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1

Toxins targetting the plasma membrane (6)

S. aureus alpha toxin
S. aureus leukocidine
Perfringolysin
E. coli alpha toxin
C. perfringens enterotoxin
V. parahemolyticus hemolysin.

2

Toxins targetting protein synthesis (3)

Diphtheria toxin
Pseudomonas exotoxin A
Shiga toxin.

3

Toxins targetting the cytoskeleton

C. botulinum C2 toxin (actin ADP-ribosylating
C. perfringens a (?) toxin
V. cholerae RTX (catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments)

4

Toxins that target the cell membrane - general types

Phospholipases
Pore forming cytotoxins.

5

Phospholipase effects - esp Clostridium perfringens alpha toxin

Membrane disrupting alpha toxin has broad tropism, kills cells.
Results: release of nutrients, formation of dead tissue which has no blood supply for the delivery of oxygen, antibiotics or immune defence.

6

Structure of clostridium perfringens alpha toxin

Ctd; membrane binding, like eukaryotic C2 domains.
Ntd: catalytic, homologous to other phospholipases.

7

Clostridium perfringens alpha toxin action

Ca++ dependent insertion --> conformational change --> cleavage. At sublytic concentrations, diacylglycerol produced may lead to signalling leading to inflammatory response, vascular permeability and platelet aggregation.

8

General action of pore-forming toxins

Bind receptor. Oligomerise. Insert to form pore. Alter ion concentrations.

9

Clostridium perfringens pore forming toxin, PFO.

Bind to the membrane to form arcs and rings. Regulated by VirR-VirS and PfoR systems. Prepore --> pore.

10

Pore forming toxins results

Lytic concentration: ion conc changes --> osmotic potential --> lysis.
Sublytic: increase in Ca++, decrease in K+ --> alters MAPKKK, alters Sek, alters MAPK signalling.

11

Clostridium perfringens pore forming toxin, PFO.

Bind to the membrane to form arcs and rings. Regulated by VirR-VirS system. Prepore --> pore.

12

Clostridium perfringens pore forming toxin, PFO. Prepore --> pore collapse

D2 undergoes vertical collapse. D3 insertes beta-hairpins.

13

Clostridium perfringens toxins co-operation.

PFO needs to bind cholesterol, hidden by phospho head groups. Alpha toxin removes these.

14

PFO pathogenesis

Tissue destruction, absence of inflammatory cells, intravascular blockage, cardiovascular collapse.

15

Staph aureus toxins

Hyaluronidase (spreading factor), coagulase (clot formation), staphylokinase, lipase (penetration of fatty tissue), collagenase.
Under control of Agr system.
RNA III needed for translation of alpha hemolysin.

16

Staph aureus pore forming toxins.

alpha-hemolysin - disruption of epithelial barrier.
Leukocidins - immune cell death and dysfunction.

17

Short form of staphylococcal alpha hemolysin

Hla

18

Short form of staphylococcal leukocidins

Luk

19

HlyE pore formation

Hydrophobic B-tongue region inserts. Helix A swings up to make an extension of helix B. Oligomerisation occurs, stimulating insertion of helix A.

20

Mechanism of Hla and Luk

Structural rearrangement of amino latch to contact neighbouring monomer leads to extension of beta hairpin stem into lipid bilayer.
Forms mushroom shaped toxin with B-stem.

21

Mechanism of Hla and Luk

Structural rearrangement of amino latch to contact neighbouring monomer leads to extension of beat hairpin stem into lipid bilayer.

22

HlyE

35 angstrom pore. Dodecameric alpha-helical pore.

23

HlyE pore formation

B-tongue region inserts. Helix A swings up to make an extension of helix B. Oligomerisation occurs, stimulating insertion of helix A.

24

Common features between HlyE and S. aureus alpha hemolysin.

Soluble monomers.
Oligomeric pre-pore complex.
Structural rearrangements lead to hydrophobic domain swinging out.

25

RTX toxins

E. coli hemolysin.
Multiple nonapeptide repeats which bind calcium.

26

Effect of binding specific receptors

lowering diffusion space from 3D to 2D means much more likely to contact each other. Pre-clustered receptor exacerbrate this effect.

27

Structures and mechanisms by which pores are formed

o β-PFTs
o α-PFT

28

β-PFTs - types

Cholestrol dependent cytolysins.
α-haemolysin and leukocidins
Aerolysin .

