Lecture 2 - Delivery of VFs + Journal Club (Vettiger/Basler)

Question 1

What are the three fates of proteins that are exported from the bacterial cell?

Answer 1

1) secreted into HOST CELLS
2) secreted into EXTRACELLULAR MEDIUM
3) expressed on BACTERIAL CELL SURFACE

*this is how several pathogenic organisms exert their effects on their host cell (i.e. secretion of virulence factors)

Question 2

Give examples of secreted substrates from the bacterial cell.

Answer 2

1) Protein toxins
2) Proteases
3) Lipases
4) Adhesins

*what do each do?

Question 3

How does the secretion of proteins differ between gram-positive and gram-negative bacteria?

Answer 3

Secretion of proteins by gram-positive bacteria requires that they be transported across the plasma membrane. Depending on the protein, it can then pass through the porous peptidoglycan or become embedded/attached to the peptidoglycan.

Gram-negative bacteria have more hurdles to pass - secreted factors must escape the attack from protein-degrading enzymes in the large periplasmic space and they must pass the inner and outer membranes.

Question 4

What is the major method for transporting proteins across the plasma (inner) membrane in gram positive and gram negative bacteria?

Answer 4

Sec-dependent (secretion-dependent) pathway.

Question 5

The sec dependent pathway _____ substrates across the _____ membrane or may promote substrate _____ into the membrane.

Proteins that utilize the Sec pathway are synthesized as ______ ______ that have a __-terminal ______ ____ that is recognized by the Sec machinery.

Answer 5

Translocates; plasma; integration

Unfolded; pre-proteins; N; signal sequence

Question 6

Match the following components of the Sec-dependent machinery to their functions:

1) SecYEG
2) SecA
3) SecB

A) binding to this component keeps the pre-protein in an unfolded state that renders it competent for translocation and prevents aggregation of protein in the cytoplasm.

B) forms a channel in the plasma membrane which facilitates translocation of the pre-protein.

C) forms a complex with SecYEG; acts as an ATP-dependent motor to translocate the pre-protein through the channel via ATP-hydrolysis.

Answer 6

SecYEG: forms a channel in the plasma membrane which facilitates translocation of the pre-protein.

SecA: forms a complex with SecYEG; acts as an ATP-dependent motor to translocate the pre-protein through the channel via ATP-hydrolysis.

SecB: binding to this component keeps the pre-protein in an unfolded state that renders it competent for translocation and prevents aggregation of protein in the cytoplasm.

*there are slight variations of this pathway depending on the protein substrate to be secreted.

Question 7

For the Sec-dependent pathway, the signal sequence is located at the ___-terminal of the pre-protein.

Answer 7

N

Question 8

What are the three steps of delivering virulence factors using the Sec dependent pathway?

Answer 8

1) Targeting
2) Translocation
3) Release

Question 9

The targeting step of the Sec-dependent pathway is different for membrane bound and periplasmic/extracellular targets.

1) Describe the steps of membrane bound targets.

2) Describe the steps of periplasmic/extracellular targets.

Answer 9

1) Membrane-bound targets are recognized by SIGNAL RECOGNITION PARTICLE (SRP - CO-translation) as they are being translated. FtsY directs the SRP-ribosome complex to SecYEG.

2) Periplasmic/extracellular targets are recognized by SecB (POST-translation), ensuring the protein is unfolded and allowing for shutting to SecA. The SecB-target is recruited to the SecYEG-SecA complex.

Question 10

(T/F) For the Sec-dependent machinery, both SRP and SecB targets require SecA.

Answer 10

False!

SRP targets generally do not require secA as TRANSLATION drives secretion. SecB targets require SecA and ATP hydrolysis for secretion.

Question 11

After targeting, there is the translocation and release of the targets in the Sec-dependent pathway. Briefly describe these steps.

Answer 11

Translocation: Pre-proteins are translocated via the SecYEG channel.

Release: In both cases, the signal sequence is cleaved by a SIGNAL PEPTIDASE (found in the plasma membrane). Proteins are FOLDED and either released in the periplasm or integrated in the plasma membrane.

Question 12

1) What is the Tat (twin arginine translocation) pathway?

2) How does it differ from the Sec pathway?

Answer 12

1) Another type of system used by gram-negative and gram-positive bacteria to translocate proteins across the plasma membrane.

