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lec 20 - listeria II

Terms in this set (41)

What is the function of the PEST-like sequence?
Hypothesis: The LLO PEST-like sequence targets LLO for degradation in the host cytosol
--> the idea is that the PEST sequence is protecting LLO from killing host cell once listeria gets into cytosol
--> this hypothesis is not correct
The amino terminus of mature LLO contains three MAP kinase consensus target sites
the left is the N terminus, underlined is what we think is the PEST sequence. the three boxed sequences are perfect consensus sequences for recognition by MAP kinases
--> so its thought that these portions may be targeted by host kinases (maybe the PEST sequence is phosphorylated [by the kinase] which leads to its inactivation, either by degradation or simply phosphorylation)
--> it's bc they have serine-threonine residues, which is what kinases tend to target
-we have various mutants (single, double, & triple) compared to Δ26 (where entire PEST sequence gone)
why do phosphorylated sites (map kinase target sites) affect LLO activity?
the thought is that the PEST sequence might be phosphorylated, and that might lead to its degradation
-or maybe phosphorylation itself affects its activity
S44 (serine #44) within the PEST region is the most important MAP kinase site for intracellular growth
-typically if you change a serine or threonine to an alanine, it will prevent it from being phosphorylated
-the Δ26 results in death of bug (obvious) bc the bug makes a lot of LLO and the LLO (which breaks host cell membrane) allows gentamicin in which results in death of the bug
-there is a 100 fold difference in CFUs between LLO that lacked the three kinase recognition sites (brown line) vs. wild type
-S44 by itself (green line) was significantly different from wildtype as well
Is the toxic phenotype caused by S44A mutation due to the amino acid change or the codon?
S44 = serine 44 (codon TCT)
S44A = serine changed to alanine (codon GCT)
S44S = serine changed to a serine by three nucleotide changes (codon AGC) but still serine
L. monocytogenes LLOS44S is toxic!
turns out that S44S (serine changed to serine of diff codon) phenotype is toxic.
we made a synonymous change that resulted in a large change.
-WT listeria (first block) causes v little cell death (3% cells die)
-listeria missing S44A (serine -> alanine) causes more death (47% cells die)
-listeria w/ conservative AA change (S44A, serine -> serine) causes even MORE death (68% die) -> bizarre
-> Δ26 kills almost all cells (expected)
The PEST region regulates LLO synthesis under conditions that simulate cytosolic growth
Δ26 and S44A (and S44S as it turns out) make a lot more LLO protein under cytosolic conditions
The PEST-like region regulates LLO synthesis by controlling protein synthesis, not RNA synthesis
the amount of protein is changing for each mutation but the amount of RNA for each isn't changing
--> so the PEST sequence is affecting translation (not transcription) of LLO
How is LLO activity compartmentalized to avoid killing host cells?
•PEST-like sequence stops LLO translation during growth in the cytosol
-the mRNA of LLO forms a stemloop
•A single base pair mutation in the predicted mRNA stem causes an increase LLO translation that results in toxicity to host cell and consequent loss of virulence
--> so it looks like the part of the mRNA where there's the S44 codon forms a stem-loop; this stem loop was responsible for stopping protein synthesis (bc the stem stalls the ribosome) ; when you mutate the stem, stem doesn't form, translation isn't stopped, & LLO continues to be translated
--> by affecting mRNA structure we're affecting translation
--> now it makes sense that a conservative mutation didn't matter. it was the sequence that mattered bc that is what allows proper base pairing of stem loop
The multiple ways that the PEST-like sequence compartmentalizes LLO activity?
the PEST sequence does a lot of things:
•Prevents translation of LLO in the cytosol; synonymous codon mutations of PEST (e.g. S44S) don't stop translation i.e. are toxic to host
•PEST affects LLO pH optimum (to a small extent)
•PEST mediates phosphorylation of cytosolic LLO (we don't know if this is important)
•PEST may mediate proteolysis (breaking down) of LLO
•PEST mediates endocytosis of LLO pores that form on the cytosolic membrane by binding to a component of clathrin-dependent endocytosis (not shown) (LLO helps cell heal membrane holes)
--> all of these help prevent toxicity to the host cell
Cell mediated immunity to L. monocytogenes (technically the next lecture)
•Mice immunized with a sub-lethal infection of L. monocytogenes are resistant to L. monocytogenesand other intracellular pathogens, (Salmonella or BCG) at 1-2 weeks after infection.
