Repetition

Question 1

Where are PRRs located, and give an example of its function in that location.

Answer 1

PRRs can be transmembrane proteins and located on the surface of many types of host cells where they recognise extracellular pathogens. For example, on macrophages and neutrophils they can mediate the uptake of pathogens into phagosomes to destroy the pathogens. PRRs can also be located intracellularly, where they can detect intracellular pathogens such as viruses. These PRRs are either free in the cytosol or associated with the membranes of the endolysosomal system.

Question 2

How do bacteria defend themselves against viruses?

Answer 2

Bacteria defend themselves from viruses using intracellular proteins called restriction factors, which block viral propagation.

Question 3

What is the function of natural killer (NK) cells?

Answer 3

NK cells, which can be enhanced by type 1 interferons (interferon-a and interferon-b) are recruited to the site of inflammation and destroy virus-infected cells by inducing apoptosis.

Question 4

What happens if a pathogen is too large to be phagocytosed by phagocytic cells?

Answer 4

Instead of phagocytosing the pathogen, phagocytic cells like macrophages, neutrophils, and eosinophils will gather around the invader. They secrete defensins and other damaging agents and release the toxic products of the respiratory burst. This barrage is often sufficient to destroy the pathogen.

Question 5

What is xenophagy? How does it function?

Answer 5

Xenophagy is the autophagy of microbial invaders. Xenophagy functions in the direct elimination of invaders, activation of immune responses, inflammasome control, and antigen presentation.

Question 6

Give the pathway of xenophagy. How does this compare to viral phagocytosis?

Answer 6

In xenophagy, phagocytic cells engulf a pathogen, and the pathogen is sequestered in a double membrane vesicle named the autophagosome. Subsequently, the autophagosome is fused with a lysosome containing antimicrobial toxins, and the pathogen is degraded in this autolysosome. In xenophagy with viral pathogens, a virus is engulfed by a phagocytic cell and viral nucleic acids are then sequestered in an autophagosome. The autophagosome containing viral nucleic acid is then turned into an endosome and the viral nucleic acid can be recognised to induce expression of type 1 interferons (interferon-a and interferon-B).

Question 7

How do selective autophagy receptors (SLRs) function in xenophagy.

Answer 7

Salmonella can escape phagocytosis and end up in the cytosol, after which it is recognised by the ubiquitin machinery, after which the bacterium can be sequestered in an autophagosome and lysed. SLRs, like Atg8, recognise ubiquitin and/or galectins on bacteria or damaged phagosomes and target them to LC3-II. SLRs also deliver neo-antimicrobial peptides derived from ubiquitinated proteins. These ubiquitinated proteins are often useful precursors for new antimicrobial peptides. These neo-antimicrobial peptides and the ubiquitinated pathogen end up in the same autophagosome, where the antimicrobial peptides can degrade the pathogen.

Question 8

What is Persephone and how is it activated?

Answer 8

Persephone is a serine protease that belongs to a danger pathway activated by abnormal proteolytic activities, and results in the activation of the Toll pathway. The Persephone pathway can be activated by the exogenous proteases of a range of different microorganisms, including Gram-negative bacteria. Persephone itself is an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns.

Question 9

Explain the Ca2+-dependent CAX1/3 activation pathway.

Answer 9

The Ca2+ sensor CBL couples with CBL-interacting protein kinases (CIPKs) and form a complex. CBL-CIPK modules can then activate CAX1/3 by phosphorylating the conserved S-cluster in the auto-inhibitory domain. CAX1/3 may also serve as a convergent point of other Ca2+ signalling events in response to abiotic stress factors.

Question 10

Explain the Ca2+-independent CAX1/3 activation pathway.

Answer 10

Ca2+-independent activation of CAX1/3 occurs through pattern-triggered immunity (PTI). In response to MAMPs, the immune receptor complex of flagellin sensitive 2 (FLS2) and brassinosteroid insensitive 1-associated kinase 1 (BAK1) is assembled. Downstream cytoplasmic kinases BIK1 and PBL1 can then phosphorylate the conserved S-cluster in the auto-inhibitory domain of CAX1/3.

Question 11

When is a plant protected against a pathogen.