2) It is able to translocate proteins that are already in their FOLDED state.

Question 13

Match the steps of the Tat pathway:

1) Step 1
2) Step 2
3) Step 3
4) Step 4

A) Binding of substrate to TatBC leads to TatA polymerization to form a channel (facilitates the passage of proteins).

B) Signal peptide cleavage, releasing passenger domain and TatA depolymerization.

C) TatBC (complex within the plasma membrane) binds to the signal peptide of the folded substrate.

D) Passenger domain crosses the membrane via TatA (translocation mediated by portion motive force).

Answer 13

1. TatBC (complex within the plasma membrane) binds to the signal peptide of the folded substrate.
2. Binding of substrate to TatBC leads to TatA polymerization to form a channel (facilitates the passage of proteins).
3. Passenger domain crosses the membrane via TatA (translocation mediated by portion motive force).
4. Signal peptide cleavage, releasing passenger domain and TatA depolymerization.

Question 14

(T/F) For the Tat pathway, the signal peptide contains “twin” arginine residues in the signal motif S-R-R.

Answer 14

True!

Question 15

Which part of the TatBC complex binds to the substrate?

Answer 15

TatC contains binding group that recognizes the ss of the substrate in the cytoplasmic surface.

Question 16

Once a secreted protein gets beyond the inner membrane (via Sec or Tat systems), how does it get through the periplasm and outer membrane?

Answer 16

Bacteria have evolved sophisticated nanomachines called SECRETION SYSTEMS to export proteins (i.e. virulence factors) beyond the inner membrane.

Question 17

1) Which protein secretion systems of gram-negative bacteria are sec-independent?

2) Which protein secretion systems of gram-negative bacteria are sec-dependent?

Answer 17

1) Type I and Type III (proteins are directly translocated from cytoplasm through to the cell exterior).

2) Type II and Type V (proteins are first translocated across plasma membrane).

Question 18

List each statement as true or false.

1) Type IV is sometimes linked to the Sec pathway, but often functions on its own.

2) There are only 5 protein secretion systems of gram-negative bacteria.

3) Pathogenic bacteria may express several types of secretion systems.

Answer 18

1) True.

2) False! More than 5!

3) True.

Question 19

Briefly answer the following questions regarding the T5SS:

1) How many steps of a process is it?

2) Is it sec-dependent or sec-independent?

3) Where is the signal sequence located in the protein susbtrates?

4) What are autotransporters?

Answer 19

1) Occurs as a TWO-STEP PROCESS.

2) Sec-dependent process.

3) Protein substrates have an N-terminal SS.

4) Most common types of T5SS use AUTOTRANSPORTERS. These are MULTIDOMAIN proteins that have a N-terminal PASSENGER domain and a C-terminal translocator domain linked by a linker region.

Question 20

1) What are the 3 parts of the T5SS?

2) What is the role of the periplasmic chaperones?

Answer 20

1) a) C-terminal translocator B-barrel domain, b) Linker region, c) N-terminal passenger domain.

2) helps to keep the passenger domain in an unfolded state after the Sec pathway.

*protein (T5SS) is already secreted to the periplasm by the Sec pathway; its N-terminal passenger domain then gets secreted.

Question 21

Describe the mechanism of action of the hairpin model of the T5SS.

Answer 21

Before these steps occur, the multidomain autotransporter crosses the inner membrane via the Sec pathway.

The flexible linker forms a HAIRPIN LOOP that promotes the passenger domain to migrate through the translocator channel (the passenger domain folds as it exits; not sure about the energy source).

The secreted passenger is cleaved and released or remains embedded in the outer membrane.

Question 22

Give an example of a pathogenic bacterium that uses T5SS.

Answer 22

Neisseria gonorrhoeae secretes IgA protease.

IgA protease secreted BLOCKS host-mediated neutralization (cleaves the IgA antibody that prevents pathogen colonization), allowing for bacterial adhesion, internalization, and infection.

Question 23

Briefly answer the following questions regarding the T3SS:

1) How many steps of a process is it?

2) Is it sec-dependent or sec-independent?

3) Where is the signal sequence located in the protein substrates?

4) Briefly describe its structure and what it is referred as.

