6: Virulence factors - Intracellular trafficking & survival

Question 1

How does the Enterohermorrhagic E.coli (EHEC) modulate the host cell?

Answer 1

The EHEC removes the host cell’s filli, then a type 3 secretion system inserts Tir (transloated Intimin Receptor) into the host cytoplasma. These receptors will bind with the EHEC Intimin on the plasma membrane. The Tir is then phosphorylated and this activates the actin polymerization cascade.

Question 2

What is the Type 3 secretion system (T3SS)?

Answer 2

It’s a needle-like complex which secretes effector proteins (which interfere with host signal transduction cascades) directly from the bacterial cytoplasm into the cytoplasm of the host cell.

Question 3

How does actin polymerization work?

Answer 3

There are phosphoinositides in the membrane which bind Rac or CDC42 (small GTPases). When they are on they bind nucleation promoting factors (NPFs) like N-WASP. N-WASP activates Arp2/3 and then there will be actin polymerization.

Question 4

What are the advantages of living inside host cells?

Answer 4

You are shielded from humoral antibodies and there are less competing bacteria for the same resources.

Question 5

What are the disadvantages of living inside of host cells?

Answer 5

Endocytosed bacteria are en route to the lysosome, there are nutritional limitations (e.g. iron), there is antigen presentation of MHC class II and you are in close contact with innate immune receptors.

Question 6

What are differences between living in the phagosome vs. cytosol?

Answer 6

In the phagosome the nutrients are low, in the cytosol they are high.
In the phagosome are hydrolytic enzymes, radical, antimicrobial peptides and innate immunity receptors. In the cytosol is autophagy, are PAMP receptors and innate immunity receptors.

Question 7

How does Salmonella enter a host cell?

Answer 7

via the trigger method, where it injects SopE/SopB/SptP proteins through a T3SS into the host cell, which then will regulate actin polymerization. The actin will push the membrane up so it will engulf the bacteria.

Question 8

What are SPI-1 and why are they important?

Answer 8

Salmonella Pathogenity Islands-1 (SPI-1) encode the T3SS in Salmonella enterica, which is required for invasion of epithelial cells.

Question 9

Which small GTPases are important for actin?

Answer 9

CDC42 and RAC1

Question 10

Which small GTPases are important for trafficking/fusion of the fagosome?

Answer 10

All Rab proteins encode for trafficking/fusion, but importantly RAB7 and RAB9.

Question 11

What is a small GTPase?

Answer 11

They are Ras-like proteins that binds a GTP, so it can bind to its effector protein in the ‘on’ state. Or it binds a GDP in the ‘off’ state.
A GAP protein can turn the GTPase off, a GEF protein can turn the GTPase on.

Question 12

What is the SopE molecule?

Answer 12

Its a GEF protein, so it can switch the RAC1 from it’s GDP bound state to the GTP bound state. Activating actin polymerization.

Question 13

What is the SptP molecule?

Answer 13

Its a GAP protein which inhibits the GTPases, so there is no more actin polymerization.

Question 14

SopE and SptP are secreted at the same time, how can they act sequencely?

Answer 14

SopE has a higher affinity, but has lysines so it gets ubiquitinated and will get degraded by the proteasome, and the SptP doesn’t. So the SopE wil work first because of a high affinity, but will then get degraded so SptP will do its job.

Question 15

What are the functions of phosphoinositides?

Answer 15

Phosphoinositides regulate recruitment of proteins involved in the organization of vesicular trafficking (e.g. Rab proteins).

Question 16

Which endocytic compartments do the phosphoinositides PI(3)P and PI(3,5)P2 label?

Answer 16

PI(3)P labels the early-endosome and PI(3,5)P2 labels the lysosome.

Question 17

How does Salmonella regulate membrane trafficking?

Answer 17

It secretes SopB proteins which trough a positive feedback loop induce production of Rab5. This binds the membrane of the Salmonella containing vacuole and prevents the fusion with lysosomes and so it stays in an early endosome.

Question 18

When are T3SS-1 and T3SS-2 important?

