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Similar a virulence factors F. noatunensis
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virulence factors F. noatunensis
- 1. Virulence factors in Francisella noatunensis: pdpA Karina Ray Hansen Lab
- 2. Overview Francisella introduction Virulence factors Gene cloning Future directions
- 3. N L 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
- 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 Francisella tularensis Francisella noatunensis
- 6. 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
- 7. Gene Cloning Transform vector plasmid into F. noatunensis via electroporation pdpA flanks KANAMYCIN resistance AMPICILLIN resistance sacB suicide vector Pir-dependent ORI AMPr
- 8. XX X Amp resistance Sucrose sensitivity Mutant allele of target gene target gene Resolution Co-integrate Step 1 Step 2 “Knockout—loss of function”
- 9. KAN KAN + 7% sucrose 10-1 10-2 10-3 Counter-selection for resolution on sucrose
- 10. pdpA ∆pdpA v1.0 Kanamycin 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
- 11. ∆pdpA v. 1 100 bp 1kb 100 bp 1kb Wt 5’ flank fwd Wt pdpA rev Wt pdpA fwd Wt 3’ flank rev KANA fwd Wt 3’ flank rev Wt 5’ flank fwd KANA rev WT
- 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 Zebrafish challenges (to test for attenuation) pdpA knockouts iglC knockout (from Soto lab) Wild type Genetic complementation To restore function of pdpA
- 14. Thank you Mary Gates Research Foundation
Notas del editor
- Mutagenesis has been a valuable tool for elucidating the functions of different genes in an organism of interest. As we are interested in finding the genes responsible for Francisella noatunensis virulence and by extension ones that would cause attenuation during infection we chose the tried and true method of mutagenesis. There are various mutagenesis techniques that can be used but the question being asked drives which one should be utilized. Directed allows you to target one specific gene of interest while leaving all others unaffected through homologous recombination for example. This would be a good approach if we already had some leads as to what genes we suspected were involved in virulence. Undirected or random method transposons can be inserted randomly into the genome and then the sites affected mapped to the original genome. Using this generated randomly mutagenized library a host selective method can be used uniquely tagged transposon mutagenized bacteria which are then used to infect a host organism. After infection has set up but before it is cleared, single celled suspensions can be made of the affected organs and the bacteria recovered and plated. The recovered pool would then be compared to the original pool of mutants introduced into the host and any bacteria that are missing would be presumed to have a virulence defect.