Answer 23

1) Single-step process

2) Sec-independent

3) Protein substrates have an N-terminal signal sequence

4) T3SS consists more THAN 20 PROTEINS that form a SUPRAMOLECULAR structure that delivers effector proteins from the bacterial cytoplasm DIRECTLY into the host cell cytoplasm. It can also be referred to as INJECTOSOMES (needle-like).

Question 24

What are the three main components of the T3SS? Briefly describe the function of each.

Answer 24

Translocator: creates pores

Needle: protein structure that extends to the plasma membrane of the host. contact of needle with host cell triggers secretion of translocator proteins which forms a pore in the host membrane.

Base/basal body: inner rings, neck, outer rings. effectors enter via portal. ATPase is part of the base.

Question 25

(T/F) The effectors secreted by the T3SS are in a folded state.

Answer 25

False!

Effectors are in an unfolded state so they can pass through needle.

Question 26

Give an example of a pathogenic bacterium that uses T3SS.

Answer 26

Enteropathogenic E.coli uses T3SS to secrete Tir protein.

*recall: Tir is an effector protein that is secreted to the host cell to be embedded on the membrane. once embedded on host cell membrane, it forms a receptor like structure to bind to intim (another effector protein but is not secreted), allowing 4 pedestal formation n engulfment.

Question 27

Briefly answer the following questions regarding the T6SS:

1) How many steps of a process is it?

2) Is it sec-dependent or sec-independent?

3) Briefly describe its structure and mechanism of action.

4) Which types of cells does the T6SS secrete effector proteins into?

Answer 27

1) Single-step process

2) Sec-independent process

3) It is composed of AT LEAST 13 PROTEINS. It resembles an INVERTED BACTERIOPHAGE TAIL (works like a ‘speargun’). Sheath CONTRACTION drives system through target cell membrane.

4) It secretes effector proteins into both EUKARYOTIC and BACTERIAL cell targets (roles in pathogenesis and bacterial competition).

Question 28

Match the parts of the T6SS to their definitions:

1) Shaft
2) Membrane complex
3) Baseplate
4) Sheath

A) composed of TssE, TssK. it provides a platform for assembly of the contractile sheath and shaft. attaches to inner membrane.

B) hollow tube composed of Hcp rings. begins with a tip composed of VgrG trimer and effector proteins.

C) contractile sling composed of VipA/VipB. this structure surrounds the shaft. can exist in a contracted or extended state.

D) composed of TssJ, TssL, TssM and is required for anchoring entire apparatus to the bacterial membrane.

Answer 28

Shaft: hollow tube composed of Hcp rings. begins with a tip composed of VgrG trimer and effector proteins.

Membrane complex: composed of TssJ, TssL, TssM and is required for anchoring entire apparatus to the bacterial membrane.

Baseplate: composed of TssE, TssK. it provides a platform for assembly of the contractile sheath and shaft. attaches to inner membrane.

Sheath: contractile sling composed of VipA/VipB. this structure surrounds the shaft. can exist in a contracted or extended state.

Question 29

(T/F) In an extended state, the shaft is projected through the inner and outer membrane of the gram (-) bacteria and into the host membrane.

Answer 29

False!

In an extended state, shaft is within the sheath.

In a contracted state, the shaft is projected through the inner and outer membrane of the gram (-) bacteria and into the host membrane.

Question 30

Match the steps of assembly and function of the T6SS to the right order:

1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5

A) VgrG and effectors are recruited and assemble into the structure. Hcp proteins may be recruited.

B) The contracted sheath is recognizes by the ClpV ATPase which uses ATP hydrolysis to remodel the sheath, promotes Hcp tube disassembly; components may be recycled.

C) Conformational change in sheath causes it to contract which launches the Hcp shaft out of the cell and into the target cell membrane. effectors are delivered into target cell.

D) Baseplate assembles in the IM; membrane complex associates with baseplate and assembles in the IM which spans the periplasm and is anchored in the OM.

E) Hcp tube (shaft) polymerizes from the VgrG proteins and the VipA/VipB sheath polymerizes around it.

Answer 30

Step 1: Baseplate assembles in the IM; membrane complex associates with baseplate and assembles in the IM which spans the periplasm and is anchored in the OM.

Step 2: VgrG and effectors are recruited and assemble into the structure. Hcp proteins may be recruited.

