L11 - Bacterial exploitation of host cytoskeleton and intracellular trafficking Flashcards
What are the three principal components of the cytoskeleton?
Microtubules, intermediate filaments, and microfilaments.
Which protein is the main constituent of microtubules?
Tubulin.
What is the approximate diameter of microtubules?
About 25 nm.
What are the approximate diameters of intermediate filaments and microfilaments?
Intermediate filaments are ~10 nm and microfilaments (actin) are ~7 nm.
Which Shigella effector mentioned on this slide modulates the cytoskeleton?
VirA.
Provide an example of an E. coli effector that modulates host cell processes.
EspG (also referenced as orf3).
What types of pathogens are known to produce factors that manipulate the host cell’s cytoskeleton?
Bacteria and Viruses
Why is it significant that multiple bacterial species employ these tactics?
It highlights the evolutionary advantage of subverting host cell dynamics for invasion and survival.
What are the two principal mechanisms of bacterial invasion?
The trigger and zipper mechanisms.
How does the “zipper” mechanism facilitate bacterial entry?
By engaging adhesin/receptor interactions that induce localised host cell responses.
What characterises the trigger mechanism of bacterial invasion?
The injection of bacterial proteins into host cells that actively induce cytoskeletal rearrangements.
Which pathogens are typically associated with each trigger and zipper mechanism?
Zipper is linked to Yersinia and Listeria; trigger is linked to Salmonella and Shigella.
Which cellular structure is central to both the trigger and zipper mechanisms?
The actin cytoskeleton.
How do adhesin/receptor interactions contribute to bacterial uptake?
They initiate host cell responses that promote the engulfment of bacteria.
What role do translocated bacterial effectors play in invasion?
They trigger rearrangements in the actin cytoskeleton that aid in internalisation.
What dynamic cellular structure is induced during Salmonella invasion?
Membrane ruffles.
What is the primary cytoskeletal component in membrane ruffles?
Actin.
How can membrane ruffling be visualised experimentally?
Through the use of GFP-tagged actin in cultured epithelial cells such as MDCK cells.
What is the role of membrane ruffles in the process of bacterial invasion?
They remodel the plasma membrane to facilitate bacterial entry.
What role do Rho GTPases play in the cell?
They act as molecular switches that regulate actin cytoskeleton dynamics.
What cellular changes occur when constitutively active Rho GTPases are introduced?
There is stimulation of stress fibres, lamellipodia, or filopodia depending on the specific GTPase involved
Which actin structure is stimulated by active Rho?
Stress fibers.
What does GEF stand for, and what is its role?
Guanine nucleotide exchange factor; it activates GTPases by facilitating the exchange of GDP for GTP.
What is the function of a GAP in the regulation of GTPases?
A GTPase-activating protein (GAP) accelerates the hydrolysis of GTP, inactivating the GTPase.
How does a GDI influence Rho GTPase activity?
It binds to the GTPase and prevents the release of GDP, thereby maintaining the inactive state.
Why is the regulation of the GTP-bound state of Rho GTPases important?
It ensures that signalling is precisely controlled and transient.
Name a Salmonella effector that functions as a GEF.
SopE (and SopE2).
What role does the Yersinia effector YopE play in modulating Rho GTPases?
It acts as a GAP to inactivate Rho GTPases.
How do bacterial proteins that mimic GDP dissociation inhibitors affect host GTPases?
They maintain the GTPases in an inactive state by preventing GDP release.
Can you name additional bacterial effectors that modulate Rho GTPases?
Burkholderia pseudomallei BopE and Shigella IpgB1/IpgB2
Which two Salmonella effectors are responsible for activating Rac1 and Cdc42?
SopE and SopE2.
What are the distinct cellular outcomes of activating Rac1 versus Cdc42?
Activation of Rac1 induces lamellipodia (membrane ruffles), while Cdc42 activation leads to the formation of filopodia
Which bacterial species are well known for exploiting host cytoskeletal functions?
Salmonella, Shigella, Yersinia, Helicobacter, Listeria, and Neisseria.
How is the activity of SopE regulated following its initial function?
It is rapidly removed via ubiquitination and proteolysis, ensuring that the activation of GTPases is transient.
Which enzyme from Clostridium modifies actin by ADP‑ribosylation?
The C3 exoenzyme.