29

Example of B-PFT cholestrol dependent cytolysin

perfringolysin
ILY

30

Structure of PFO

• Four domains per monomer. Each monomer secreted as soluble monomer by Sec. D1 - D4

31

Structure of PFO; D4

D4 tryptophan-rich undecapeptide anchors in perpendicular position. Anchors to non-classical receptor cholesterol in lipid rafts.

32

Structure of ILY; D4

Changes in D4 mean ILY doesn’t bind all cholesterol containing membranes, but is specific to those containing CD59 instead; it has the single function of initiating pre-pore to pore conversion.

33

Structure of PFO; D3

o D3 also important in oligomerisation to form prepore – after membrane binding by D4 leads to conformationally coupled change in small loop in D3.

34

Example of B-PFT cholestrol dependent cytolysin

perfringolysin
Intermedilysin

35

Structure of PFO; D3

o D3 also important in oligomerisation to form prepore – after membrane binding by D4 leads to conformationally coupled change in small loop in D3.
Has beta hairpins (two per monomer in PFO) which probably form pre-insertion beta-barrel.

36

Cholesterol dependent cytolysins - cholestrol dependence.

For unknown reasons, CDCs seem most dependent on cholesterol on pre-pore to pore stage, not initial association stage.

37

Cholesterol dependent cytolysins - pore size.

20-30 nm.

38

α-PFT

HlyE
RTX toxins such as HlyA

39

Unique CDCs

LLO
Streptolysin
PLY

40

Uniqueness of LLO

o LLO activity is pH dependent.
o LLO PEST sequence causes degradation in cytoplasm

41

Uniqueness of streptolysin

Translocation through pore of active domain.
NAD+ glycohydrolase is transported in. Inhibits internalisation of bacteria, and promote keratinocyte death.

42

Uniqueness of PLY

A CDC, but contains a domain which activates complement as well.

43

Aerolysin

Binds lipid-anchored proteins, requires propeptide cleavage to initiate oligomerisation. Since protease furin is restricted to cell surface, so is oligomerisation.

44

HlyE oligomerisation

Oligomerisation occurs with tight specific interactions between protomers, more than 1/4 of surface involved in interface

45

RTX repeats

In pore forming toxins, but also in others e.g. P. aeruginosa alkaline phosphatase also has this motif.

46

Use of PFTs in biotech

Α-haemolysin used in biotech sequencing.

47

Expression of HlyA

rfaH promotes expression.

48

rfaH

rfaH promotes expression of long operons encoding cell surface and secreted proteins including HlyA operon.
Enhances elongation since ops element induces transcriptional pause: rfaH binds both ops and RNAP to prevent termination and increase speed of elongation.

49

Proteins using pore forming toxins for translocation

Not AB toxins, where the pore doesn't count.
Pertusis CyaA toxin is RTX toxin fused with calmodulin dep. cyclase.
Streptolysin translocates NAD+ glycohydrolase.

50

Pertussis CyaA

RTX domain allows translocation and causes ion fluxes. Latter is primary function of RTX.
calmodulin dependent AC makes cAMP.

51

Anthrax toxin.

PA binds anthrax toxin receptor. Activation requires cleavage. Oligomerises. LF or EF bind PA. complex is endocytosed, pH change leads to PA insertion and translocation of LF/EF.

52

Pertussis CyaA

RTX domain allows translocation and causes ion fluxes.
calmodulin dependent AC makes cAMP.

53

cAMP synthesis and breakdown

Synthesised from ATP by cya.
Broken down by phosphodiesterases to AMP.

54

G proteins affecting cAMP

GPCRs release active G proteins when stimulated. Gas stimulates AC, Gai inhibits. Alpha subunit GTPase activity terminates G protein activity.

55

cAMP affects

Many, including ion channels and PKAs.

56

cAMP toxins - entry

Retrograde transport and ERAD (cholera).
Pore forming toxin translocation - Pertussis CyaA.
Endocytosis, B forming pore, A translocates. Anthrax edema factor.
T3SS - ExoY.

57

4 protein toxins which are adenylate cyclases.

Cya, EF, ExoY and AC of Y. Pestis

58

Cholera toxin activity

ADP ribose from NAD+ transferred onto R (arginine) on GTPase so no GTP hydrolysis so stimulation permanently active.

