Transcript What is Francisella? - Oregon State University

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
- 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
•
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 homologues
mglA- transcription factor
pdpD- no homologues.
Challenges of Francisella
• Slow growth, requires supplements
to survive (freeze dried hemoglobin, MuellerHilton Broth)
• Most known vectors don’t replicate
in Francisella
Francisella Growing On
Francisella
on Chocolate
Chocolate
Agar Plateagar
• Difficult to introduce foreign DNA
> electroporation very low efficiency
> conjugation- possible
• Much of the genome is still undetermined
Method to
Identify 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
mviN operon
gene 0369c
An operon that is homologous to a known Coxiella virulence factor
mviN
Operon
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)
gene 0369c
SalI
1
ATG
AvrII
3
Flanking 1300 bp
Flanking 1500 bp
AvrII
2
Fragment 1-2
Stop
SalI
4
Fragment 3-4
1500bp
Making Knock-Out Mutant
2nd Step: Clone the Fragments independently into Topo TA pCR 2.1 cloning vector.
SalI
1600bp
Fragment 3-4
in Topo TA
pCR 2.1
AvrII
AvrII
1400bp
Fragment 1-2
in Topo TA
pCR 2.1
SalI
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
SalI
AvrII
Fragment 3-4
in pCR 2.1
Fragment 1-2
in pCR 2.1
~3 kb
4 kb
3 kb
RsrII
RsrII
~ 4kb
Step B: Once separated, they are excised from the gel and purified out of agarose.
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
SalI
Flanking 1500 bp
ATG
AvrII
∆ AvrII
Stop
Truncated gene 0369c
Flanking 1300 bp
SalI
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
Δ0369c
3 kb
fragment
Sal I
Sal I
+
pPV
pPV suicide cloning vector
Step B: Transform into DAP- E. coli
=
pPV-Δ0369c
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
~200 bp
~2000 bp
Truncated
0369c
pPV
vector
ATG
1061 bp
Wild-type
0369c
Stop
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
~200 bp
~2000 bp
Truncated
0369c
pPV
vector
ATG
1061 bp
Stop
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
~200 bp
~2000 bp
Truncated
0369c
pPV
vector
ATG
1061 bp
Stop
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
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)
ATG
Stop
Wild-type
Stop codon
primer
mutant
Start codon
primer
Start codon
primer
ATG
~200 bp
mutant gene
Stop codon
primer
Stop
~ 1000 bp
Wild-type gene
1000 bp
*Representation of gel electrophoresis
200 bp
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
• 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