

Mutant

RawSequence

MutID

SampleID

RawSeqID

PlateID

Library

MutantID

Well Position

ChromatPath

MutantID

Sequence


How will the database analyze the data l.jpg

C plates)hromatogram

Phred

Raw Sequence

Quality Scores

Trimmed Sequence

How will the Database Analyze the Data?


How will the database analyze the data49 l.jpg

Processed Sequence plates)

Trimmed Sequence

How will the Database Analyze the Data?

Remove transposon, vector, and/or other contaminant sequences.

BLAST PAO1 genome

BLAST PAO1 annotated ORFs


How will the database analyze the data50 l.jpg
How will the Database Analyze the Data? plates)

  • 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 l.jpg
How will we Retrieve/View the Contents of the Database? plates)

  • 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? plates)

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? plates)

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 l.jpg
Future Steps plates)

  • 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 plates)

  • 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 l.jpg

Phred plates)

Chromatogram

Remove transposon, vector, and/or other contaminant sequences.

Raw Sequence

Quality Scores

BLAST PAO1 genome

BLAST PAO1 ORFs

Processed Sequence

Trimmed Sequence

Trouble Shooting:Example Test Case

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


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A: plates)aroE

B: braB

C: coxA

D: dnaA

E: exoT

F: fabF

G: galE

H: hmgA

Trouble Shooting:Example Test Case


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A: plates)aroE

B: braB

C: coxA

D: dnaA

E: exoT

F: fabF

G: galE

Plate 2

Plate 3

H: hmgA

Trouble Shooting:Example Test Case

Plate 1


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Example Test Case: plates)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 : ---


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“System Requirements” plates)

  • 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 l.jpg
Database Summary plates)

  • 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.


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Section V plates)

CF Mouse Oropharynx Colonization Model


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Utility of transgenic CF mice for identifying novel plates)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 acquisition of

  • 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 acquisition of 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 acquisition 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

P < .001

0

mucD mutant

complemented

in trans

mucD

mutant

Wild-type

PA14

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.


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The acquisition of 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


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Another mutant of acquisition 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


Summary of cf mouse model l.jpg
Summary of CF Mouse Model acquisition of

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.


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