•However, after a month or more, the mice remain resistant to L. monocytogenes, but not to Salmonella and BCG: Discuss
Cell mediated immunity to L. monocytogenes
•Immunity is initially non-specific (innate immunity) and can be attributed to macrophage activation mediated by IFN-g. Activated macrophages can kill L. monocytogenes, BCG and Salmonella.
•After additional time, innate immunity subsides, but specific anti-L. monocytogenes immunity persists. •Where as wild-type L. monocytogenes induces a potent CD8+ T-cell response, killed L. monocytogenes fail to induce protective immunity.
•Antibody play no role in immunity to L. monocytogenes
-immunity in listeria is mediated by cytotoxic t cells (CD8 cells)
What is it about the Listeria monocytogenes intracellular life-cycle that makes it a potent inducer of cell-mediated immunity?
listeria, because it gets into the cytosol, secreted its antigens directly into the cytosol of the host cell, which is the site @ which antigens are processed & presented on the surface of APC cells to induce (in the context of an MHC class I antigen) CD8 T cells
--> so basically cells are producing antigens at a high rate so they invite lots of CD8 T cells to come & bind
Cytosolic entry is required for protective immunity to L. monocytogenes
1. you immunize a mouse with listeria:
ΔactA can get out of vacuole well but can't spread
Δhly can spread but can't get out of vacuole
2. challenge the mouse a month later with WT listeria (lethal dose)
3. analyze # of bugs in spleen
--> the ΔactA strain did the best at preventing further infection (this is only strain that could get into host cytosol)
--> so something about getting to the cytosol is critical for immunity
Possible reasons that L. monocytogenes induces potent and long-lived cell-mediated immunity?
•Entry and growth within antigen presenting cells (DCs and macrophages) (so listeria enters into the right cell to induce immunity)
•Delivery of antigens directly into the cytosolic pathway of antigen processing and presentation
•Induction of innate immunity (necessary for induction of adaptive immunity)
•Can macrophages/DCs discriminate between vacuolar and cytosolic bacteria?
-LLO minus bugs don't induce immunity (bc they end up dead)
Listeria monocytogenes intracellular life-cycle
L. monocytogenes secreted proteins are introduce into the MHC Class 1 pathway of antigen processing and presentation (class I pathway is essentially where the antigens start from the cytosol rather than extracellularly)
Can host cells discriminate between vacuolar and cytosolic bacteria?
Macrophages discriminate between vacuolar and cytosolic L. monocytogenes
here we are infecting bone marrow derived macrophage with either LLO minus bacterium that's stuck in a vacuole (--> which triggers MyD88 response & get lots of cytokines)
or if the bacterium gets out of the vacuole it is seen differently (IRF3-dependent response)
- so yes, macrophages can discriminate between bugs in vacuoles & in cytosol
-northern blot is a way to look at whether an mRNA is made.
-WT makes TNF & IFN
-LLO(-) makes IL12 & TNF (no IFN)
How are intracellular L. monocytogenes detected?
MyD88 are toll like receptors which can see listeria in a vacuole
--> but what about in the cytosol? what does listeria make that is seen by the host, and what receptors are there to recognize what it makes? --> STING does
-STING is known for detecting DNA, so we would think that it is detecting listeria DNA... but it's not, so what is it? --> do forward genetics (phenotype -> genotype)
--just found out, and in fact STING is detecting cyclic-di-AMP from listeria after it gets pumped out of listeria by a MDR
Forward genetic screen for L. monocytogenes (listeria) mutants that induce altered levels of cytosolic signaling (IFN-b)
L. monocytogenes transposon libraries (10,000 mutants) --> infection of macrophages (infected 30,000 macs) --> the macrophage will then make IFN (can be detected with ELISA) or can have a reporter cell line --> addition of macrophages supernatant to ISRE cells (basically just a process that allows you to measure presence of IFN by light emission)
--> if you have LLO minus, they can't get out of vacuole, and no IFN is induced, so no light
mutation site and IFN-B induction
first mutant induces 20x more IFNB. the TetR gene had homology to a family of repressor genes
--> so the transposon hit a repressor protein, resulting in upregulation in the gene nextdoor (MdrT)
-Mdr = Multidrug Resistance Efflux pump
Multidrug-Resistance Transporters- MDRs
•Membrane proteins that actively transport a wide variety of structurally unrelated compounds.
•Found in all bacterial species.
•Play a role in active efflux of antibiotics and other toxic compounds.
•The natural substrates of MDRs are mostly unknown.