59

Pertussis toxin target

Targets Gia cysteine residue, preventing exchange of GDP for GTP. Inhibitory pathway blocked.

60

Cholera toxin activity

ADP ribose from NAD+ transferred onto R on GTPase so no GTP hydrolysis so stimulation permanently active.

61

ExoY

Produced by P. Aeruginosa. Probably enters cells by T3SS. Doesn't depend on calmodulin, but does depend on unknown eukaryotic factor. Heat labile.

62

Things to consider with cAMP toxins

How they get into the cell
Outline cAMP pathways
Action of toxins
Effect of location within cell
Effect of location outside cell.

63

How do toxins affecting cAMP work

1) Modulate G proteins
2) Deliver adenylate cyclases.

64

Labile toxin

Labile toxin like cholera toxin - produced by enterotoxinogenic E. Coli.

65

ExoY

Produced by P. Aeruginosa. Probably enters cells by T3SS. Doesn't depend on calmodulin, but does depend on unknown eukaryotic factor.

66

Retrograde transport for cholera toxin.

Binds GM1 via B-subunits. Retrograde transport leads to entering Golgi. Binds KDEL on ERD2 for retention to ER. Protein disulphide isomerase releases A1. Binds ERAD so is transported into the cytoplasm but is not degraded as no exposed lysine residues for ubiquitination.

67

Results of cAMP toxins

Water loss, chemotactic expression, preventing phagocytosis, altering cytokine response.

68

AC domain of CyaA

N-terminal AC domain translocated by RTX domain. Binds calmodulin like EF but with much higher affinity - probably competing with different proteins.
 Kcat = 2000 s-1 conversion of ATP  cAMP

69

Edema factor

Part of bacillus anthracis toxin.
Needs to be activated by calmodulin. Prior to this, it is disordered; binding of CaM leads to ordering of switch B, to form an ATP binding section and stable catalytic residues.

70

Results of cAMP toxins

Water loss, altering inflammatory response, preventing phagocytosis, altering cytokine response.

71

CyaA effects - which cells.

o Primarily acts on myeloid phagocytes as it binds αMβ2
• Ciliated epithelial cells - difficult to differentiate from RTX action.

72

CyaA action in myeloid phagocytes.

o Supresses superoxide production, chemotaxis or phagocytosis.
o Changes cytokine profile by acting on dendritic cells. Supresses synthesis of pro-inflammatory cytokines.

73

CyaA action in ciliated epithelial cells.

o Induces pro-inflammatory IL-6
o Prevents bacterial uptake
Difficult to differentiate RTX and AC action.

74

Pertussis toxin effects

o Impaired chemotaxis
o Impaired phagocytosis
 Reduced oxidative response
 Decreased killing capacity.
May induce apoptosis of some immune cell lines.

75

EF effects.

• Inhibits phagocytosis, superoxide production.
• Chemotactic response
• Cytokine expression
• Altered immune signalling (TNFa and IL-6 esp) leads to edema?
• Apoptosis in lymphocytes, dendritic cells (avoids stimulation of adaptive immune response).

76

Effect of Yersinia pestis AC

• Cytotoxic effect on macrophages. Not much known.

77

V. cholerae colonisation of small intestine

Depends on toxin co-regulated Type IV pili (TCP) which can form bundles like E. cole long bundle forming pili and which can both extend and retract.

78

Cholera toxin bacteriophage

Carries CtxAB (cholera toxin), zonula occludens toxin and accessory cholera toxin. Can integrate into the host genome at specific sites. Infects bacterial cell by binding TCP.

79

Regulation of cholera toxin expression

ToxRS and TcpPH systems. These respond to pH, and activate ToxT, which is a transcription factor activating ctxAB among other genes. Quorum sensing converging on AphA, and cAMP-CRP signalling alters transcription of TcpPH.

80

Secretion of cholera toxin

T2SS. Sec to cross IM, then enters Type II pathway which shares functional features with T4SS.

81

Secretion of cholera toxin

T2SS. Sec to cross IM, then enters Type II pathway which shares functional features with T4SS. Channel in OM occupied by pseudopilus which is used for translocation, to push substrate out.

82

Exit of cholera toxin phage

Hijacks T2SS, using OM pore to exit.

83

Cholera toxin general

AB5 structure. B is pentameric. A is cleaved, but A1 and A2 units are linked by disulphide bond. Reduced after internalisation.

