virulence factors in francisella noatunensis : pdpa
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Virulence factors in Francisella noatunensis : pdpA. Karina Ray Hansen Lab. Overview. Francisella introduction Virulence factors Gene cloning Future directions. Francisella tularensis. Gram-negative, fastidious Highly infectious Tularemia, aka “rabbit fever” Intracellular life cycle. - PowerPoint PPT PresentationTRANSCRIPT
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