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Determining the role of the SpiC protein in Salmonella typhimurium infection of the model organism, C. elegans: a phenotypic assay
 

Determining the role of the SpiC protein in Salmonella typhimurium infection of the model organism, C. elegans: a phenotypic assay

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  • Hello, my name is Ajay Singh. I go to pine Crest School and conducted my research Determing the blah bhlabh at Florida Atlantic university
  • Pb; tomate; pet hedgehogs; chicken what do they all have in common? SAL outbreaks
  • A pathogenic gram-negative bacterium that can cause infection if consumed; humans come into contact with bacteria through animals and contaminated food; symptoms--; symptoms are more severe in those with compromised immune systems which may lead to dehydration and deathPathogenic capability of gram (-) due to lipopolysaccharide layer (endotoxin) FOR FJAS:Talk more about global problems of salmonella;. Don’t need patghonec on the bacteria Cantaloupes; peanut butter; ground beef; tomatoes;Diarrhea fever abdominal cramps for 12-72 hrsaftr infection; illness lasts 4-7 days; severe infections in old/infants/bad immune systems (may cause death w/o antibiotics)Salmonella – gram negative bacterium (cell wall); Persisent intestinal infection: achieved by two step process involving SPI 1&2; one is invasion; the other internal replication/survivability; once invaded, resides in SCVs; T3SS2 = SPI2 effector proteins are transferred to cytoplasm; help propagate reproduction and survivabilitySpiC: Part of SPI 2 –Translocon export virulence factors into target cells; Sifs- tubules on SCVs; protect SCVs from interaction with lysosomes (helps keep salmonella alive in cell)Previous studies in mouse models (lack of sifs = attenuated virulence)
  • Enters lumen through consumption; causes inflammation which makes it easier for the bacteria to invade epithelial; Proteins invovled for invasion and inflammation are encoded by SPI 1LOOK UP:how does the inflammation allow
  • Once inside, bacteria is encapsulated in a vacuole from the host cell; the structure created is a Salmonella containing vacuole or SCV.
  • once in the SCV, Salmonella induced filaments are created by the salmonella and extend from the SCV. The SIFs protect the SCV from being destroyed by the host cell’s lysosomes
  • the Type 3 Secretion System 2 is a protein appendage on the salmonella that secretes effector proteins from the salmonella into the eukaryotic host cell; the effector proteins help propagate infection by helping the Salmonella survive and by helping the bacteria internally replicate within the cellFIGURE OUT HOW REPLICATION ACTUALLY OCCURS
  • One protein that seems to have a very important part in the salmonella infection process is SpiCIt regulates translocon proteins which allows salmonella to take effector proteins secreted from its t3ss2 and have them pass through the vacuole into the host cell’s cytosol. The Sifs as previously mentioned, protect the Salmonella containing vacuole from destructionThe roles that SpiC have are important because the t3ss2 protein appendage secretes effector proteins that help with internal replication and survivability of Salmonella within a host cell in previous mamallian studies, salmonella strains that lacked the ability to create Sifs had attenuated virulence in the organism that it infectedSifs- used for protection of SCVs; helps keep SCV and the Salmonella inside intact Salmonella entericaharbours two Salmonella pathogenicity islands (SPIs) each encoding a type III secretion system for virulence proteins. SPI1 is required for invasion, while systemic infections and intracellular accumulation of Salmonella are dependent on SPI2 function. This review will describe and compare the genetic organisation, evolution, regulation and molecular functions of SPI1 and SPI2.
  • So overall, I wanted to PURPOSEMy hypothesis was that HYPOTHESISControl-uninfected on non pathogenic e. coli (common lab food)WT salmonella- use that to compare the differnceExposed for 48 hrs to salmonella; then placed on non-pathogenic e. coli and lifespan experiment of the infected group was conducted. These data indicate that, in contrast toP. aeruginosa, a small inoculum of S. typhimurium canproliferate in the C. elegans intestine and establish apersistent infection.Genetic analysis of host–pathogen interactions hasbeen hampered by the lack of genetically tractablemodels of such interactions.