84

Cholera toxin binding GM1

Each monomer binds a GM1 molecule on apical membrane of intestinal epithelial cells.

85

Adenylate cyclase pathway

G proteins or toxins activatae adenylate cyclase, which makes cAMP, which activates PKA, which activates various including CFTR.

86

Importance of Bordetella pertussis adenylate cyclase and pertussis toxin

Mostly in impairment of the immune response.

87

Bordetella pertussis virulence gene expression - general

Bvg- is environmental survival, so vir repressed genes.
Bvg(i) in respiratory transmission with class 2 then class 3 genes expressed. Bvg(+) phase is colonisation and pathogenesis, with class 1 genes expressed.

88

Bordetella pertussis virulence gene expression - Bvg(-)

Vir repressed genes on at 25 degrees.

89

Bordetella pertussis virulence gene expression - class 2.

Resp transmission stage. Early genes. Remains on throughout infection as depends on bvgA binding high affinity promoters.

90

Bordetella pertussis virulence gene expression - class 3

Intermediate genes, only expressed in respiratory transmission stages. Depends on bvgA binding high affinity promoters, but RNA processivity is blocked by bvgA binding low affinity promoters as occurs later in infection, when there is more bvgA.

91

Bordetella pertussis virulence gene expression - class 1.

Pathogenesis, includes cyaa and ptx. Depends on there being enough bvgA to activate low affinity promoters.

92

Activating BvgA

37 degrees centigrade activates BvgS, whereas low temperatures, MgSO4 and nicotinic acid deactivate. BvgS activates BvgA, a response regulator.

93

Secretion of pertussis toxin.

Type IV pathway.

94

T4SS uses

Conjugation (E. coli)
Toxin secretion (Bordetella)
Pseudopodia formation (H. pylori)
Ti plasmid transfer (Agrobacterium).
Vacuole modification (Rickettsia and Legionella).

95

Pertussis toxin general

AB5 toxin, with Bs as different proteins although subunit 4 repeats. Binds a sialoglycoprotein receptor.

96

How anthrax kills.

Non-systemic intestinal.
Systemic from cutaneous or pulmonary infection.

97

Non-systemic intestinal infection, anthrax.

Low level germination leads to effusion, mucosal edema and necrotic lesion.

98

Anthrax - systemic.

Spores infect. Bacterial virulence factors allow migration to lymph node, leading to regional hemorrhagic lymphadenitis, and hence pulmonary blockage, septicemia and toxemia (latter 2 can lead to meningitis) and death.

99

Anthrax toxin receptor

Extracellular von Willebrand factor A domain with 3 N-linked glycosylation sites. Interspecies conserved.

100

Anthrax toxin LF.

Lethal factor. Zinc dependent metalloprotease, high homology with EF, 7 Nt residues vital for PA binding.

101

Mechanisms of neurotransmitter release.

v-SNARES associatted with vesicle, t-SNAREs with target membrane.
Synaptobrevin is a v-SNARE which is anchored in the vesicle membrane by a hydrophobic carboxy terminus. A central, 70 amino acid residue, region forms an alpha helix which
complexes with two other proteins, SNAP-25 and syntaxin. This structure is known as the core complex (see Figure 5) and consists of 4 alpha helices - one each from synaptobrevin and syntaxin and two from SNAP-25. These
helices lie parallel to each other and form a leucine zipper which brings the vesicle and
plasma membranes close together.

102

Botulinum toxin

preferentially exerts its effects on cholinergic neurons. The C-terminus of its heavy chain binds to a ganglioside "receptor" (e.g. GT1b) and the N-terminus translocates
the light chain into the cell through a channel; botulinum anti-toxin only works if given within 30 minutes of adsorption of the toxin.
The light chain has peptidase activity and, once inside the cell, it cleaves the target SNARE.

103

Tetanus toxin

Does not act directly on the motor neuron, but is retrogradely transported to the
cell body. It then transfers to an inhibitory interneuron which is then unable to release its
transmitter and so the motor neuron becomes more excitable.

