Virulence factors in Francisella noatunensis : pdpA

1. Virulence factors in Francisella noatunensis: pdpA Karina Ray Hansen Lab

2. Overview • Francisella introduction • Virulence factors • Gene cloning • Future directions

3. Francisella tularensis • Gram-negative, fastidious • Highly infectious • Tularemia, aka “rabbit fever” • Intracellular life cycle Clinical symptom of tularemia • Category select A agent by CDC • Vaccine ~30% effective N L Electron microscopy Francisella

4. Francisella noatunensis • Emerging disease: farm and wild fish • Atlantic cod, tilapia, Atlantic salmon, Hybrid Striped bass • Causes large economic loss in fishing industry • Genetically similar to F. tularensis Ottem et al, 2007 Granulomas from Fran+ cod

5. Virulence Factors • Francisella Pathogenicity Island (FPI) • Genes required for bacteria to cause disease • Intracellular growth locus (iglC) • Pathogenicity determining protein (pdpA) • >90% sequence identity • Two directions anmK pdpA pdpD fbaA pdpB pdpC treA iglD iglA iglC iglB pgk Francisella tularensis Francisella noatunensis

6. Hypothesis: loss of function of pdpA gene in F. noatunensiswill cause attenuation • analogous to the attenuation observed in pdpA knockouts of F. tularensis • Identify pdpA as potential vaccine target

7. Gene Cloning • Transform vector plasmid into F. noatunensisvia electroporation • pdpA flanks • KANAMYCIN resistance • AMPICILLIN resistance • sacB suicide vector • Pir-dependent ORI pdpA KANr AMPr sacB

8. Amp resistance X Sucrose sensitivity Step 1 target gene Mutant allele of target gene Co-integrate Step 2 X X Resolution “Knockout—loss of function”

9. Counter-selection for resolution on sucrose KAN + 7% sucrose KAN 10-1 10-3 10-2

10. Genotyping candidate knockouts pdpA ATG wt-5’-Flank-Fwd wt-pdpA-Rev wt-pdpA-Fwd wt-3’Flank-Rev ∆pdpA v1.0 ATG Kanamycin Cassette ∆pdpA v2.0 wt-5’-Flank-Fwd wt-5’-Flank-Fwd Kana-“Fwd” Kana-Rev ATG Kanamycin Cassette Kana-“Rev” Kana-Fwd wt-3’Flank-Rev wt-3’Flank-Rev Confirm by amplifying the entire region using the wt flanking primers and sequence

11. ∆pdpA v. 1 Wt 5’ flank fwd Wt pdpA rev Wt pdpA fwd Wt 3’ flank rev WT 100 bp 1kb Wt 5’ flank fwd KANA rev KANA fwd Wt 3’ flank rev 100 bp 1kb

12. Project Summary • Ligate insert into plasmid • Two directions • Electroporate Francisella with plasmid • Identify recombinant colonies using selective media • CHA+KAN, CHA+KAN+7% Sucrose • Confirm PCR, sequencing

13. Future Directions • Zebrafishchallenges (to test for attenuation) • pdpAknockouts • iglC knockout (from Soto lab) • Wild type • Genetic complementation • To restore function of pdpA

14. Thank youMary Gates Research Foundation