  • To test my hypothesis: I conducted two trials of my experiment the design of my experiment was using 3 groups of wild type C. elegans, a multicellular nematode commonly used in in vivo experiments involving salmonella infections as it has distinct phenotypic responses, to test my hypothesis. 1 group was exposed to a salmonella strain that has a SpiC protein deletion 2nd group of c. elegans was exposed to wt salmonella 1 group was used as a control as it remained uninfected and stayed on OP50 E. coli food plates throughout the experiment. I will refer to this group as the OP50 controlI sterilized them worms in order to make sure that the worms that I was observing were only the original experimental worms that I had started with and not their progeny. FIX THOSEANImation48 hrs of exposure to infect with Salmonella strainsRelocated to OP50 food plates and lifespan experiment was conductedWhy C. elegans?C. Elegans – multicellular nematode; commonly used for salmonella in vivo experiments; distinct phenotypic ressponsces, increased transparency, decrease in food consumption and motility; intestines of C. elegans become infected and distended (similar to mammalian)Scoring = everyday until the last subject diedFUdR – helped increase accuracy of data by making sure that no progeny of the experimental worms were produced and counted as experimental worms; also decreased the handling of the worms2 trials of experiment were run; experiment consisted of OP50
  • On the first trial, there was a noted differnce in the median and maximum lifespan values between the control and the two salmonella groups. Both salmonella groups had similar and shorter lifespans in comparison to the control groups
  • On the second trial, similar results were found that supported the previous trial as the salmonella groups again had similarly shorter lifespans in comparison to the control group
  • This figure is the survival curve for the 1st trial. The green line is the control, the red line is the wt salmonella group, and the blue line is the spic deletion group. When the surivial curves were compared to each other, there was a significant difference between the survival curves of the control and the two salmonella groups as the p values are much less than .01. when the two salmonella groups were compared to each other, a p valuemuch greater than .5 was found indicating that there is no significant differnce in the surivial curves between the two groups. The second trial supported the first trial’s results as there was signfiicantdiffernce between the salmonella groups and the control surival curves but not a signficinatdiffernce between the two salmonella groups.
  • So in conclusion, my experiment did not support my hypothesis as the deletion of the spic protein did not affect the virulence of Salmonella typhimurium; the spic deleted salmonella strain still caused lethatlity in c. elegans which occurred at similar rate as wtsal, which indicates that it did not attenuate the degree of virulence Another interesting find was that SifsMay not be necessary in the infection of c. elegans because spic has previously been associated with the formation of sifs and sifs have also been shown to be very important in the vireulenceDeleted- still caused death significantly faster than controls; Those on SpiC had similar median and maximum lifespans as well as p values greater than .5; indicating no significant difference in lifespanContradicting previous studies of mammalian testing- either not necessary in C. elegans or Sif production is not fully knocked outCOMBINE THE TWO BULLETS UNDER THE FIRST POINT BECAUSE THEY ARE THE SAME
  • The implications of this research are such that it may help with the development of medicines that can prevent infection or treat infection more effecively. Also there is a focus on a further understanding of Salmonella infection especially focusing on the p-h protein interaction. By learning how these salmonella protiens interact with model orgaminsms such as c. elegans, we will gain a better understanding of how the salmonella interacts within humans. DOUBLE NULL MUTANT KNOCKDOWNWORD CHOICE “observable delay” –c. elegans undergoes a phenotypic response to the infection. As salmonella infection runs its course, the worms became increasingly transparent. During the trials, there was an observable delay in body transpency in the spic salmonella exposed c. elegans group in comparison to the group exposed to the wild type salmonella. Since salmonella infection
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Determining the role of the SpiC protein in Salmonella typhimurium infection of the model organism, C. elegans: a phenotypic assay Determining the role of the SpiC protein in Salmonella typhimurium infection of the model organism, C. elegans: a phenotypic assay Presentation Transcript

  • Determining the role of the SpiC protein in Salmonella typhimurium infection of the model organism, C. elegans: a phenotypic assay Ajay Singh Pine Crest School Florida Atlantic University S
  • Salmonella Outbreaks!