Step 3: Hcp tube (shaft) polymerizes from the VgrG proteins and the VipA/VipB sheath polymerizes around it.

Step 4: Conformational change in sheath causes it to contract which launches the Hcp shaft out of the cell and into the target cell membrane. effectors are delivered into target cell.

Step 5: The contracted sheath is recognizes by the ClpV ATPase which uses ATP hydrolysis to remodel the sheath, promotes Hcp tube disassembly; components may be recycled.

Question 31

Which proteins are needed for sheath assembly in V. cholerae? How did researchers discover this?

Answer 31

V. cholerae needs vgrG2 and Hcp for sheath assembly to occur.

They fused vipA to GFP - when VipA is expressed, there is green fluorescence. In WT, you can see vipA proteins forming a sheath.

When there is a deletion of the vgrG2, hcp, or both, there is no detection of the sheath assembly.

There is still vipA production but no sheath formation in these mutants.

*vgrG2: same as vgrG (tip of T6SS)
*Hcp: Hcp tube polymerizes from the VgrG proteins
*vipA: forms sheath around Hcp tube

Question 32

Are Hcp and VgrG exchanged and reused for new T6SS assembly between sister cells?

Describe the experiment done to figure this out.

Answer 32

Yes! Hcp and VgrG are exchanged and reused between sister cells!

The authors took a RECIPIENT with negatively functioning T6SS that is unable to form a sheath (ΔHcp/ΔVgrG/both) with its VipA fused to GFP.

They took a DONOR with a wildtype T6SS with clpV (a T6SS protein) fused to a red fluorescent protein (mCherry2).

They mixed donor and recipient cells in a ratio of 1:4 and monitored the localization of VipA-GFP in recipient cells by microscopy to see if THERE WAS GOING TO BE SHEATH FORMATION IN RECIPIENT CELLS.

Sheath assembly was significantly restored in recipient cells (they got the Hcp and VgrG2 from donor cells)!

Question 33

Which statement is true regarding the assay used to determine if Hcp and VgrG2 are exchanged between cells and reused for new T6SS assembly?

1) More than 1 in 10 cells assembled at least one sheath structure within 5 minutes of microscopy imaging!

2) Researchers could have picked another T6SS protein to label besides clpV as they just wanted to label the donor cells.

Answer 33

2!

For 1) More than 1 in 20 cells assembled at least one sheath structure within 5 minutes of microscopy imaging

Question 34

After using donor cells that had WT T6SS, the authors used donor cells that were T6SS deficient.

1) How did they make the donor cells T6SS deficient?

2) What kind of recipient cells were used?

3) Why did they do this experiment?

4) What did they discover?

Answer 34

1) The donor cells were ΔvipB (a protein involved in sheath formation). Donors could not be ΔHcp/ΔVgrG because we want them to give to these proteins to recipient cells to restore sheath formation.

2) ΔHcp/ΔVgrG/both (same as other experiments)

3) The authors wanted to test if their observations (significant restoration of sheath assembly in recipient strain when using WT donor strain) were dependent on a functional T6SS of the donor cells.

4) Little to no sheath assembly was detected in recipient cells that were mixed with donor cells lacking T6SS (ΔvipB).

Question 35

Do recipient cells require de novo protein synthesis for the assembly of a new T6SS?

Describe how this was discovered.

Answer 35

Interbacterial protein complementation does not require de novo protein synthesis for the initiation of a new T6SS!!

The researchers were unsure if RNA transcripts or actual vgrG2 and hcp proteins were being sent to recipients.

Thus, they inhibited the protein synthesis in the recipient cells by pre-treating them with chloramphenicol (Cam). Sheath formation should occur if proteins are being transferred. If sheaths are not forming, the donor might be delivering mRNA transcripts.

The researchers saw SHEATH FORMATION in Cam pre-treated recipient cells!!!

Question 36

What are the functions of these proteins?

1) TseL
2) tsiV1
3) CPRG

Answer 36

TseL: virulence factor that causes lysis in cells.

tsiV1: corresponding immunity protein of TseL that protects bacteria from its own toxin.

CPRG: cell impermeable substrate of lacZ

Question 37

How was the LacZ reporter assay used to determine if the newly assembled T6SS in recipient cells
(Δhcp/ΔvgrG2/both) were functional?