How do Clostridium difficile toxins affect actin?
They glucosylate actin, leading to its inactivation.
What modification is performed by the E. coli toxin CNF on actin regulators?
Deamidation or transglutamination.
What is the effect of Yersinia YopT on actin regulation?
It proteolytically inactivates Rho GTPases, thereby affecting actin dynamics.
Which Salmonella effector directly ADP‑ribosylates actin?
SpvB
How does Shigella manipulate actin without involving Rho GTPases?
Through IpaC, which stabilises actin by recruiting vinculin.
Which Vibrio cholerae effector contributes to actin organisation?
MARTX (and VgrG1).
What is the role of E. coli SPATE toxins in actin organisation?
They cleave fodrin, thereby indirectly affecting the actin cytoskeleton.
What is the main function of the Arp2/3 complex?
It nucleates new actin filaments and creates branched actin networks.
Which protein family is responsible for activating the Arp2/3 complex?
The WASP family, including N‑WASP.
How is N‑WASP normally regulated?
It remains auto‑inhibited until activated by signals such as active Cdc42 and PtdIns(4,5)P₂.
Why is the formation of branched actin networks important?
They provide the structural basis for dynamic cellular processes like motility.
Which intracellular bacterium is famous for its actin-based motility within host cells?
Listeria.
Name another bacterium that utilizes actin for intracellular movement.
Shigella.
What advantage does actin-based motility confer on intracellular pathogens?
It enables rapid movement within cells and facilitates spread between adjacent cells.
Aside from bacteria, which type of pathogen also exploits actin for propulsion?
Certain viruses, such as Vaccinia virus.
What does live-cell imaging of intracellular bacteria typically reveal?
The dynamic movement of pathogens like Listeria within host cells.
Which imaging technique is commonly employed to study these intracellular dynamics?
Live-cell fluorescence microscopy.
By what mechanism does Listeria achieve intracellular motility?
It hijacks the host’s actin polymerisation machinery.
What distinctive structure is induced on host cell surfaces by EPEC?
Actin pedestals.
Which host proteins are recruited to form these pedestals?
N‑WASp and the Arp2/3 complex.
How do actin pedestals assist EPEC during infection?
They secure bacterial attachment and can modulate host cell signalling.
What do these structures indicate about host–pathogen interactions?
They exemplify how pathogens directly reprogramme the host actin machinery.
Which two type III secretion systems (T3SS) does Salmonella employ?
SPI‑1, which facilitates invasion, and SPI‑2, which is crucial for intracellular survival and systemic spread.
What is the primary function of SPI‑1?
It mediates the initial entry of Salmonella into host cells.
What role does SPI‑2 play in Salmonella infection?
It supports the bacterium’s survival within host cells and assists in systemic dissemination.
What is the critical function of phosphoinositides in host cells?
They delineate intracellular compartments and regulate the recruitment of proteins for vesicle trafficking.
How do phosphoinositides influence vesicle fusion and maturation?
By controlling the binding of specific proteins that are involved in trafficking pathways.
Why is the regulation of phosphoinositides important during infection?
Pathogens can manipulate these lipids to modify vacuolar membranes and create niches for survival.
Which two bacterial enzymes serve as inositol phosphatases?
Salmonella SigD and Shigella IpgD.
What substrate do these enzymes target?
They cleave PI(4,5)P₂.
What is observed when Salmonella lacks SigD during infection?
Vacuolar membranes remain enriched in PI(4,5)P₂ and actin.
What does the detection of PI(3)P indicate about the vacuole?
It suggests that the vacuole is undergoing maturation and engaging in trafficking pathways.
What effect do the Salmonella effectors SigD and SopB have on host membranes?
It cleaves PI(4,5)P₂, altering the phosphoinositide composition.
How does the alteration of PI(4,5)P₂ levels affect the host cell?
It disrupts actin dynamics and promotes membrane remodelling.
What drives the movement of vesicles along microtubules?
Motor proteins such as kinesin and dynein.
Which factors regulate vesicle trafficking and fusion?
Phosphoinositides, small GTPases (Rab and Arf proteins), and SNARE proteins.
How can bacterial proteins exploit the host’s vesicle trafficking machinery?
By modifying trafficking pathways to deliver toxins or alter the vacuolar environment.
Why is the control of vesicle trafficking critical for pathogens?
