Virulence factors

in Francisella

noatunensis:

pdpA

Karina Ray

Hansen 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

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

AMPr

XX

X

Amp 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 v2.0

Kanamycin 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 fwd

Wt pdpA revWt pdpA fwd

Wt 3’ flank rev

KANA fwd

Wt 3’ flank rev

Wt 5’ flank fwd

KANA 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