Lecture 7 - Bacterial Effector Proteins

Question 1

Bacterial effector proteins are a specialized class of ______ factors.

L. Pneumophila and S. Enterica rely heavily on effector proteins for ______.

Answer 1

Virulence

Infection

Question 2

What are the 4 stages of infection?

Answer 2

1) Exposure to pathogen
2) Adherence to skin/mucosa
3) Invasion through epithelium
4) Colonization and growth

this leads to toxicity and invasiveness, causing tissue damage and disease.

Question 3

Which statement is true regarding virulence factors?

1) Production of virulence factors occurs during the Colonization and Growth stage.

2) Virulence factors function by interacting with the host; often modify host function for the benefit of pathogen.

3) Expression is static; same virulence factors are made at different timepoints of infection.

Answer 3

2!

1) Production of virulence factors occurs during ALL FOUR STAGES OF INFECTION (entire course)!

3) Expression is DYNAMIC; different virulence factors are made at different timepoints of infection for different purposes. Effectors made immediately after infection are often different than those made during late infection.

*VFs are complex
*bacteria have evolved clever ways to take advantage of the host

Question 4

Bacteria can establish infection using diverse strategies.

What are the three typical strategies used by pathogens?

Answer 4

1) Extracellular
2) Intracellular (vacuoles): remain in compartments
3) Intracellular (cytosolic): break free from compartments

*all secrete virulence proteins to mediate infection

Question 5

Intracellular bacteria are first internalized into ___________.

Answer 5

Compartments

*can either remain or break free

Question 6

Why are the most successful virulence factors often not the most toxic?

Answer 6

Bacterial infection is a SYMBIOTIC RELATIONSHIP.

Successful pathogens balance host damage and host.

Pathogens need to MANIPULATE THE HOST to establish infection (to benefit the pathogen). But host death would lead to END OF INFECTION; therefore an INTRICATE BALANCE is needed!

Question 7

What are the two major pathogenesis tool used to cause infection by pathogens?

Answer 7

1) Toxins (endo and exotoxins)
2) Other virulence proteins -> effectors

Question 8

Endotoxins are normal constituents of ______?

When are they active?

Compared to exotoxins:
They exhibit relatively _____ toxicity. They exhibit more ______ effects.

Answer 8

Bacterial cell wall (LPS)

They are active only after released: during CELL DIVISION or bacterial CELL LYSIS.

low (but can be fatal at high doses); non-specific effects (fever, aches, shock)

Question 9

Exotoxins are synthesized and ______ by the pathogen.

What is their molecular structure?

What do they do?

Answer 9

Secreted

Protein

Cause damage to host cells

Question 10

Which statement regarding exotoxin is false?

1) Exotoxins have limited diversity in structures.

2) A pathogen may secrete more than one different toxin.

3) They are susceptible to antibodies; therefore highly immunogenic (robust immune response).

Answer 10

1!

Exotoxins have DIVERSE structures.

Question 11

How are effectors same as toxins?

How do they differ from toxins?

Answer 11

Like toxins, effectors are SECRETED.

UNLIKE toxins, effectors MANIPULATE host pathways to mediate infection.

UNLIKE toxins, they are usually NOT OVERLY toxic!

Question 12

Bacterial effectors are specialized virulence proteins that are ______ into host cells using specialized bacterial ______ _____.

They function to manipulate host cell pathways to facilitate infection. They evolved to function in the ____ cell not the _____ cell.

Answer 12

Injected; Secretion Systems

host; bacterial

Question 13

Which SS does Salmonella use to infect host cells?

Answer 13

T3SS

Question 14

How do bacterial effectors function at a molecular level?

Answer 14

They usually target a host protein or protein complex; host pathogen protein-protein interaction ALTERS function of HOST PROTEIN.

Question 15

The legionella effector ____ targets host regulatory GTPase _____.

Answer 15

SidM; Rab1

Question 16

Effectors target _____ _____ processes.

Effectors are often _________ (ex, several co-evolve to target different host proteins in ____ pathway).

Effectors can also have ____ mechanisms. Give examples.

Answer 16

Diverse cellular

Co-operative; same

Complex
- some can MIMIC MAMMALIAN proteins to manipulate host cell processes
- some can have ENZYMATIC ACTIVITY on host proteins
- some can have MULTIPLE DOMAINS with different functions

Question 17

What is the general purpose of effectors?

Answer 17

Usually, to aid bacterial survival and/or replication in the host, without causing death.

