Salmonella II Flashcards
What are the characteristics of the late SCV in salmonella?
-pH=5 -low Ca2+ -lacks certain lysosomal proteases -Sifs! (Salmonella induced filaments=MTs) formed inside host cells -contain vacuolar ATPase (endosomal markers) -associated with cytoskeleton structures
How does the salmonella-containing vacuole (SCV) mature?
Entry into epithelial cell through splash/trigger mechanisms➡️early SCV➡️intermediate SCV➡️late SCV (sifs= salmonella induced filaments…membrane tubules)➡️MT cytoskelatal elements
How is the late phagosome different than the late SCV?
SCV doesn’t have cathepsin D, which is a protease that degrades insulin
How can you determine the genetic requirements for intracellular growth?
Screen for mutant that grow in broth but not in mice: -signature tagged mutagenesis (STM) -transposon each with unique signature tag….get 1000 strains -each into a well of 96 well plate…pool together, inject into mouse, recover bacteria from spleen, pcr and see which mutants didn’t grow in mice.
Where in the genome were the signature tagged mutagenesis (STM) mutants that tell us which genes are required for intracellular growth?
SPI2! -the T3SS functions in maintaining SCV. -SPI2- mutant = higher LD50 in both oral and IP
How is the T3SS of SPI2 regulated?
Translocon secreted at pH=5 (effectors= SseB, SseC, SseD) Degradation of inhibitory regulator complex at pH=7 (Effectors= SsaL, SsaM, SpiC Degradation of inhibitory complex releases effectors for secretion: SseF, SseJ, etc
What’s An example of a SPI2 effector?
SifA, which encodes for sifs (salmonella induced filaments of MTs)
What is the function of SifA (effector of SPI2)?
SifA forms complex with SKIP (SifA and kinesin interacting protein) SKIP binds kinesin motor protein = MT Sifs of the SCV bind to the MT via kinesin and moves it along the MTs. This is the SifA-SKIP complex.
How is the expression of SPI2 genes regulated?
Two component system! Found out through transcriptional fusion of SPi2 gene promoter to GFP. Infect macrophages. SsrA= sensor kinase SsrB=response regulator Low pH: bafikomycin inhibits SPI2 expression Low Ca2+: chelators of Ca2+ stimulate SPI2 expression
What happens if there’s an SsrA- mutant?
No sensor kinase of two component system….no SPI2 genes expressed.
How does EnvZ/OmpR regulate gene expression of SPI2?
It’s a two component system that encodes for a TF that regulates the two component system of SsrA and SsrB which is the two component system for expression of SPI2