--> so listeria is pumping something out that turns on IFN
Hypothesis: L. monocytogenes multidrug efflux pumps (MDRs) secrete (export) some ligand that activates STING
ligand shown in red
Induction of IFN-b by L. monocytogenes mutants varies by 60-fold!
ΔTetR caused the largest influx of IFN
this makes sense bc TetR --x--> represses MDR, which ---> makes a ligand that ---> induces IFN
how do we find the molecule (ligand) that induces IFN?
Purification of active fraction by reverse-phase HPLC and introduction into cells by digitonin permeabilization
Mass spec analysis reveals c-di-AMP
it was previously known that bacteria can make cyclic-di-GMP, but now were figuring out that they can make cyclic-di-AMP
--> turns out c-di-AMP is what induces IFN β
--> it binds to STING & activates it
now we know that these strains are secreted different amounts of c-di-AMP
turns out that the bottom left light blue gene encoded a phosphodiesterase for c-di-AMP & breaks it down. so when you knock out light blue gene, bacteria have 8x more IFN bc they're not breaking down c-di-AMP
Synthesis, degradation, and secretion ofL. monocytogenes c-di-AMP
PdeA = a phosphodiesterase that breaks down c-di-AMP
Summary and Questions
•Cyclic-di-AMP is a secreted listerial molecule that activates STING in the cytosol of infected cell, leading the the host expression of IFN-band co-regulated genes
--> so we think: ok so STING must be necessary for immunity bc it's what invokes IFN. but no
•What is the role of STING and it's downstream genes in the induction of CMI?
Listeria-immunized mice lacking STING and its signaling pathway are still protected against lethal challenge
note that the x axis labels imply that it is "label (minus)"
--> this graph tells us that you don't need STING for immunity. if you don't have it the mice still immunize
Possible reasons L. monocytogenes induces potent and long-lived cell-mediated immunity?
•Induction of innate immunity
•Delivery of antigens directly into the cytosolic pathway of antigen processing and presentation
•Lack of cell death
L. monocytogenes induces low levels of cell death compared to some other bacterial pathogens
L. monocytogenes mutants that undergo bacteriolysis in the macrophage cytosol activate host cell death
all the mutants that they found that induced more inflammasome activation had death phenotype
--> turns out the bugs that lyse in the cytosol release DNA that leads to AIM2 -> ASC -> Caspase-1 -> host pyroptotic cell death
Induction of innate immune pathways by L. monocytogenes
maybe listeria is such a good inducer of immunity bc it doesn't kill the host cell
Question from previous final exam
15. You want to evaluate the role of inflammasome activation in L. monocytogenes and decide to use the Legionella pneumophilaflagellin (Fla) as the ligand.
a. Based on the method used to express antigens in L. monocytogenes, describe how you would construct a strain of L. monocytogenes that expresses L. pneumophila flagellin.
answer: make a fusion of actA and flagellin so that when listeria gets into the cytosol it secretes actA & flagellin & activates cell death
b. What is your hypothesis on the virulence of this strain? Propose a Genetics Squared experiment to test this. Include the genotypes of the mice and strains of L. monocytogenes used to test your hypothesis.
answer: listeria that secretes flagellin & induces inflammasome will be avirulent.
use WT & WT(flagellin) & infect WT & WT (-inflammasome)
c. What is your hypothesis for the ability of this strain to induce adaptive immunity? Justify your hypothesis and describe an experiment to test it.
one example: by killing cells that phagocytose listeria you won't generate immunity bc you killed the APC
Why does Listeria monocytogenes induce potent and long-lived cell-mediated immunity?
•Delivery of antigens directly into the dendritic cell cytosolic pathway of antigen processing and presentation
•Induction of innate immunity
•Lack of cell death
•Recall, while WT induces immunity, LLO-minus does not; Which is dominant; WT or LLO-minus?
Which is dominant: Live replicating bacteria or killed/vacuolar bacteria?
...
Immunization with ΔActA + Δhly:
...
CFU/spleen from immunized mice
LLO-minus suppress the host inflammatory response to wild-type L. monocytogenes
Why does Listeria monocytogenes induce potent and long-lived cell-mediated immunity?
•Delivery of antigens directly into the dendritic cell cytosolic pathway of antigen processing and presentation
•Induction of innate immune pathways that promote CMI and perhaps more importantly, lack of pathways that suppress it.
a transposon library
when u think about it, transposons are just jumping genes that randomly (kinda randomly) disrupt genes. so when you make a transposon library your essentially just making random mutations and storing them to figure out what affects the diff mutations make
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