targeted gene mutation of the mvin locus homolog in francisella tularensis lvs l.
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Targeted gene mutation of the mviN locus homolog in Francisella tularensis LVS. Jeffrey Hall Mentor: Dr. Malcolm Lowry Department of Microbiology. What is Francisella ?. Gram (-) coccobacillus Facultative intracellular pathogen Zoonotic disease - Tularemia

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Targeted gene mutation of the mviN locus homolog in Francisella tularensis LVS


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targeted gene mutation of the mvin locus homolog in francisella tularensis lvs
Targeted gene mutation of the mviN locus homolog in Francisella tularensis LVS

Jeffrey Hall

Mentor: Dr. Malcolm Lowry

Department of Microbiology

what is francisella
What is Francisella?
  • Gram (-) coccobacillus
  • Facultative intracellular pathogen
  • Zoonotic disease - Tularemia
    • Rabbit fever, Deer fever
  • Category A bioterrorism agent
    • can be easily disseminated or transmitted from person to person;
    • result in high mortality rates and have the potential for major public health impact;
    • might cause public panic and social disruption; and
    • require special action for public health preparedness.
history of f tularensis
History of F. tularensis
  • Isolated by Edward Francis in 1911 in Tulare county, CA
  • Reported to be part of several countries biological warfare arsenal, including the United States
  • Aerosolization of F. t. by Russia; used against German advancement in WWII
  • Live Vaccine Strain (LVS) - attenuated strain
    • -In 1960’s the US used LVS as vaccine for those
    • in military at highest risk of contracting Tularemia
francisella tularensis method of infection
Francisella tularensisMethod of Infection
  • Francisella infects mainly macrophages and replicates to high numbers intracellulary
  • Ability to infect with as few as 10 CFU
  • Francisella can also infect epithelial cells - mechanism of entry is unknown
  • Molecular basis for evasion of immune response is unknown
  • Three potential virulence genes have been identified:

iglC- no homologues

mglA- transcription factor

pdpD- no homologues.

challenges of francisella
Challenges of Francisella
  • Slow growth, requires supplements to survive (freeze dried hemoglobin, Mueller-Hilton Broth)
  • Most known vectors don’t replicate in Francisella
  • Difficult to introduce foreign DNA
      • electroporation very low efficiency
      • conjugation- possible
  • Much of the genome is still undetermined

Francisella Growing On Chocolate Agar Plate

Francisella on Chocolate agar

method to identify virulence factors
Method toIdentify Virulence Factors

Targeted Gene Mutagenesis

Purpose: To create a knock of the gene 0369c in the mviN loci via a double homologous recombination event

choosing a knock out target
Choosing A Knock-Out Target

mviN operon gene 0369c

An operon that is homologous to a known Coxiella virulence factor

mviN Operon

slide9

Making Knock-Out Mutant

1st Step: Using 4 custom primers and PCR, create 2 fragments of the gene that omit the middle part of the gene

Result:

Lane 1: Gene Ruler 1kb

Lane 2: Gene 0369c Fragment 1-2 (1400bp)

Lane 3: Empty

Lane 4: Gene 0369c Fragment 3-4 (1600bp)

1500bp

gene 0369c

3

1

SalI

ATG

AvrII

Flanking 1300 bp

Flanking 1500 bp

AvrII

Stop

SalI

2

4

Fragment 1-2

Fragment 3-4

slide10

Making Knock-Out Mutant

2nd Step: Clone the Fragments independently into Topo TA pCR 2.1 cloning vector.

1600bp

1400bp

AvrII

AvrII

SalI

SalI

Fragment 3-4 in Topo TA pCR 2.1

Fragment 1-2 in Topo TA pCR 2.1

slide11

Making Knock-Out Mutant

3rd Step A: Using a unique restriction site in the vector, RsrII along with the AvrII restriction site, the plasmids are digested and assayed on a 1% agarose gel.

