Lecture 4

Question 1

What are the two major types of toxins produced by bacteria?

Answer 1

Endotoxins which are cell bound and released due to cell lysis and includes substances like lipopolysaccharide, lipotechtoic acid and Neisseria Lipooligo-saccharide
Exotoxins which are secreted, soluble and of which there is a large variety

Question 2

How do Lipopolysaccharide and Lipotechtoic acid function as toxins?

Answer 2

Both of these compounds bind to CD14 and Toll-Like receptor 4 on antigen presenting cells resulting in a very strong inflammatory cytokine response which potentially leads to septic shock

Question 3

What are the different types of bacterial exotoxins?

Answer 3

Membrane damaging toxins which includes pore forming toxins, toxins that enzymatically damage the membrane, toxins with detegent like effect
Intracellular toxins such as AB toxins and injected toxins (type III and type 4 secretion)
toxins that act fro mthe cell surface such as superantigens
toxins that interfere with the immune response such as complement inhibitors and Ig-proteases

Question 4

How do pore forming toxins function?

Answer 4

Secreted as soluble monomers they then oligermise with the host membrane and open a central pore

Question 5

What are the classes of pore forming toxins?

Answer 5

Alpha pore forming toxins such as colchins, P.aeruginosa exotoxin A and Diptheria toxin
Beta Pore Forming Toxins such as Aerolysin, alpha haemolysin, Anthrax protective antigen, two-component Panton-Valentine Leukocidin, Leukocidin, gamma haemolysin and cholesterol dependent cytolysins

Question 6

How do beta pore forming toxins function?

Answer 6

The pore is built from beta strand domains
Transition from water soluble to amphipathic beta barrel
Use certain host proteins as receptors (such as ADAM-10 metalloprotease)
the oligomers form voltage gated channels
permeation of water, ions, and small organic molecules
Can lead to osmotic lysis of the cell

Question 7

How do Cholesterol-dependent cytolysins function?

Answer 7

Secreted as monomers by some gram positive species ans insert as a pre-pore complex using cholesterol as a receptor, forms a large pore of 30-50 sub-units which is larger than that of pore forming toxins
Uses beta sheets when in water soluble monomeric form which change to alpha helices when in the host membrane

Question 8

What are some examples of cholesterol dependent cytolysins?

Answer 8

Pneumolysin
Listerolysin
Perfringolysin

Question 9

What is the clinical relevance of pore-forming toxins?

Answer 9

This is often not easy to establish as many pathogens produce many cytolysins
however pneumolysin deficient pneumococci are cleared from lungs
Neutralising antibodies against this toxin protect from pneumococcal challenge
Pneumolysin destroys ciliated epithelial cells and phagocytes this cytoltyic activity increases neutrophil infiltration into the lungs thus increasing inflammation
Pneumolysin is therefore an essential virulence factor for the development of pneumonia
This toxin can also induce inflammation in the brain if the bacteria breach the blood-brain barrier

Question 10

What is an example of a cytolysin with enzymatic activity?

Answer 10

The alpha toxin of clostridium perfringens also known as Zn-metallophosphoplipase C

Question 11

What are the effects of Zn-metallophosphoplipase C at differing concentrations?

Answer 11

Low concentrations of Zn-metallophosphoplipase C results in limited hydrolysis of PC, generatin of DAG, signal transduction pathways
High concentrations result in massive degradation of PC, membrane disruption and cell lysis

Question 12

What is the relationship between C. Perfringens alpha toxin and disease?

Answer 12

The toxin is the major virulence factor of C. Perfringens and targets erythrocytes, platelets, leukocytes, and endothelial cells resulting in membrane damage of these cells and lysis releaseing secondary messengers such as DAG and IP3 which initiates Ca2+ efflux resulting in increased vascuar permeability and edema leading to tissue destruction known as clostridial myonecrosis or gas gangrene

Question 13

What is the functional importance of poreforming toxins for bacteria?

