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 homologues mglA- transcription factor pdpD- 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 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 An operon that is homologous to a known Coxiella virulence factor mviN operon gene 0369c mviN Operon

gene 0369c SalI AvrII Making Knock-Out Mutant 1 st 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 1-2Fragment 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 bp AvrIISalI Fragment 1-2 in Topo TA pCR bp

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 SalI AvrII Fragment 3-4 in pCR 2.1 AvrII SalI Fragment 1-2 in pCR 2.1 RsrII ~ 4kb ~3 kb 4 kb 3 kb Step B: Once separated, they are excised from the gel and purified out of agarose.

∆ AvrII Flanking 1500 bp Flanking 1300 bp SalI Making Knock-Out Mutant 4 th Step: The separate pieces are then ligated together to re-create a 7 kb vector AvrII RsrII SalI 3 kb truncated gene 0369c SalI Truncated gene 0369c ATG Stop

Making Knock-Out Mutant 5 th 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 + = pPV suicide cloning vector Step B: Transform into DAP- E. coli pPV Δ0369c 3 kb fragment pPV-Δ0369c

Making Knock-Out Mutant SalI Δ replication ATG Stop ATGStop Truncated 0369c pPV vector Wild-type 0369c ~200 bp~2000 bp1061 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 ATGStop ATGStop Truncated 0369c pPV vector Wild-type 0369c ~200 bp~2000 bp1061 bp Grow without selection (Allows for 2nd homologous recombination) Plate on 10% sucrose (Selects for loss of plasmid, carrying sacB) ATGStop ATGStop Truncated 0369c pPV vector Wild-type 0369c ~200 bp~2000 bp1061 bp This 2 nd recombination event will result in the Δ0369c being left in the chromosome and the vector and wild-type gene being removed This 2 nd 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: ATGStop 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 mutant 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