Question 18

What are effectors being used in:

1) Salmonella

2) Listeria

Answer 18

Salmonella uses effectors for INVASION in many different types of cells for infection. They turn on uptake process in cells that are normally off (for example epithelial cells).

Listeria uses a SINGLE effector to POLYMERIZE HOST ACTIN to give it MOTILITY (for replication and spread). The effector mimics a mammalian protein that polymerizes actin.

Question 19

Are bacterial effectors just important in human infection? If not, give examples.

Answer 19

No! They are important for infection of PLANTS and ANIMALS too.

P. syringae infects agriculturally important crops such as tomato, tobacco, and beans. Effectors help EVADE host immunity.

Pathogenic E. coli strains cause respiratory and reproductive diseases in chickens, pigs and cows. Effectors help mediate infection.

Question 20

(T/F) Effectors sole role lies in infection.

Answer 20

False!

Effectors play important roles OUTSIDE infection. They help mediate SYMBIOTIC RELATIONSHIPS between organisms.

Root nodules are important for nitrogen fixation in legumes. Some nitrogen-fixing bacteria use effectors to promote Nodulation.

T6SS effectors often play roles in killing competing microbes in MICROBIAL COMPETITION.

Question 21

What are some examples of targets of effector proteins in host cells?

Answer 21

Bacterial effectors target diverse cell processes.

1) Tight junctions
2) Cytoskeleton
3) Cellular defense pathways (endocytic, autophagy, immune cell function)
4) Biochemical activities
5) Protein degradation (proteasome)
6) Organelle function

Question 22

1) What are small GTPases? What do they help define and what does this allow for?

2) Small GTPases are subclassified into 5 families. What are they?

3) What do the GTPases that belong to the Ras superfamily control?

4) Which families of GTPases are most targeted by effectors?

Answer 22

1) Small GTPases are regulatory proteins often targeted by effector proteins. Within host cells, compartment membranes have UNIQUE GTPase compositions; this helps define their MEMBRANE IDENTITY. This allows researchers to identify and study them.

2) Ras, Rab, Rho, Arf, and Ran. There’s over 150 members.

3) Control important host cell processes that include RESPONSE PATHWAYS to infection, INTRACELLULAR TRAFFICKING, and CYTOSKELETAL REORGANIZATION.

4) Effectors can target all 5 families of GTPases but Ras superfamily are frequently targeted by bacterial effectors to mediate infection.

Question 23

How do host Ras-superfamily GTPases work?

Answer 23

Ras-superfamily GTPases are small G-proteins that function as MOLECULAR SWITCHES for signaling pathways.

They cycle between active (GTP-bound) and inactive (GDP-bound) forms on biological membranes.

When active, they interact with HOST BINDING partners to mediate cellular effectors.

Question 24

GTPases are fine tuned in the host using regulatory proteins. What are the two main regulatory proteins?

Answer 24

1) GEF (guanine nucleotide exchange factor) –> activate

2) GAP (GTPase activating protein enzyme) –> deactivate

Question 25

Membrane lipids such as __________ are important to organelle function.

Intracellular compartments have ____ membrane lipid compositions; this helps define __________ ______.

Answer 25

Phosphoinosides (PIs)

Unique; membrane identify

*bacterial effectors often bind or manipulate processes that involve membrane lipids

Question 26

Within host cells, PIs can recruit host proteins to mediate specific functions.

What are these functions?

Answer 26

1) Organelle trafficking
2) Secretion
3) Compartment fusion

Question 27

How do bacterial effectors control what is happening in the host cell using PIs?

Answer 27

1) Can bind PIs for effector recruitment to compartments
2) Can manipulate PI content at compartments
3) Can manipulate host proteins that are endogenously recruited to membranes by PIs

Question 28

An important host target of effectors is the innate and adaptive immunity.

How?

Answer 28

Bacterial pathogens use effectors to either reverse or finely tune immune responses for the benefit of the pathogen.

Question 29

1) What is the cytoskeleton essential for?

2) What other important roles does it play?

Answer 29

1) Cytoskeleton essential to maintain the STRUCTURE of the cell.

2) Endocytosis (uptake from extracellular environment), trafficking (transport/movement of intracellular compartments), and cell division

Question 30

What are the three components of the host cell cytoskeleton?