  • BackgroundS Salmonella typhimurium = problem for humans S Pathogenic gram negative bacterium S Persistent intestinal infection if consumed S Symptoms: S Diarrhea, vomiting, fever, abdominal cramps S Life threatening to those with compromised immune systemsS How does infection occur?
  • Salmonellacontainingvacuole (SCV) Vacuole
  • SIF Vacuole LysosomeSIF
  • Vacuole Effector proteins
  • BackgroundS SpiC S Regulator of T3SS2 translocon proteins S Has a role in formation of Salmonella induced filaments (SIFs)S Type 3 Secretion System 2 (T3SS2) S Helps with internal replication and survivability of Salmonella within a host cellS Lack of SIFs = attenuated virulence in mammalian studies
  • Purpose and HypothesisS Purpose: To determine the importance of the role of SpiC protein in Salmonella tyhpimurium infection using C. elegans as a model organismS Hypothesis: The deletion of SpiC from Salmonella typhimurium will decrease the virulence and lethality of the Salmonella infection.
  • Materials and MethodsS Design S 3 groups of N2 (Wild type) C. elegans S ∆SpiC Salmonella S Wild-type Salmonella S OP50 (control) (remained uninfected)S Scoring: determining the viability of each wormS Application of Fluorodeoxyuridine (FUdR): sterilization of experimental worms to control population
  • Results Data Table for Trial 1Strain Subjects at risk Median lifespan Maximum (# of subjects (days) lifespan (days) censored)N2 OP50 62 (18) 23 32N2 ∆SpiC 72 (8) 20 28SalmonellaN2 WT Salmonella 63 (17) 20 29
  • Results Data Table for Trial 2Strain Subjects at risk Median lifespan Maximum lifespan (# of subjects (days) (days) censored)N2 OP50 53 (27) 27 40N2 ∆SpiC 51 (29) 25 36SalmonellaN2 WT Salmonella 65 (15) 26 36
  • Trial 1 P-Value N2 OP50 vs. N2 0.0001* ∆SpiC Salmonella N2 OP50 vs. N2 <0.0001* WT Salmonella N2 WT 0.5381 Salmonella vs. N2 ∆SpiC * Denotes significance SalmonellaTrial 2 P-ValueN2 OP50 vs. 0.0016*N2 ∆SpiCSalmonellaN2 OP50 vs. 0.0008*N2 WTSalmonellaN2 WT 0.9810Salmonella vs.N2 ∆SpiC * Denotes
  • ConclusionsS Deletion of SpiC = unaffected virulence of Salmonella typhimurium S Deletion does not affect infection’s lethality in C. elegans S Similar rate of death as WT Salmonella groupS Salmonella induced filaments (Sifs) S Possibly not necessary in infection of C. elegans S Formation of Sifs may not be solely reliant on SpiC gene
  • Future Work and ImplicationsS Test ∆SifA and ∆SopD2 Salmonella strains on C. elegans S Proteins related to Sif formationS Development of medicines and further understanding of Salmonella infection S Focused on the pathogen-host protein interaction
  • Works CitedS Alejandro Aballay, Peter Yorgey, Frederick M. Ausubel, (2000) Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans, Current Biology, Volume 10, Issue 23, Pages 1539-1542S Freeman JA, Rappl C, Kuhle V, Hensel M, Miller SI. 2002. SpiC is required for translocation of Salmonella pathogenicity island 2 effectors and secretion of translocon proteins SseB and SseC. J. Bacteriol. 184:4971–4980.S Ibarra, J Antonio & Steele-Mortimer, Olivia. (2009). Salmonella--the ultimate insider. Salmonella virulence factors that modulate intracellular survival. Cellular microbiology, 11.S Kailiang Jia, Collin Thomas, Muhammad Akbar, Qihua Sun, Beverley Adams-Huet, Christopher Gilpin, and Beth Levine (2009) Autophagy genes protect against Salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance PNASS Labrousse, A., Chauvet, S., Couillault, C., Kurz, C. L., Ewbank, J. J., (2000) Caenorhabditis elegans is a model host for Salmonella typhimurium, Current Biology, Volume 10, Issue 23, Pages 1543-1545
  • Thank You!