Presenter : Jennifer Bratt

G-Proteins? ( Wasn’t that from, like, Cell Bio? ) What are these G-proteins and what do they do? G proteins are guanine nucleotide binding proteins. They are often associated with transmembrane receptors called G-protein-coupled receptors (GPCRs). It’s a heterotrimer (Ga, Gb, Gg ).

G-protein Activity http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/G_Proteins.html

G-proteins, there’s even more! There are different forms of G-protein G-a subunits. GaS = Stimulatory G-protein. Activates adenylyl cyclase. Gai = Inhibitory G-protein. Inhibits adenylyl cyclase. Gaq and Gat

GTPases The G-Protein Control Mechanism G-proteins are signaling molecules. They have a very slow rate of hydrolysis on their own. GTPases increase the rate of G-protein deactivation by hydrolysis of the bound GTP to GDP. No bound GTP, no signal transduction.

Neisseria gonorrhoeae A Very Bad Thing Obligate human pathogen Colonize mucosal surfaces Very sneaky bacteria Antigenic variation of pilus Adheres to cell surfaces and invades epithelial cells Induces signal transduction cascade in host Has numerous effects on host in piliated form, including cytokine release and Ca2+ release from intracellular stores.

In normal piliated N. gonorrhoeae, Opa proteins bind to CD66 and heparan sulfate proteoglycan receptors. Induces cytoskeletal rearrangement by activation of a signaling cascade.

Non-piliated (P-) N. gonorrhoeae Avirulent Does not cause characteristic inflammation Able to colonize the male urethra Still able to adhere to epithelial cells

What are we trying to figure out with this study? What is the cell signaling pathway that non-piliated N. gonorrhoeae uses to mediate adherence to epithelial cells?

Experiment 1 Q. Does the interaction between the host cell and Neisseria during adhesion require GTP hydrolysis? GDP-b-S = nonhydrolyzable guanine nucleotide analogue. Blocks G-protein activity. Binds to the G protein and cannot be exchanged for GTP.

Experiment 1: Results P+ vs. P- adhesion to ME180 cells cells post 1H treatment with GDP-b-S P+ adhesion to ME180 cells post 1H treatment with GDP-b-S

Experiment 1: Results GTP hydrolysis is a required step of the cell signaling pathway that results in the adhesion of P- Neisseria to ME180 cells. Remember that hydrolysis of G Protein bound GTP is a function of GTPases.

Experiment 2 Q. Can G-protein activators get around the inactivation of G proteins by the GDP analogue? AlF4- (fluroaluminate) is a GTPase inhibitor. It increases G protein activity by blocking GTP hydrolysis.

Experiment 2: Results The treatment of ME180 cells with 5mM AlF4- resulted in a increase in P- Neisseria adhesion 20x greater than control. G protein activation is a step in the cell signaling pathway that results in the adhesion of P- Neisseria to ME180 cells.

Experiment 3 Q. What Ga subunit G protein is the pathway dependent upon? Gai or GaS Cholera Toxin (CTX) specifically activates GaS G proteins via ADP ribosylation of the a subunit. Pertussis Toxin (PTX) specifically activates Gai

Experiment 3: Results The P- Neisseria cell adhesion pathway is 10 minute pretreatment of ME180 cells with CTX resulted in a significant increase in P- Neisseria adhesion to the cells. 10 minute pretreatment of ME180 cells with PTX resulted in no significant change in P- Neisseria adhesion to the cells. The P- Neisseria cell adhesion pathway is dependent upon the activation of Gas G protein.

Experiment 3: Results Immunofluorescent images taken of the different Neisseria groups. The P+ and P- bacteria adhered to the ME180 cells are labeled with FITC.

Experiment 4 Q. Are the activities of Gs G protein and GTP hydrolysis we see on the same pathway? And if so, in what order are they? Pretreatment with GDP-b-S. Does addition of CTX or AlF4- cause a recovery of Neisseria adhesion properties?

Experiment 4: Results Surprise! Data not shown! G protein activation and subsequent hydrolysis of GTP is a required step in the cell signaling pathway that results in the adhesion of P- Neisseria to ME180 cells.

Where Are We Now in this Cell Signaling Pathway Model?

Experiment 5 Q. Does the CTX or AlF4- incubation result in Ca2+ release from intracellular stores? In previous experiments, it was seen that AlF4- can act as a Ca2+ dependent ATPase inhibitor. It causes an increase of intracellular [Ca2+]. Fura-2/AM is a Ca2+ chelator. It can be used to visualize intracellular Ca2+ levels by the change in the emissions wavelength of Fura-2 when it has calcium bound to it. Used in combination with either CTX or AlF4- to assess levels of intracellular [Ca2+].

Experiment 5: Results ME180 cells pretreated with AlF4- showed a spike in Ca2+ levels at 400 seconds post exposure. ME180 cells pretreated with AlF4- showed no increase in Ca2+ levels post exposure. CTX probably does not follow the same pathway as AlF4-.