Answer 11

If an avirulent (Avr) allele in a pathogen responds to an R allele in the host plant, the host plant will have resistance, making the pathogen avirulent.

Question 12

Name some examples of PAMPs/MAMPs.

Answer 12

Flagellin in bacterial flagella, peptidoglycan in bacterial cell walls, EF-Tu (bacterial elongation factor for protein translation), chitin in fungal cells walls, and oligosaccharides in fungal and plant cell walls.

Question 13

What is the signalling cascade of flagellin recognition in plant immunity.

Answer 13

The PAMP flagellin can be recognised by the FLS2 PRR, after which it dimerises with BAK1, which is a co-receptor of several PRRs. Subsequently, BIK1, a receptor-like cytoplasmic kinase (RLCK) is recruited and mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs) are activated.

Question 14

How do specialised pathogens, like certain bacteria or fungi, affect PTI in plants?

Answer 14

With specialised pathogens, like certain bacteria or fungi, the fungi grows inside the plant cell and secretes effector proteins, or the bacterium secretes effector proteins inside the plant cell using a pilus. These effector proteins inhibit the PTI response and can be recognised by an R-proteins’ nucleotide-binding leucine-rich repeats (NB-LRR), resulting in an ETI response. These effector proteins have an effect on a lot of different parts of the PTI. For example, the effector proteins can inhibit PRRs and BAK1, MAPK, and GRP7, which are important in mRNA translation.

Question 15

On which responses is plant immunity based?

Answer 15

Plant immunity is based on a hypersensitive response (cell death), and the production of pathogen-related proteins (toxic proteins and enzymes that degrade bacterial/fungal cell walls).

Question 16

Plant responses can be divided into two categories. Give these two categories and explain what they entail.

Answer 16

Plant responses can be divided into a local defence and a systemic defence. The local defence, which happens at the site of inflammation, consists of a hypersensitive response (HR) and the secretion of antimicrobial secondary metabolites (e.g. alkaloids in some plant species, glucosinolates in Arabidopsis), antimicrobial proteins (e.g. defensins, pathogenesis-related (PR) proteins), and antimicrobial cell wall-degrading enzymes (e.g. chitinases and B-glucanases). The systemic defence consists of systemic acquired resistance (SAR; depends on salicylic acid and ethylene) and induced systemic resistance (ISR; depends on jasmonic acid and ethylene).

Question 17

What are the 5 key requirements of the immune system?

Answer 17

1. Rapidly and efficiently eliminate dangers
2. Be able to recognise a diverse range of molecules
3. Be tightly controlled (up- and downregulated)
4. Be tolerant to own cells and tissues, and commensals
5. Memorise and transfer protection to progeny

Question 18

Name all types of phagocytic cells and their function.

Answer 18

Neutrophils, macrophages, and dendritic cells are all phagocytic cells. Of those, macrophages and dendritic cells function also as antigen presenting cells (APCs). Proteins that are eaten by APCs are broken down to small pieces (peptides), which are loaded on special receptors (MHCs) and transported to the cell surface. A peptide-MHC complex can be recognised by a T cell and that interaction can lead to an adaptive immune response.

Question 19

What does activation of a Toll-like receptor do?

Answer 19

TLR signalling activates the inflammatory response. A signalling cascade leads to NFkB inducing the expression of cytokines (TNF, IL-1, and IL-6), chemokines (CCL2, CXCL8, and others), endothelial adhesion molecules (E-selectin), and costimulatory molecules (CD80 and CD86).

Question 20

Macrophages can polarise into two states. Explain these states.

Answer 20

Macrophages can differentiate into different phenotypes, of which the main extremes are M1 and M2, due to activation by certain compounds. The M1-macrophage phenotype has a pro-inflammatory, bactericidal, and phagocytic function. The M2-macrophage phenotype has an anti-inflammatory, matrix producing, pro-angiogenesis, and pro-wound healing function. Macrophage polarisation goes along with metabolic change. In the inflammatory M1-macrophage phenotype, due to two breaks in the TCA cycle, the production of antimicrobial nitric oxide is activated due to an excess of citrate, and due to an excess of succinate, IL-1B and IL-5 are produced. In the anti-inflammatory M2-macrophage phenotype, the TCA cycle is not broken at two points, resulting in an absence of nitric oxide, and IL-1B and IL-5 production.