Answer 18

T3SS-1 is important for entry of the bacteria into the host cells, T3SS-2 is important after that for the growth (replication) inside the host.

Question 19

What is the function of SPI-2?

Answer 19

SPI-2 is activated at an acidic pH (inside the phagosome) and it secretes effectors that regulate membrane recruitment to the Salmonella containing vacuole and avoid degradation, and also effectors that downregulate MHCII to prevent detection.

Question 20

What does Rab9 do?

Answer 20

Rab9 is essential for delivery of Cathepsins to lysosomes from the Golgi. Rab9 binds to the Manose 6 phosphate receptor (M6PR) which then takes along the Cathepsins from the Golgi to the lysosomes where they become active enzymes (proteases) that degrade bacteria.

Question 21

How does Samonella avoid degradation with SifA?

Answer 21

Salmonella excretes the SifA effector molecule that binds to host proteins SKIP and Rab9 to reduce lysosome activity. The SKIP/SifA complex catches all Rab9 away from the cell, so there is no Rab9 left to bring the Cathepsins to the lysosome and so Salmonella won’t be degraded by the proteases.

Question 22

How does Salmonella avoid degradation with SopD?

Answer 22

FYCO1 and RILP normally bind to Rab7 to form proper lysosomes. Salmonella excretes SopD2 which acts as an effector of Rab7. It binds to Rab7 in its active state, so it blocks binding of FYCO1 and RILP so there wont be proper lysosomes and the Salmonella stays in the early lysosome.

Question 23

What is autophagy?

Answer 23

It means ‘self-eating’ and is normally used for degration of e.g. mitochondria. Organelles or bacteria get ubiquitinated (marked with ubiquitin which is the eat-me signal) which binds to LC3 on the phagosomal membrane through adaptor proteins. There will be full enclosure of the membrane around the particle which then fuses with mature lysosomes containing Cathepsins that degrade the particles.

Question 24

How does Salmonella escape autophagy?

Answer 24

It produces SseL that removes ubiquitin form the Salmonella containing vacuole, so it can’t bind to a phagosome and won’t be destroyed. (encoded on SPI-2)

Question 25

How does MHCII antigen presentation exactly work?

Answer 25

A bacterium engulfed by a macrophage is encased in a vacuole. Then lysosomes fuse with the vacuole and digest the bacterium. The antigens from the digested bacterium are presented with MHCII on the cell surface.

Question 26

How does Salmonella avoid recognition with SteD?

Answer 26

SteD is secreted by Salmonella and acts like a E3 ligase, so it recruites a host protein that ubiquitinates the MHCII molecule and targets it for degradation. So Salmonella removes MHCII.

Question 27

How does Listeria enter a host cell?

Answer 27

Via the zipper method, it binds E-cadherin receptors on cells with InlA or Met receptors (brain/placenta) with Inlb to be internalized without having to inject anything into the cell. It ubiquitinates the receptor, which leads to recruitment of the Chathrin machinery followed by actin remodeling (Arp2/3) and endocytosis.

Question 28

What are the advantages of living inside host cells?

Answer 28

You are shielded from humoral antibodies, there are less competiting bacteria for the same resources and it’s a nutritional heaven.

Question 29

What are the disadvantages of living inside host cells?

Answer 29

There is autophagy and the innate immune system.

Question 30

How does Listeria escape the phagosome?

Answer 30

They secrete LLO, a pore forming protein and bacterial phospholipase A and B (PlcA, PlcB) that destroy the membrane of the phagosome.

Question 31

How does Listeria move to a new host cell?

Answer 31

Listeria mimics a NPF (e.g. N-WASP) so they directly have actin polymerisation on their surface. They have ActA on one pole of the cell and here there is direct actin polymerization with the Arp2/3 complex, so no CDC42 and Rac is involved.

Question 32

What are examples of bacterial regulated actin dynamics?

Answer 32

TIR, SopE and ActA.

Question 33

How does Listeria escape autophagy?

Answer 33

It has Inlk proteins on its surface which are bound by host MVP preventing p62 (adaptor) recruitment. There will be no binding of the phagosome.