Experiment 6 Q. Does extracellular [Ca2+] affect the ability of the P- Neisseria to adhere to ME180 cells? And how does exposure time affect adhesion? In previous experiments, it has shown that the influx of Ca2+ into the host cell can result in the depletion of host intracellular stores.

Experiment 6: Results Ca2+ has a significant effect on P- Neisseria adhesion to ME180 cells. Adding 5mM of EGTA prior to infection negated most of extracellular calcium’s effect on Neisseria adhesion. (Adding EGTA post infection had no effect of adhesion.) Most P- Neisseria cells adhered to the ME180 cells within the first 10 minutes of infection

Experiment 7 Q. Does extracellular [Ca2+] enter the cell via calcium channels resulting in the increase in cytoplasmic [Ca2+] or is there a signaling mechanism that causes the intracellular Ca2+ stores to be released? Verapamil is a calcium channel blocker. BAPTA/AM is an intracellular Ca2+ chelator.

Experiment 7: Results Data not shown! Treatment with Verapamil did not affect calcium-induced adherence of P- Neisseria to ME180 cells. Treatment with BAPTA/AM was able to reduce calcium induced adherence of P- Neisseria to ME180 cells. The extracellular Ca2+ probably acts through a cell membrane receptor that results in G-protein activation.

Experiment 8 Q. Does the depletion of intracellular or extracellular Ca2+ affect adhesion if you already have G protein activation? Is it an upstream or downstream effect? Thapsigargin is an intracellular Ca2+ chelator. EGTA is an extracellular Ca2+ chelator. Used in combination with AlF4- to determine the effects of Ca2+ levels on P- Neisseria adhesion

Experiment 8: Results EGTA is an extracellular divalent cation chelator. When added to the media, it does not affect the activation of G proteins by AlF4-. Thapsigargin, an intracellular chelator, does decrease the amount of P- Neisseria adhesion to ME 180 cells induced by AlF4-. The activation of G proteins is upstream of the release of Ca2+ from intracellular stores but downstream of or on a different pathway as extracellular calcium ion activation in the cell adhesion signaling pathway.

Where Are We Now in this Cell Signaling Pathway Model? Part 2

Experiment 9 P- Neisseria interacts with ME180 cells via G protein activation. Q: Does it act on the Rho GTPase post G protein activation specifically to create this signal transduction cascade? C. botulinum toxin (C3) is an ADP ribosyltransferase and ribosylates Rho, inactivating it.

Experiment 9: Results The addition of C3 decreases the number of P- cells that can adhere to ME180 cells. Rho inactivation blocks the signaling mechanism. The G protein’s GTP is not hydrolyzed by Rho. Can this adherence phenotype be saved by CTX, AlF4-, or increased extracellular Ca2+?

Experiment 10 – Part 1 Q. Can the adherence phenotype be saved by CTX, AlF4-, or increased extracellular Ca2+? Are they all a part of the same pathway? C. botulinum toxin (C3) is an ADP ribosyltransferase and ribosylates Rho, inactivating it. G protein bound GTP will not be hydrolyzed. Use C3 in combination with AlF4-, CTX, or increased extracellular Ca2+ to determine the effects on P- Neisseria adhesion.

Experiment 10: Results C3 blocked AlF4- and CTX mediated adherence of P- Neisseria but did not affect Ca2+ mediated adherence. Extracellular [Ca2+] mediated adherence is therefore either on a separate signaling pathway or further downstream in the pathway from CTX and AlF4- which require Rho activation.

Experiment 10 - Part 2 P- Neisseria interacts with ME180 cells via G protein activation. Q: Does it act on the Rho G Protein specifically to create this signal transduction cascade? Cytotoxic Necrotizing Factor 1 (CNF1) activates Rho. Note it has the opposite effect on Rho as C3.

Experiment 10: Results Preincubation with CNF1 increased the number of adherent P- Neisseria cells. The Ca2+ independent signaling pathway is Rho G Protein dependent.

Conclusions GTP hydrolysis and Ga-s protein activation are required steps of the cell signaling pathway. CTX does not follow the same pathway as AlF4-. The extracellular [Ca2+] probably acts through a cell membrane receptor causing a release of intracellular Ca2+ stores. This pathway is probably independent of the AlF4- and CTX pathway. The Ca2+ independent signaling pathway is Rho GTPase independent.

The Final Pathway

What to do now? What are the bacterial ligands and other signaling intermediates involved in this signal transduction pathway? Adenylyl cyclase? Do changes in extracellular [Ca2+] play a significant role in vivo for nonpilus mediated cell adhesion of Neisseria? Rho regulates a number of cell functions through the reorganization of the actin cytoskeleton of the host cell. Could that be a mechanism by which adhesion occurs?

What’s this Pathway Trying to Do? It looks suspiciously like this pathway is trying to turn off the adenylyl cyclase activity. GTPase activity turns off the G protein signal transduction G-s is the activator of adenylyl cyclase and the hydrolysis of the bound GTP turn off the G protein activation function. Increased level of intracellular Ca2+ also has a negative regulatory effect on adenylyl cyclase activity.

You made it! THE END!