Question 21

With insect immunity, the innate immune system is split into two parts. What are these parts and what function do they have?

Answer 21

The innate immune system of insects is split into the systemic/humoral part and the local part. With the systemic/humoral part, the fat body secretes antimicrobial peptides (AMPs) upon infection into the haemolymph that circulates through the whole insect, and reactive oxygen species (ROS) are produced. The local system consists of a cellular response with haemocytes.

Question 22

What is the function of plasmatocytes in insect immunity, and which proteins play an important role in this function?

Answer 22

Plasmatocytes can conduct phagocytosis. In this phagocytosis, scavenger receptors, eater proteins, and DSCAM play a very important role.

Question 23

What is the function of crystal cells in insect immunity, and which protein plays an important role in this function?

Answer 23

Crystal cells can conduct melanisation, where prophenoloxidases play a very important part.

Question 24

What is the function of lamellocytes in insect immunity?

Answer 24

Lamellocytes can conduct encapsulation. Lamellocytes differentiate and migrate to the infection.

Question 25

Which two main signalling pathways are present in the insect innate immune system, and how are they activated?

Answer 25

The Toll pathway and the IMD pathway. The Toll pathway recognises the lysine in the peptidoglycan in the cell wall of Gram-positive bacteria, but is also activated by fungi and yeast. The IMD pathway recognises the mesodiaminopimelic acid in the peptidoglycan of Gram-negative bacteria. However, they don’t exclusively activate one pathway or the other; bacilli, Gram-positive bacteria, synthesise mesodiaminopimelic acid peptidoglycan, which activates the IMD pathway, but their microbial patterns activate the Toll pathway.

Question 26

How does the IMD pathway signalling cascade work?

Answer 26

Upon activation, PGRP-LC recruits IMD, DFADD, and Dredd. Dredd activates dTAK1 and its dTAB2, which activate Relish. Dredd is also a caspase that cleaves activated Relish from its inhibitory IkB domain. The NFkB domain of Relish can enter the nucleus as a transcription factor, which results in the synthesis of AMPs, prevention of self-tissue damage, haemocyte proliferation/differentiation, and induction of phagocytosis.

Question 27

How does the Toll pathway signalling cascade work?

Answer 27

After recognition, a proteolytic cascade leads to the cleavage of Spaetzle, that can bind to and activate the Toll-receptor. The intracellular pathway then leads to the degradation of Cactus to free the Dorsal/Dif for nuclear translocation. These NFkBs can enter the nucleus as transcription factors, resulting in the synthesis of AMPs, prevention of self-tissue damage, haemocyte proliferation/differentiation, and induction of phagocytosis.

Question 28

How does insect Toll differ from vertebrate Toll?

Answer 28

In comparison to vertebrates, Toll is not a pattern recognition protein, but it is activated by a ligand. The pattern recognition proteins in the Toll or IMD pathway do not recognise lipopolysaccharides, like they do in vertebrates, but peptidoglycans.

Question 29

What are macrophages?

Answer 29

Macrophages are mononuclear phagocytes that act as tissue-immune sentinels and perform critical homeostatic functions, including phagocytosing spent cells, recycling nutrients, remodelling tissues, and resolving inflammation. Macrophages mediate key antimicrobial responses against bacterial pathogens.

Question 30

Stimulation by which compounds results in M1-like macrophage polarisation? And stimulation by which compounds results in M2-like macrophage polarisation?

Answer 30

The M1-state occurs upon recognition of PAMPs, such as LPS, and inflammatory immune signalling, such as interferon-gamma. The M2-state entails macrophage activation from type-2 immunity-associated cytokines such as IL-3 and IL-4 or IL-10.

Question 31

How do intracellular bacteria persist in macrophages?

Answer 31

Intracellular bacteria, like Salmonella enterica, expresses macromolecular secretion systems to inject virulence effector proteins into the host cell cytoplasm to co-opt cellular activities. Manipulation of macrophage polarisation is an important bacterial pathogenesis mechanism facilitating tissue persistence despite host immune responses.

Question 32

When is immunoresponsive gene 1 (IRG1) expressed, where is it expressed, and by what is it expressed?

