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

Jeffrey Hall

Mentor: Dr. Malcolm Lowry

Department of Microbiology

• 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.

What is Francisella?

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

Transmission

Francisella tularensis Method 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 homologuesmglA- transcription factorpdpD- no homologues.

Challenges of Francisella

• Slow growth, requires supplements to survive (freeze dried hemoglobin, Mueller-Hilton Broth)

• Most known vectors don’t replicate in Francisella

Francisella on Chocolate agar

• Difficult to introduce foreign DNA> electroporation very low efficiency> conjugation- possible

• Much of the genome is still undetermined

Francisella Growing On Chocolate Agar Plate

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

An operon that is homologous to a known Coxiella virulence factor

mviN operon gene 0369c

mviN Operon

gene 0369c1

2

3

4SalI

AvrII

AvrII

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

Flanking 1500 bp

Flanking 1300 bp

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

SalI

Fragment 1-2 Fragment 3-4

ATG

Stop

Making Knock-Out Mutant

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

SalI AvrII

Fragment 3-4 in Topo TA pCR 2.1

1600bp AvrII SalI

Fragment 1-2 in Topo TA pCR 2.1

1400bp

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.

Making Knock-Out Mutant

SalIAvrII

Fragment 3-4 in pCR 2.1

AvrII SalI

Fragment 1-2 in pCR 2.1

RsrIIRsrII

~ 4kb

~3 kb

4 kb

3 kb

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

∆ AvrIIAvrII

Flanking 1500 bp

Flanking 1300 bpSalI

Making Knock-Out Mutant

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

AvrII

RsrII

SalI

3 kb truncated

gene 0369c

SalI SalI

Truncated gene 0369c

ATG Stop

Making Knock-Out Mutant5th 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

=

pPV suicide cloning vector

Step B: Transform into DAP- E. coli

pPV

Δ0369c 3 kb

fragmentpPV-Δ0369c

Making Knock-Out Mutant

SalI SalIΔ

replication

ATG

ATG

ATG

Stop

Stop

Stop

ATG Stop

Truncated 0369c

pPV vector

Wild-type 0369c

~200 bp ~2000 bp 1061 bp

Wild-type 0369c

pPV-Δ0360c vector

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)

Making Knock-Out Mutant

ATG Stop ATG Stop

Truncated 0369c

pPV vector

Wild-type 0369c

~200 bp ~2000 bp 1061 bp

Grow without selection (Allows for 2nd homologous recombination)

Plate on 10% sucrose (Selects for loss of plasmid, carrying sacB)

ATG Stop ATG Stop

Truncated 0369c

pPV vector

Wild-type 0369c

~200 bp ~2000 bp 1061 bp

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

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

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)

Making Knock-Out Mutant

Final Steps:

ATG Stop

Start codon primer

Stop codon primer

~200 bp

mutant gene

ATG Stop

Start codon primer

Stop codon primer

~ 1000 bp

Wild-type gene

200 bp

1000 bp

mu

tan

t

Wild

-type

*Representation of gel electrophoresis

• 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

Conclusion

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

• 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

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