Answer 13

Used in nutrition as lysed cells (particularly red blood cells) can release iron which is essential for many pathogens growth
Can be used as a spreading factor as the tissue destruction will allow the bacteria to spread to colonize other tissues
Can be important for immune evasion as they destroy immune cells or they allow the bacteria to escape the phagosome (for example listerolysin)

Question 14

What is the largest family of intracellular toxins?

Answer 14

AB toxins

Question 15

What is the structure of AB toxins

Answer 15

They are made of two different types of subunit, a B subunit which binds to target regions on cell membranes and an A subunit which possesses enzymatic activity which affects intracellular bio-mechanisms

Question 16

What are examples of AB toxins?

Answer 16

Cholera toxin, pertussis toxin and shiga toxin

Question 17

How do AB toxins function?

Answer 17

Bind to the host cell and taken up via endocytosis (may be cholesterol dependent/independent or potentially clatherin mediated)
The endosome will then be acidified this results in a pH drop triggering a conformational change in the protein to seperate the A and B subunits the A subunit may then leave directly from the endosome or travel through the endosome to the golgi to the Endoplasmic recticulum via the endoplasmic recticulum associated degradation route and enter the cytosol at this point

Question 18

What are the most common intracellular targets for A subunit?

Answer 18

elongation factor EF-2 (diphteria toxin, Pseudomonas exotoxin A)
G proteins (cholera toxin, pertussis toxin, E. coli heat-labile enterotoxin)
Rho proteins (large clostridial cytotoxins)
Actin (Clostridium C2 toxin)
nucleus (cytolethal distending toxins)
28S rRNA (shiga toxin)

Question 19

What are examples of AB toxins where the A subunit leaves at the endosome?

Answer 19

Diphtheria toxin
Clostridial neurotoxins
Anthrax

Question 20

What are examples of AB toxins where the A subunit leaves via the endoplasmic recticulum associated degradation route?

Answer 20

Shiga toxin
Cholera toxin
Pertussis toxin
Pseudomonas exotoxin A

Question 21

What is the enzymatic activity triggered by cholera toxin and LT?

Answer 21

The A subunit is a globular enzyme that ADP-ribosylates the G alpha unit of Gs proteins this leads to constant activation of the cyclase causing increased leavles of cAMP and therefore active Protein Kinase A
Protein Kinase A then phosphorylates Cystic Fibrosis Trans-mambrane conductance regulator resulting in active Cl- secretion triggering osmotic water loss and diarrhea

Question 22

What is LT?

Answer 22

Heat-labile enterrotoxin produced by enterotoxigenic E.Coli
It is plasmid encoded
Induces diarrhea though typically this is less serious than the diarrhea induced by cholera toxin
causes disease known as traveler disease

Question 23

What are the four genera that use injected toxins?

Answer 23

Salmonella spp.
Shigella spp.
Yersinia spp.
E. coli!

Question 24

What are the salmonella typhimurium effector proteins?

Answer 24

Proteins that are injected into the host cell via a type 3 secretion mechanism
Sip A acts as a molecular staple while Sip C promote actin nucelation and filament bundling
Sop E mimmcks G nucelotide exchange factor leading to the activation of small G proteins of the Rho family triggering actin nucleation and branching
these combine to cause membrane rufflng and allows the microorganism to be taken up into non-phagocytic cells

Question 25

What are toxins that act from the cell suface?

Answer 25

Superantigens which are only found in 4 genera
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus disgalactiae
Streptococcus agalactiae
Yersinia pseudotuberculosis
Mycoplasma arthritidis

Question 26

What are super antigens?

Answer 26

Very powerful T cell mitogens, these bind to MHC-II outside the antigen binding region and to the Tcell Receptor VBeta chain
They lead to overstimulation of T cells (can stiumlate about 20% of the T cell population) resulting in a massive release of proinflammatory cytokines which can induce shock like conditions in animals
this potentially leads to staphylococcal/streptococcal toxic shock syndrome which involves systemic inflammation