AvrII

SalI

~3 kb

SalI

AvrII

Fragment 3-4 in pCR 2.1

Fragment 1-2 in pCR 2.1

4 kb

3 kb

RsrII

RsrII

~ 4kb

Step B: Once separated, they are excised from the gel and purified out of agarose.

slide12

Making Knock-Out Mutant

4th Step: The separate pieces are then ligated together to re-create a 7 kb vector

AvrII

SalI

SalI

3 kb truncated gene 0369c

RsrII

ATG

AvrII

AvrII

Stop

SalI

Flanking 1300 bp

Flanking 1500 bp

Truncated gene 0369c

SalI

slide13

Making Knock-Out Mutant

5th Step A: Once the fragments are ligated together, the vector is restricted with SalI to remove the 3 kb piece, gel separated, cut and purified out of the agarose gel, and then ligated with the pPV vector, which is also has restricted with SalI

Sal I

Sal I

Sal I

Sal I

Sal I

Δ0369c 3 kb fragment

=

+

pPV

pPV-Δ0369c

pPV suicide cloning vector

Step B: Transform into DAP- E. coli

slide14

Making Knock-Out Mutant

  • Conjugate E. coli with Francisella LVS
    • (Transfer of plasmid)
  • Harvest and plate on chloramphenicol & Polymyxin B
    • (Selection for Francisella with integrated plasmid,
    • i.e., single cross-over via homologous recombination)

Wild-type 0369c

ATG

Stop

replication

ATG

Stop

Δ

SalI

SalI

pPV-Δ0360c vector

ATG

Stop

ATG

Stop

~200 bp

~2000 bp

1061 bp

Truncated 0369c

pPV vector

Wild-type 0369c

slide15

Making Knock-Out Mutant

  • Grow without selection
    • (Allows for 2nd homologous recombination)
  • Plate on 10% sucrose
    • (Selects for loss of plasmid, carrying sacB)

ATG

Stop

ATG

Stop

~200 bp

~2000 bp

1061 bp

Truncated 0369c

pPV vector

Wild-type 0369c

  • This 2nd recombination event will result in the Δ0369c being left in the chromosome and the vector and wild-type gene being removed

ATG

Stop

ATG

Stop

~200 bp

~2000 bp

1061 bp

Truncated 0369c

pPV vector

Wild-type 0369c

  • This 2nd recombination event will result in the Δ0369c and the pPV vector being removed and the wild-type gene being left behind
slide16

Making Knock-Out Mutant

Final Steps:

  • Replicate plate onto Chloramphenicol plates and no-selection plates
    • (Confirms efficiency of sucrose “counter-selection”)
  • Check for deletion of gene by PCR
    • (Ideally, 50% are WT and 50% are mutants)

Start codon primer

Stop codon primer

Start codon primer

Stop codon primer

Wild-type

mutant

ATG

Stop

Stop

ATG

~200 bp

mutant gene

~ 1000 bp

Wild-type gene

1000 bp

*Representation of gel electrophoresis

200 bp

conclusion
Conclusion
  • The Δ0369c gene construct was created and maintained successfully in E. coli
  • Unsuccessful in transferring the truncated gene into the pPV mutagenesis plasmid
  • Electroporation of Topo-Δ0369c unsuccessful
long term goals
Long Term Goals
  • Create and screen for an 0369c mutant in Francisella tularensis LVS
  • Assess role of the F. tularensis gene 0369c and the mviN operon in its ability to evade and infect macrophage cells
  • Assay will compare mutant vs. LVS, looking at multiplicity of infection (MOI) and length of infection.
  • Infection rate will be analyzed using the Enzyme-Linked ImmunoSorbent Assay (ELISA) technique.
future research
Future Research
  • Focus on continued screening for mutant LVS colonies
  • Generate a greater understanding of Francisella’s virulence mechanisms
  • Possibility for design of a new vaccine against Tularemia
acknowledgements
Lowry Lab

Dr. Malcolm Lowry

Lindsay Flax

Edward Lew

Häse Lab

Dr. Claudia Häse

Markus Boin

Acknowledgements
  • Dr. Kevin Ahern – Program Director
  • Department of Microbiology
  • Howard Hughes Medical Institute