It creates an intracellular niche that supports replication and evasion of host defences.
What cellular process do Rab and Arf GTPases primarily regulate?
Vesicle trafficking and membrane remodeling.
Which Legionella effector acts as an ArfGEF to modulate Arf1?
RalF.
Which Rab GTPase is modulated by Legionella effectors SidM and LepB?
Rab1.
How does Salmonella influence Rab GTPases?
It employs the proteolytic cleavage of Rab32 via the effector GtgE.
Which pathogen employs a type IV secretion system (T4SS) to modify its intracellular compartment?
Legionella pneumophila, utilising the Dot/Icm system.
Approximately how many effectors does the Legionella T4SS deliver?
Over 100 effectors.
Which bacterium uses a type IV secretion system known as the VirB system?
Brucella.
How do these secretion systems benefit the pathogen?
They remodel the vacuole to interact with endoplasmic reticulum exit sites, thus creating a niche for intracellular survival.
How does the exploitation of actin benefit these pathogens?
It supports both intracellular mobility and cell-to-cell dissemination.
How does actin-based motility contribute to a pathogen’s virulence?
It facilitates rapid intracellular spread and helps evade host immune responses.
What does the widespread use of actin-based movement suggest about pathogen evolution?
It indicates that subverting the host’s cytoskeleton is a highly advantageous strategy for infection.
What common strategy is observed among pathogens that utilise the host’s actin cytoskeleton for movement?
They all manipulate actin to propel themselves and promote intercellular spread.
How do these systems complement each other in the infection process?
They enable both the initial invasion and subsequent intracellular persistence of the pathogen.
How does the probe function in the context of infection?
It binds to PI(3)P, thereby indicating the localisation of this phosphoinositide.
What is the composition of this PI(3)P probe?
It consists of a fusion protein containing a 2×FYVE domain linked to EGFP.
What role do these phosphatases play in bacterial infection?
They alter the membrane composition to facilitate infection and vacuolar remodelling.
Why is a PI(3)P-specific probe employed in experimental studies?
To detect and monitor the recruitment and presence of PI(3)P during infection.
What is the overall consequence of this enzymatic activity during infection?
It facilitates vacuolar remodelling and supports bacterial intracellular survival.
Which factors regulate vesicle trafficking and membrane fusion?
Phosphoinositides, small GTPases (including Rab and Arf proteins), and SNARE proteins.
What does the active (GTP-bound) state of a Rho GTPase signify?
It indicates the protein is signalling and capable of initiating downstream cellular responses.
How do bacterial pathogens exploit host cytoskeletal components?
They manipulate microfilaments (actin) and microtubules to facilitate invasion and survival within host cells.
What are the two main mechanisms bacteria use to enter host cells?
Trigger Mechanism: Causes major membrane ruffling and extension (e.g., Salmonella, Shigella). Zipper Mechanism: Involves minimal membrane changes via bacterial adhesion (e.g., Listeria, Yersinia).
What is the role of small GTPases in bacterial entry?
They regulate actin polymerization and cell shape. Bacteria mimic or inhibit these GTPases (e.g., Salmonella alters GTPase activity to manipulate the host cytoskeleton).
How do bacteria manipulate the endocytic pathway to avoid degradation?
They alter phosphoinositide metabolism, disrupting membrane trafficking. They use type III or IV secretion systems to inject effector proteins that interfere with host processes.
How does Salmonella evade the immune response after entering a host cell?
It modifies the complement system and recruits host proteins to maintain a stable intracellular environment.
How does Listeria monocytogenes use actin polymerization for survival?
It produces ActA protein, which triggers actin polymerization, allowing movement through the cytoplasm and cell-to-cell spread.
How do Brucella and Legionella establish intracellular niches?
They use type IV secretion systems to resemble the endoplasmic reticulum, preventing detection and degradation.
What role does actin polymerization play in bacterial motility?
Bacteria like Listeria and Shigella induce actin tails to propel themselves through the cytoplasm, aiding rapid spread.
Why is understanding bacterial manipulation of host cytoskeletal dynamics important?
It provides insights into bacterial survival strategies, helping researchers develop targeted therapeutics against infections.
What host processes do bacterial pathogens exploit for survival?
They target receptors, GTPases, and phosphoinositide metabolism to manipulate cytoskeleton dynamics and evade immune detection.
