Virulence factors in Francisella noatunensis: pdpA

Karina RayHansen Lab

Overview Francisella introduction Virulence factors Gene cloning Future directions

NL

Francisella tularensis Gram-negative, fastidious Highly infectious

Tularemia, aka “rabbit fever” Intracellular life cycle

Electron microscopy Francisella

Clinical symptom of tularemia

Category select A agent

by CDC Vaccine ~30% effective

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

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

Francisella tularensis

Francisella noatunensis

pdpC

pdpA

pdpB

iglDigl

CiglBigl

Apd

pD treAfba

Apgk

anmK

Hypothesis: loss of function of pdpA gene in F. noatunensis will cause attenuation

analogous to the attenuation observed in pdpA knockouts of F. tularensis

Identify pdpA as potential vaccine target

Gene Cloning Transform vector plasmid into F. noatunensis via electroporation

pdpA flanks KANAMYCIN resistance AMPICILLIN resistance sacB suicide vector Pir-dependent ORI pdpAKANr

AMPr

sacB

XX

XAmp resistance

Sucrose sensitivity

Mutant allele of target gene

target gene

Resolution

Co-integrate

Step 1

Step 2

“Knockout—loss of function”

KAN

KAN + 7% sucrose

10-1 10-2 10-3

Counter-selection for resolution on sucrose

pdpA

∆pdpA v1.0Kanamycin Cassette

Genotyping candidate knockouts

Confirm by amplifying the entire region using the wt flanking primers and sequence

wt-5’-Flank-Fwd wt-pdpA-Rev

wt-pdpA-Fwd wt-3’Flank-Rev

Kana-Revwt-5’-Flank-Fwd

wt-3’Flank-RevKana-Fwd

∆pdpA v2.0Kanamycin Cassette

Kana-“Fwd”wt-5’-Flank-Fwd

wt-3’Flank-RevKana-“Rev”

ATG

ATG

ATG

∆pdpA v. 1

100 bp 1kb

100 bp 1kb

Wt 5’ flank fwdWt pdpA rev

Wt pdpA fwdWt 3’ flank rev

KANA fwdWt 3’ flank rev

Wt 5’ flank fwdKANA rev

WT

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

Future Directions

Zebrafish challenges (to test for attenuation) pdpA knockouts iglC knockout (from Soto lab) Wild type

Genetic complementation To restore function of pdpA

Thank you

Mary Gates Research Foundation