Answer 32

IRG1 expression is rapidly induced within macrophage-lineage cells in response to live bacterial challenge and TLR agonist stimulation.

Question 33

What is the function of immunoresponsive gene 1 (IRG1)?

Answer 33

IRG1 localises to mitochondria and functions during catabolism of the short-chain fatty acid propionate.

Question 34

What does C/EBPB do?

Answer 34

The primary-responsive transcription factor C/EBPB helps drive infection-responsive irg1 expression within macrophage lineage-cells.

Question 35

What is CHX and how does it affect C/EBP? Is C/EBP controlled?

Answer 35

CHX is a protein synthesis inhibition protein that enhances C/EBP expression in infected zebrafish larvae and control larvae. C/EBP is possibly controlled by a transcriptional repressor.

Question 36

What is the effect of glucocorticoid receptor (GR) agonists on C/EBPB and IRG1?

Answer 36

Stimulation with GR agonists is sufficient to activate C/EBPB, but not IRG1.

Question 37

How does STAT3 function in the IRG1 signalling cascade?

Answer 37

Activation of STAT3 is crucial for inducing IRG1. Inhibition of JAK/STAT signalling abolishes IRG1 expression during inflammatory responses. However, STAT3 depletion does not affect the expression of C/EBPB.

Question 38

Which pathways regulate IRG1 expression?

Answer 38

The C/EBPB, GR, and JAK/STAT signalling pathways.

Question 39

What happens to mitochondria that lack IRG1?

Answer 39

Mitochondria of IRG1-depleted macrophage-lineage cells are deficient in their ability to utilise fatty acids as an energy substrate for mitochondrial oxidative phosphorilation and mitochondrial ROS (mROS) production, resulting in decreased bactericidal activity.

Question 40

What is a requirement for IRG1-dependent mROS production?

Answer 40

IRG1-dependent mROS production requires fatty acid B-oxidation.

Question 41

How and where is itaconate synthesised?

Answer 41

Itaconate is synthesised by IRG1 out of aconitate and citrate in the mitochondrial matrix.

Question 42

Give the functional pathway of itaconate.

Answer 42

In response to LPS, in part through type 1 IFNs, itaconate is synthesised by IRG1. Subsequently, itaconate moves across the mitochondrial inner membrane to activate Nrf2 in the cytosol by alkylation of KEAP1 cysteine residues. KEAP1 normally associates with and promotes the degradation of Nrf2, but alkylation of KEAP1 cysteine residues allows newly synthesised Nrf2 to accumulate, migrate to the nucleus and activate a transcriptional anti-oxidant and anti-inflammatory program. This limits further inflammatory gene expression, and its own production by downregulating the IFN response.

Question 43

Explain how recognition of effector proteins by NLR receptors leads to a hypersensitive response in plants.

Answer 43

Intracellular NLR receptors recognise pathogen effectors. When they recognise an effector, they induce oligomeric NLR complexes called resistosomes. Activation of NLR resistosomes induces effector-triggered immunity (ETI), often including a hypersensitive response (HR). The TIR-NLR (TNLs) resistosomes function as holoenzymes of TIR-encoded NADase. The NADase activity is required for the activation of two immune signalling nodes, EDS1-SAG101 and EDS1-PAD4 heterodimers, and the helper NLRs (RNLs), NRG1s, and ADR1s. Once TNLs are activated, EDS1-SAG101 forms a hetero complex with NRG1s and EDS1-PAD4 forms a hetero complex with ADR1s, inducing Ca2+-channel activity of the RNLs. This then leads to cell death and disease resistance.

Question 44

What is the Krebs cycle and where does it take place?

Answer 44

The Krebs cycle is a metabolic cycle that takes place inside the mitochondrial matrix, whereby its primary function is the harvesting of high-energy electrons from carbon fuel sources.

Question 45

What happens to the Krebs cycle upon infection?

Answer 45

In response to inflammation, the Krebs cycle is rewired to provide compounds that are used in immunity in M1 macrophages: breaks in the Krebs cycle result in an excess of citrate and succinate, which is used in the production of the antimicrobial nitric oxide and IL-1B and IL-5, respectively. In M2 macrophages, the Krebs cycle remains intact and functions as normal.