Lecture 3: host factors Flashcards
Name three reasons why so few drugs pass clinical trials.
- “The low hanging fruits have already been plucked” → the easiest drug targets have already been found.
- There’s no money to be made. Only when there’s so much resistance to old/current antibiotics, new drug will be used.
- Bacteria have several ways to inactivate drugs that target specific pathways that researchers have found (low hanging fruits). So drugs can be modified, but it’s really hard to find a drug that won’t be inactived by bacteria or to find a drug that targets new found pathways.
What’s the alternative to targeting the bacteria?
Targeting the host so that bacterial growth is limited. This is because pathogens need nutrients and other factors from the host.
Plerixafor and maraviroc are two drugs that are used in the clinic against HIV. They target respectively CXCR4 and CCR5 (co-receptor proteins) in the life cycle of HIV infection. What’s the downside of these drugs and what does this tells us?
Plerixafor can be used against HIV, but only through IV-administration and it has major side effect liks cardiac failure. Maraviroc is not effective on its own and can only be used in combination with other drugs. This tells us that targeting the host to prevent/treat infection is very challenging, due to (amongst others) the risk of side effects.
Bacteria can live in host cell under different circumstances: obligate, facultative and opportunistic. What is the difference between these pathogens and describe for obligate and facultative pathogens whether they’re suited for host-directed treatment.
- Obligate pathogen: needs the host for survival. Very easy to treat with host-directed targeting, because they rely upon the host.
- Facultative pathogen: able to have an intracellular phase in the host, needed for pathogenicity, while also being capable of extracellular growth in vitro. Very hard to treat, lot of resistancy, because a part of the life cycle occurs within the cell and a part occurs outside the cell.
- Opportunistic pathogen: a pathogen normally wouldn’t be harmful, but takes advantages of e.g. a weakened immune system.
Describe Salmonella entry into the gut and host cell.
- Salmonella enters the gut
- Via microvilli it finds a place to adhere in the gut
- When it has found a place to adhere, a type 3 secretion system creates a pore in the host cell it’s attached to
- Toxines enter the host cell and bind and activate Rac/Cdc42
- Rac/Cdc42 recruits Arp2 complex
- Arp2 complex polymerises F-actin so that the membrane is pushed outwards for endocytosis of the bacteria
- Salmonella is in vacuole and Rac signaling pathway is stopped
- Second type 3 secretion system is started
- Synthesis of effector proteins for protection of Salmonella, which also ensures safe replication conditions.
- Name two reasons why Salmonella protects itself inside a vacuole of a cell.
- Name two things Salmonella can modify once it is in a vacuole of a cell.
- Protection from immune system and antibiotics
- It can modify transport and transcription inside the cell
Salmonella can only replicate inside a mature vacuole. What GTPase is recruited and activated when Salmonella is still in the immature vacuole and what does activation of this GTPase lead to?
Rab7, it’s important for vacuole maturation (and probably important for acidification of the vacuole).
What happens if you knock-down Rab7 in a Salmonella containing late endosome?
Then there’s no acidification and thus no replication of Salmonella inside the late endosome. So acidification is needed for replication.
After it was found that Rab7 was needed for late endosome acidification, researchers were interested in which other kinases would reduce growth of Salmonella. They first knocked-down all sorts of kinases to find kinases that would reduce S. growth (see picture). After this they used chemical inhibitors to inhibit these kinases. They found only one kinase where inhibition of the kinase lead to reduced growth. What kinase was this and is it actually a potential drug target?
Inhibition of protein kinase A (PKA) reduced Salmonella growth. But this was only the case for the H-89 PKA inhibitor and not for Rp-cAMP PKA inhibitor. So this tells us that PKA isn’t actually a possible drug target.
So with only few results, scientists took their interest in PKA inhibitor H-89. They wanted to see what kinases could be inhibited by H-89. For this they used structure-activity relationship (SAR). How was SAR used to identify kinases that were inhibited by H-89?
Here, different structures of H-89 were produced (adding extra methyl group, replacing certain groups, etc.). The variants were tested on whether they could block Salmonella replication. Next, these variants were combined with the kinase genes that reduced growth when knocked-down. Here, also the effectivity of the variants was tested.
They eventually found 3 variants of compound H-89 that worked well against Salmonella replication (outlined with light blue). Next, a couple of kinases were found that were structurally similar to PKA. Why is this important and what kinase was the most similar to PKA?
These structurally similar kinases are probably also being inhibited by H-89. Akt1 is a kinase that is structurally very similar to PKA.
Next, a specific inhibitor of Akt (ETB067), a variant of H-89, was injected into mice. What was the conclusion of this mouse study?
- ETB067 is not lethal itself (see black line)
- ETB067 is Salmonella infected mice prolonged the life span from 10 to 14 days. (So not a complete cure on its own).
- Eventually ETB067 resulted in the development of cancer cells in the lungs. This again tells us that targeting the host is not always a good treatment.
