EXPERIMENTAL PROTOCOL: This exercise will engage students in examining uptake of
ID: 196397 • Letter: E
Question
EXPERIMENTAL PROTOCOL:
This exercise will engage students in examining uptake of green fluorescent-tagged bacteria by macrophage cells and unveil the endocytic mechanism used by them. Half of the class will assess bacterial endocytosis by macrophages at different time points, while the other half of the class will test the impact of a endocytosis-altering compound on bacterial uptake.
Macrophages human cells
Latrunculin A stock solution
1. Create a summary of the overall approach you use to answer the question being addressed.
2. After you have considered the data, write 1-3 sentences below that to summarize your interpretation of the findings.
3. Include a statement about the future scientific directions suggested by your findings from the experiment (suggest future experiments using as base the results obtained in the lab)
4. Creat a grapgh with data given. STANDARD DEVIATION AND AVERAGE.
different incubation time 0 min 30 min 15 min 30 min 30 minMacrophages human cells
9.2 (group 1) 2.4 (group 4) 5.4 (group 11) 0 (group 10) 14.4 (group 12)Latrunculin A stock solution
3.4 (group 6) 3.4 (group 7) 2.8 (group 9) 9.8 (group 3) 7.0 (group 2) different incubation time 15 min 30 min 0 min 0 min 15 minExplanation / Answer
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that invades macrophages and induces caspase-1-mediated host cell death in vitro and in vivo. Caspase-1-deficient mice have a 1000-fold higher oral LD50 than wild-type animals and even with high oral doses are remarkably resistant to systemic bacterial dissemination, demonstrating that activation of caspase-1 is a crucial component of bacterial pathogenesis in vivo. Caspase-1-mediated cell death, termed pyroptosis, is a newly identified pathway of programmed cell death that inherently produces an inflammatory outcome, but the molecular mechanism of cell death is poorly understood.
Caspase-1, also known as interleukin (IL)-1-beta-converting enzyme, is a cysteine protease that cleaves and activates the pro-forms of host inflammatory cytokines, IL-1 and IL-18. Both cytokines are processed and released by S. Typhimurium-infected macrophages, but neither is required for macrophage death. Active IL-1 and IL-18 are inflammatory mediators that potently stimulate fever, recruitment of immune cells, enhancement of T and NK cell function, and production of diverse secondary cytokines. Thus, macrophage pyroptosis during Salmonella infection may contribute to the inflammation and tissue destruction observed in vivo, via caspase-1-dependent production of active IL-1 and IL-18 and release of inflammatory intracellular contents during lysis.
Results:
Nuclease- and caspase-1-dependent DNA cleavage in Salmonella-infected macrophages
Degradation of chromosomal DNA is a terminal event in the life of a cell and a hallmark of cell death. Additionally, DNA cleavage can be mechanistically coupled to cell lysis. To investigate the molecular mechanism of DNA fragmentation during pyroptosis, DNA cleavage in S.Typhimurium-infected J774A.1 macrophages was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) reaction. Macrophages infected for 4 h with wild type, but not non-cytotoxic SPI-1-deficient prgH mutant S. Typhimurium, demonstrate DNA damage. Control macrophages induced to undergo apoptosis by treatment with gliotoxin demonstrate similar levels of DNA cleavag. The nuclease primarily responsible for DNA fragmentation during apoptosis, caspase-activated DNase (CAD), is present in living cells bound to the inhibitor ICAD. Caspase-mediated cleavage of ICAD results in release and activation of CAD, and recombinant caspase-1 cleaves ICAD in vitro. We hypothesized that ICAD proteolysis may lead to DNA fragmentation during pyroptosis, and therefore examined ICAD degradation by immunoblot. Although the abundance of full-length ICAD protein decreased in apoptotic macrophages treated with gliotoxin, detectable ICAD degradation did not occur in TUNEL positive macrophages infected with wild-type S. Typhimurium. These results indicate DNA fragmentation during pyroptosis is unlikely to involve the apoptotic mechanism regulated by ICAD proteolysis.
DNA cleavage in S. Typhimurium-infected macrophages is caspase-1- and nuclease-dependent. J774A.1 macrophages were treated with PBS or uniformly infected with S. Typhimurium at a moi of 10:1 for 4 h in the presence of a specific caspase-1 inhibitor (YVAD) or a nuclease inhibitor (ATA). Adherent cells were labelled with the TUNEL reaction and visualized by fluorescence microscopy. As a positive control for nucleolytic DNA fragmentation, cells were treated with 1 × 103 U ml1 DNase I and DNA cleavage by DNase I was inhibited in the presence of ATA. As a negative control, treatment of cells with the combination of 5 mM H2O2 and 1 mM SNAP causes DNA fragmentation that is not inhibited by ATA. A minimum of 378 cells were examined for each experimental condition; means and standard deviations are shown. Results are representative of five separate experiments.Lysis of S. Typhimurium-infected macrophages is independent of PARP activity and DNA fragmentation. J774A.1 macrophages were uniformly infected with S. Typhimurium at a moi of 10:1 in the presence of the PARP inhibitor 3AB (A) or the endonuclease inhibitor ATA (B) and (C). LDH released into the supernatant by dying macrophages after 6 h of infection was quantified (A) and (C) and the means and standard deviations from triplicate samples are shown. LDH release by macrophages treated with 1 mM H2O2 requires PARP activity and is inhibited by 3AB, in contrast to S. Typhimurium-infected macrophages (A). DNA fragmentation in cells infected by S.Typhimurium for 4 h was assessed with the TUNEL reaction (B) and LDH release by infected macrophages is not prevented by ATA-mediated inhibition of DNA cleavage. Results are representative of at least three experiments for each (A, B and C).
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