Answer 37

RECIPIENT (ΔHcp/ΔVgrG/both with vipA fused to GFP) was mixed with DONOR/REPORTER (T6SS+, ΔtseL, ΔtsiV1, and plasmid that encodes for lacZ enzyme) in a media that contains CPRG (cell-impermeable substrate of LacZ).

The recipient expresses TseL (toxin) while the donor does not, and the donor expresses lacZ enzyme.

The donor transfers T6SS components (hcp/vgrG2) to recipients, building a T6SS in recipients. If the recipient T6SS is functional, it will transfer TseL to donor cells, causing them to lyse and release lacZ out of the cell. LacZ will hydrolyze CPRG substrate in the media and the media will turn red from yellow!

Lysis of T6SS+ reporter/donor cells was detected when cells were mixed with Δhcp, ΔvgrG2 or Δhcp/ΔvgrG2 recipients. Researchers measured 572nm absorbance upon CPRG hydrolysis to chlorophenol red.

Question 38

Various recipients were used in the lacZ reporter assay. Predict if the media will turn red or remain yellow when mixed with T6SS+, ΔtseL, ΔtsiV1, lacZ+ donor cells:

1) Wildtype recipients (functional T6SS)

2) ΔvipB

3) ΔtseL

4) Δhcp

5) ΔvrgG2

6) Δhcp/ΔvgrG2

Answer 38

Wildtype recipients (functional T6SS): media turns RED as there is proper T6SS that can translocate tseL to donor (lysing of cells -> release of lacZ in media -> hydrolysis of CPRG)

ΔvipB: not all T6SS proteins can be translocated within sister cells and vipB is one of them. without vipB in the recipient cells, there would be no functional T6SS; thus media remains YELLOW.

ΔtseL: can not give TseL to donor cells; no lysis of donor cells; media remains YELLOW.

Δhcp/ΔvrgG2/both: we know these proteins can be translocated from donor cells to recipient cells to create a functional T6SS that will secrete TseL to donor cells. TseL will cause lysis in donor cells; release of lacZ; hydrolysis of CPRG; media turns RED.

Question 39

Can sister cells work together to kill competition?

How did researchers figure this out?

Answer 39

Yes! Sister cells can work together to kill a competition!!

They had three types of cells mixed in a CPRG media: Reporter (T6SS- ΔvipB/ΔtseL/ΔtsiV1/LacZ+), Donor (T6SS+ ΔtseL), and Recipients (Δhcp/ΔvgrG2/both).

Donor cells translocated Hcp or VgrG2 into recipient cells that were, in turn, able to make a functional T6SS to secrete TseL into the reporter strain to promote its lysis (no lysis of donor as they have the immunity protein Tsvi1)!

Question 40

Briefly answer the questions regarding the proof of concept of cooperation in microbial communities to kill competition:

1) Why did the reporter have a non-function T6SS that could not be restored (ΔvipB)?

2 )Why can’t the recipient give TsiV1 to the reporter and protect it from lysis?

3) For controls, the researchers used recipient cells that were either wildtype or ΔvipB and all constructs including Δhcp/ΔvgrG2/both without donors. What are the expected results?

Answer 40

1) Mimics a competition that does not have a T6SS so that donors and recipients (sister cells) that have a functional T6SS work together.

2) Not all T6SS proteins are transferred between sister cells. TsiV1 is one of those proteins that is not transferred.

3)
Wildtype: already functional T6SS -> can give TseL into reporter strain -> red media (high 572nm absorbance)

ΔvipB: vipB is not transferred between sister cells; recipients can not receive it; no function T6SS; yellow media

all constructs without donors:
- WT: red media
- everything else: yellow media

Question 41

(T/F) For the proof of concept, the donor strain that is ΔtseL can receive tseL from the recipient once a functional T6SS is made and that can be given to the reporter.

Answer 41

True!

In theory, this could happen. It doesn’t matter if the recipient or donor is giving TseL to the reporter. If the donor gives TseL to the reporter, then this is simply consistent with the model that the donor must have received TseL from a functional T6SS generated from the recipient (from the Hcp/VgrG2 received from the donor).

Question 42

Amount of ____ limits the number of T6SS assemblies and ___ limits the sheath length.

Efficiency of protein exchange depends on the ______ _______ of T6SS.

Answer 42

VgrG; Hcp

Precise aiming