Answer 30

1) Actin filaments (microfilaments)
2) Microtubules
3) Intermediate filaments

Question 31

Bacterial effectors can manipulate the cytoskeleton to:

*4 functions

Answer 31

1) induce pathogen UPTAKE into host cells

2) manipulate ORGANELLE MOVEMENT (and often fusion)

3) create a means of BACTERIAL MOTILITY within host cells

4) mediate CELL-TO-CELL spread

Question 32

Listeria effector _____ mimicks a host protein that polymerizes actin.

This allows ______ within host cell which is used for _________.

Answer 32

ActA

Motility; cell-to-cell spread

Question 33

(T/F) Bacterial infection can trigger gene transcription in the host to deal with the stress of infection.

Answer 33

True!

Question 34

How do bacterial effectors manipulate nuclear function?

Answer 34

1) suppress transcription of genes for HOST RESPONSE TO INFECTION

2) induce expression of HOST PROTEINS that might BENEFIT the pathogen

3) to manipulate the CELL CYCLE to benefit the pathogen

Question 35

1) Where must shigella penetrate to cause disease?

2) What is a problem faced with this location?

3) How does shigella mitigate this?

Answer 35

1) Shigella must penetrate the INTESTINAL EPITHELIUM to cause disease

2) However, the epithelium SHEDS FREQUENTLY to eliminate pathogens.

3) The host protein Mad2L2 functions with the ANAPHASE-PROMOTING COMPLEX (APC) to allow progression to mitosis. The effector IpaB sequesters Mad2L2 and STALLS the cell cycle and BLOCKS epithelial cell SHEDDING.

Question 36

What is autophagy?

Describe the 4 steps.

Answer 36

It is a cellular pathway to remove unneeded/dysfunctional CELL COMPONENTS or invading PATHOGENS.

1) Membrane formation around target
2) Double membrane completion
3) Fusion with lysosomes
4) Degradation

Question 37

What is Xenophagy?

Answer 37

the removal or elimination of INTRACELLULAR pathogens (bacteria, viruses, fungi, parasites) by autophagic degradation

Question 38

How would a pathogen want to manipulate autophagy?

Answer 38

1) SUPPRESS antibacterial autophagy to help the pathogen survival.

For example: S. enterica SopF, AvrA, and L. pneumophila RavZ.

2) ACTIVATE autophagic degradation of cell components to gain nutrients to survive.

For example: Coxiella burnetti CvpB and B. psuedomallei BPSS0180

Question 39

C. burnetti replicate in _____ ____ that contain multiple bacteria also known as _______.

Answer 39

Large vacuoles; CCVs

*intracellular pathogen
*CCVs contain many bacteria

Question 40

Match the correct order of C. burnetti’s CvpB mechanism.

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

A) On that compartment, this allows lysosome maturation and autophagy of cellular components; fusion of autophagosomes with CCVs may provide nutrients for the bacteria.

B) CvpB localizes to compartments containing the membrane lipid PI(3)P.

C) CvpB binds PI(3)P and prevents conversion to PI(3,5)P2 by the kinase PIKfyve (changes content on compartments).

Answer 40

Step 1: CvpB localizes to compartments containing the membrane lipid PI(3)P.

Step 2: CvpB binds PI(3)P and prevents conversion to PI(3,5)P2 by the kinase PIKfyve (changes content on compartments).

Step 3: On that compartment, this allows lysosome maturation and autophagy of cellular components; fusion of autophagosomes with CCVs may provide nutrients for the bacteria.

Question 41

What are the main differences between Salmonella and Legionella?

Answer 41

Salmonella produces >30 effector proteins, while Legionella produces >300 effector proteins.

They also have very different intracellular life cycles.

Question 42

Briefly answer the following questions regarding Legionella.

1) What type of pathogen is it? Where does it inhabit?

2) How many different species are there? How many linked to human disease? Which one is most common?

3) What are the two causative agents?

4) When does illness develop?

Answer 42

1) Gram-negative OPPORTUNISTIC pathogen (gammaproteobacteria). It inhabits aquatic (freshwater) environments.

2) There are 60 different species; 26 linked to human disease; L. pneumophila is the most common.

3) Legionnaires’ disease (pneumonia) and Pontiac fever (respiratory illness - flu like)

4) ~2-14 days after infection

Question 43

(T/F) In the US, almost ~900% increase in L. pneumophila since 2000. It is problematic in the elderly, immunocompromised, and those with underlying health conditions. If hospitalized. 25% mortality rate.

Answer 43

True!

Question 44

What are the treatments of L. pneumophila?