104

Corynebacterium diptheriae

gram +ve aerobic rod bacteria
transmitted by contact with infected/through air
causes exudative pharyngitis —> forms pseudomembrane that can lead to respiratory obstruction and death by asphyxiation
can also cause myocarditis with arrhythmia and sudden death, neuropathy and paralysis

105

Diptheria toxin control

carried by β-corynephage (not on bacterial genome)
dtx gene integrates into bacterial genome and repressed by dtxR in presence of Fe (signals not in host —> in mammalian host iron tightly bound to proteins such as haemoglobin/lactoferrin/ferritin leading to low free iron conc)

106

Diptheria toxin action

archetypal ART enzyme with NAD binding pocket and catalytic His and Glu residues
A-B toxin —> B (binds to cell surface) connected to A subunit (catalytically active part) by disulphide bridge
N-terminal catalytic domain has an unusual beta+alpha fold
C-terminal R binding domain with Greek-key topology
Central translocation domain (TM domain)

107

Shiga toxin

AB5 toxin —> B subunit = pentamer that binds to host cell and A has catalytic activity
Recently shown that B-chain has the ability to induce DNA cleavage and apoptosis —> translocation to the cytosol might occur and produce additional effects

108

Diptheria host cell entry

C terminal R binding domain on B subunit binds HB-EFG on cell surface —> clathrin mediated endocytosis
Acidification of endosome by ATPase triggers conformation change and insertion of TM domain into endosomal membrane —> facilitates transfer of A into cytoplasm

109

Shiga toxin entry

B-subunit binding. Uptake by macropinocytosis, retrograde transport. Less known about crossing membrane.

110

Shiga toxin entry - B-subunit binding.

B subunit binds to R gb3 —> expressed on microvascular endothelial cells of intestine, kidney and CNS
each B binds 3 pk-trisaccharide mols = sugar component of gb3 glycolipid —> each of 15 interactions has low affinity, but grouped together the affinity increases a million role = molecular velcro
Gb3 is present in greater amounts in renal epithelial tissues, to which the renal toxicity of Shiga toxin may be attributed

111

Diptheria toxin function

Function = ADP-ribosylation of EF2 —> required for eukaryotic translation —> decreased protein synthesis
Reaction involves scission of glycosidic bond of NAD+ —> transfer of ADP-ribose to N3 of dipthamide (post-translationally modified His) residue of tip of domain IV of EF2
ADP-ribose biding to EF2 blocks rRNA binding —> hard for euk to evolve resistance as EF2 mutation would also prevent rRNA binding
Mimics cellular NAD+-diphthamide ADP-ribosyltransferase.

112

Shiga toxin function

Function = RNA-N-glycosidase
Targets SRL loop in large 60s ribosomal subunit —> removes base from single adenine of 28S RNA —> inactivates whole ribosome as cannot interact with EFs
SRL loop is highly conserved —> target for many toxins

113

α-PFT

HlyE
RTX toxins such as HlyA

114

Symptoms of botulinum toxin

Symptoms of botulinum poisoning are somatic muscle weakness (which may lead to the need for respiratory support with a ventilator) and the autonomic signs that would be associated with loss of cholinergic activity (e.g. constipation, blurred vision, dry skin, urinary retention) though heart rate may be slowed rather than increased due to actions on noradrenergic nerves.

115

Pseudomonas exotoxin A

Binds via La, probably.
Partial proteolysis by furin required. Across cell membrane after endocytosis, mechanism not fully understood.
Mono-ADP-ribosyltransferase, similar to diptheria toxin, targets EF2.

116

Bacterial endotoxn

Lipid A is the single region of LPS that is recognized by the innate immune system. Picomolar concentrations of lipid A are sufficient to trigger a macrophage to produce proinflammatory cytokines like TNF-α and IL1β. To trigger an innate immune response, the lipid A portion of LPS alone is sufficient, yet the adaptive immune response during infection is usually directed toward the O-antigen. The key pattern recognition receptor for LPS recognition is Toll-like receptor 4 (TLR4).
LPS induces inflammatory cells to express a number of proinflammatory cytokines including IL-8, IL-6, IL-1β, IL-1, IL-12, and IFNγ; however, TNFα seems to be of critical importance during endotoxic shock and causes tissue damage.

117

Superantigens

SAgs bind directly to the outer leaflet of MHC-II molecules specific domains of the variable portion of β-chain of the T-cell receptor. This allows for bypassing the processing by antigen presenting cells and stimulates most T cells. In addition to binding to MHC-II and the Vβ-chain, it has been recently shown that SAgs also engage a third receptor, CD28, which is a costimulatory molecule on T cells. SAg bind directly at the homodimer interface of CD28 to cause toxicity by inducting a cytokine storm