How does an inhibitor of Akt block Salmonella replication?
Uptake of Salmonella in vacuole inside host cell → H-89/Akt inhibitor blocks maturation process i.e. acidification.
Researchers then wanted to confirm that Salmonella activates/needs Akt during infection. How was this confirmed?
By visualizing activated/phosphorylated Akt in normal Salmonella and in sopB deficient Salmonella. sopB is a effector protein of Salmonella with phosphatase activity. They saw that when sopB was inactive/absent, Akt was not phosphorylated and thus not visualized.
Fill in the amount of genes:
There are … coding genes in our genome of which … genes consist of small GTPases.
There are 18.000-20.000 coding genes in our genome of which 1500 genes consist of small GTPases.
What is a small GTPases that is known of causing cancer by uncontrolled growth?
Ras
What can you say about the function of these small GTPases based on this picture of active mutant Ras-like proteins?
You can see that these proteins do something to the cytoskeleton of cells. You can conclude that these GTPases regulate actin.
Explain the mechanism of activation and inactivation of GTPases like Ras.
GTP-bound GTPases are on and can bind to effector proteins. GDP-bound GTPases are off. When GTP is bound to a GTPase, a GAP (GTPase activating protein) can remove a phosphate of GTP to inactive the GTPase. When GDP is bound, GEF (GTPase exchange factor) can remove GDP so that GTP can bind.
The on-/off-switch of GTPases are thus mediated by GAPs and GEFs.
Explain the concept of the loaded spring mechanism of GAP (GTPase activating protein).
GAP consists of two switches that bind a phosphate group of GTP-bound GTPase. When GTP is hydrolysed into GDP, this phosphate group is released. This triggers a conformational change in GAP, thereby pushing GDP-bound GTPase off.
Salmonella can activate small GTPases through the insulin receptor pathway. Explain how.
When the insulin receptors bind insulin → Akt is activated → Akt phosphorylates and inactivates AS160 → AS160 normally acts as a GAP and inactivates Rab, but now cannot do so → Rab stays active → production Glut4 vesicles
Here, in red Salmonella is visualized and in green GFP-Rab14. What can we conclude based on this picture?
That GFP-Rab14 is recruited early-on by Salmonella to the Salmonella containing vacuole so that the vacuole can mature.
Just to summarize, answer the following questions:
- The protein that inactivates AS160 through phosphorylation is …
- By what pathway is this protein that inactivates AS160 activated?
- AS160 is a GAP for what Rab protein?
- The protein that inactivates AS160 through phosphorylation is Akt.
- By what pathway is this protein that inactivates AS160 activated? Akt is activated via the insulin signaling pathway.
- AS160 is a GAP for Rab14.
So what happens to Rab14 in sopB deficient Salmonella?
sopB phosphorylates and activates Akt. This cannot happen, so Akt cannot phosphorylate and inactivate AS160. AS160 is now constitutively active, thus Rab14 stays constitutively off. Thus Rab14 is not recruited to the Salmonella containing vacuole and the vacuole cannot mature. This is also seen in this picture.
Since Rab14 is recruited to early endosomes, Nischarin the effector protein and PI(3)P are present on early endosomes. It was thought Nischarin colocalizes with Rab14 on early endosomes by binding to PI(3)P. But this was not the case. What was found?
They found that Rab14/Nischarin complex colocalize on late endosomes.
What information about the interacting Rabs with Nischarin was found based on the phylogenetic tree of Rab GTPases?
- Rab4A was a homologue of Rab14, so it was clear why Rab4 could also bind to Nischarin.
- Rab9A had homology with Rab7, that was already known for late endosome localization.
With the knowledge that Nischarin had another binding partner Rab9, besides Rab14, it was now important to establish how Salmonella containing vacuoles could mature into late endosomes. How was this done?
They visualized Rab14, Nischarin and Rab9 expression with respectively RFP, GFP and BFP. What they saw was that Rab14 was first visualized, then Nischarin was recruited and visualized and finally Rab9 would appear. So there’s a Rab14 to Rab9 transition, also seen in the picture.
- Would Nischarin knockdown have a negative or positive effect on Salmonella replication?
- And does Nischarin knockdown affect acification?
Answer is yes for both. Nischarin knockdown prevents acidification and it affects survival.
(Try to) describe the pathway of Salmonella containing vacuole maturation, starting with bacterial uptake and insulin signaling.
- Insulin signaling pathway activates Akt.
- Akt phosphorylates and inactivates AS160.
- Inactivated AS160 results in constitutive activation of Rab14.
- Rab14 recruits Nischarin.
- Nischarin binds to PI(3)P and Rab14 on early endosomes.
- Complex of Nischarin and Rab14 has high affinity for Rab9. So Rab9 is recruited.
- Rab9-Nischarin ensures acidification and maturation of early endosomes to late endosomes.