Answer 44

Antibiotics: macrolide, quinolone, or rifamycin classes

*antibiotic resistance common to all 3 classes

Question 45

1) How do people get infected with L. pneumophila?

2) Are humans the natural host of L. pneumophila?

Answer 45

Human infection: through WATER DROPLETS from contaminated sources (AC, faucets, hot tubs).

Humans are NOT the natural host, but can cause serious illness if infected.

Natural host: Amoebae or other protozoa

Question 46

Which cell type is infected by L. pneumophila in humans?

Answer 46

Alveolar macrophages

Question 47

There are _____ infection strategies of L. pneumophila in both amoebae and humans. It targets ______ _____ processes, allowing survival in ______ hosts.

It is an ______ pathogen that survives in _______. It uses _______ to mediate infection.

Answer 47

Similar; evolutionally conserved; diverse

Intracellular; vacuoles; effectors

*many L. pneumophila in one host cell in vacuoles

Question 48

Describe the genome of L. pneumophila and how it gives clues to pathogenesis.

Answer 48

Genome: circular chromosome that is ~3.5 Mbp big with ~3000 protein encoding genes.

There are several hundred genes in its genome that encode Eukaryotic-like proteins (sequence similarity), or domains within proteins that are eukaryotic-like. Could they have evolved to target the eukaryotic host?

Question 49

L. pneumophila encodes a T_SS that is essential for ______ growth.

It is also known as type ____:_____ secretion system. It secretes over ______ diverse virulence factors for pathogenesis and many are ______ that can target mammalian cell processes.

Answer 49

4; intracellular

IVB: Dot/ICM

300; enzymes

*clues to pathogenesis

Question 50

Pathogens are internalized into compartment called a ________-________ _______.

Once inside the host cell, intracellular pathogens encounter host cellular ________ responses.

__________ pathway: uses lysosomes to target pathogens for death; lysosomes contain degradative enzymes.

Important concepts:
1. Compartment must evade:

2. Compartment must have a way of:

Answer 50

Bacteria-containing Vacuole (BCV)

Immune; endocytic

1. Compartment must evade intrinsic cellular defense mechanisms
2. Moving around inside host cells (intracellular trafficking)

Question 51

How does Legionella evade host cellular response to cause infection?

Answer 51

The membrane composition of BCV helps define its identity! Changing its identity can help disguise itself from immune responses!

Legionella recruits membrane from the ER to disguise itself from detection in the host.
1. It is first internalized into Legionella-containing vacuole (LCV)
2. Recruits ER membranes early during infection (changes identity)
3. LCV acquires ribosomes (from rough ER membranes)
4. Bacterial replication results in LCV with multiple bacteria

Question 52

How do we know that effectors from T4SS are important for Legionella infection?

Is T4SS important for infection?

Answer 52

Wild-type: recruits ER-derived membrane and is able to multiple in LCV

T4SS impaired mutant: targeted by lysosomes for degradation!

It can infect without a functional T4SS but can’t SURVIVE and REPLICATE.

Question 53

Why has Legionella evolved an incredible FUNCTIONAL REDUNDANCY in effector function (more than most pathogens)?

What implication does this have in pathogenesis research?

Answer 53

In nature, L. pneumophila relies heavily on host for survival! SYMBIOTIC RELATIONSHIP important for survival.

There is extensive co-evolution between the host and the pathogen:
Pathogen has evolved diverse strategies to evade host and the host has co-evolved new ways to target pathogen. This creates a pressure for the pathogen to further manipulate the host!

Consequently, Legionella has evolved an incredible FUNCTIONAL REDUNDANCY in effector function (more than most pathogens).

In pathogenesis research, gene knockouts are used to study effect of virulence factor on infection. Because of redundancy in function, effector gene knockouts usually result in no phenotype. Therefore, it is often difficult to study infection.

Question 54

Legionella has co-opted host genes for bacterial pathogenesis using _________ gene transfer.

Explain and give me examples.

Answer 54

Legionella has co-opted host genes for bacterial pathogenesis using HORIZONTAL gene transfer.

Legionella genome contains a large number proteins that are highly similar to eukaryotic proteins (more so than any other bacteria).

Some genomes are most similar in sequence to genes from protists which were likely horizontally acquired from host.

An example:
- Ras superfamily small GTPases are large class of regulatory proteins in EUKARYOTES.
- There are almost 200 GTPase like genes in a study of 80 legionella strains. These are unprecedented in bacteria!

Question 55

1) What is Rab1?

2) Why do many Legionella effectors target Rab1?

3) Give an example of a Legionella effector that targets Rab1.

Answer 55

Rab1 is an important regulatory GTPase in the ENDOPLASMIC RETICULUM! that controls diverse processes for organelle function such as compartment trafficking, compartment fusion, protein synthesis, and autophagy.

Manipulating Rab1 function provides Legionella with opportunities to hijack many important host processes!

SidM

Question 56

Effectors take control of the key ER regulatory GTPase _____ and bind membrane ____________.

What are some of the mechanisms effectors use to hijack Rab1?

Answer 56

Rab1; phosphoinositides

The mechanisms include diverse covalent chemical modifications.
- addition or removal of phosphocholine
- mimics host GEF
- AMP addition or removal
- mimics host GAP

Question 57

Some effectors can: ____ PIs on ER membranes and/or _______ ER membrane

Answer 57

bind; recruit

Question 58

Compartment membrane fusion
is mediated by a special class of proteins called ______ on the compartment membranes!

It is controlled by ______ proteins including ________.

Answer 58

SNAREs

regulatory; Rab GTPase

Question 59

What are the three steps of compartment membrane fusion?

Answer 59

1. SNARE proteins (donor compartment snare and acceptor compartment snare) interact
2. SNARE complex conformation change; brings membranes in close proximity
3. Membrane fusion; delivery of contents

Question 60

What does Legionella do to the host compartment fusion machinery?

Briefly describe how and for what?

Answer 60

Legionella manipulates the host compartment fusion machinery.

Legionella effector SidM mediates fusion of ER membrane with LCV; uses host SNARE complexes on LCV and Rab1 to control.

This is so LCV acquires ER membranes to shield from host immune responses.

Question 61

The acceptor compartment (LCV) snare is the _______ while the donor compartment (ER) is the _________.

Answer 61

Stx3 (syntaxin-3); Sec22b

Question 62

1) Why do bacterial effectors manipulate host cytoskeleton?

2) What is trafficking?

3) How do vacuolar pathogens move?

Answer 62

1) Bacterial effectors take advantage of the host cytoskeleton for movement within the host.

2) Organelles and other intracellular compartments MOVE along the cytoskeleton in a process called TRAFFICKING.

3) Vacuolar pathogens often use effectors to promote movement along MICROTUBULES.

Question 62

Match the steps of LCV acquiring the ER membrane by manipulated the host compartment fusion machinery:

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

A) the two SNAREs (Stx3 and Sec22b) are brought in close proximity to allow VESICLE FUSION

B) Promotion of recruitment of Stx3 which binds SidM

C) Effector SidM catalyzes AMP addition to Rab1 -> locks Rab1 into ACTIVE state

Answer 62

Step 1: Effector SidM catalyzes AMP addition to Rab1 -> locks Rab1 into ACTIVE state

Step 2: Promotion of recruitment of Stx3 which binds SidM

Step 3: the two SNAREs (Stx3 and Sec22b) are brought in close proximity to allow VESICLE FUSION

Question 63

The Legionella effector _______ binds the small GTPase _____ to induce ___________ _______ near the LCV and throughout the cell for LCV movement.

Answer 63

LegG1; Ran; microtubule polymerization

*microtubule highways!

Question 64

Legionella effectors target _______ points in the autophagy pathway to ________ function!

______ is coupled to phosphatidylethanolamine (PE) to bind ___________ membrane (important for autophagy).

_______ (effector) is a protease that __________ ___-__ and targets growing autophagosome membrane!

Answer 64

multiple; suppress

LC3; autophagosome

RavZ; deconjugates LC3-PE

Question 65

Legionella effectors target host protein for degradation by ________.

What are the two ways effectors do this?

Answer 65

Legionella effectors target host protein for degradation by PROTEASOME.

Effectors can:
A) catalyze UBIQUITINATION of host proteins (AnkB, SidE family, MavC & MvcA)

B) catalyze DEUBIQUITINATION of host proteins (DupA & DupB, Lot family, and RavD)

Question 66

Why would some effectors want to catalyze UBIQUITINATION of host proteins and some catalyze DEUBIQUITINATION of host protein?

Answer 66

Catalyzing UBIQUITINATION could provide nutrients for replication bacteria.

Catalyzing DEUBIQUITINATION could maintain homeostasis to prevent cell death: 1) suppress golgi fragmentation 2) suppress xenophagy