‫*توکسین های اثر کننده بر روی غشاء‪:‬‬
‫*فسفولیپاز و لستیناز‬
‫*توکسین های تشکیل دهنده ی پور در غشاء سلولی ‪:‬‬
‫توکسین هایی با ساختار (‪A plus B Subunit (:)A+B‬‬
‫‪)Arrangement‬‬
* The
staphylococcal alpha-haemolysin pore (pink) is made up of seven
subunits, which span the lipid bilayer as a beta-barrel, whereas ClyA
(green) forms an iris-like barrel of alpha-helices from its twelve subunits
Attachment and Entry of Toxins
1 - Direct entry
2 - Receptor-Mediated
Endocytosis (RME)
AB toxin enters cells via:
1) Receptor mediated endocytosis
2) Fusion of vesicle with lysosome
3) Acid environment of lysosome
reduces disulfide bonds and
releases A into cell
4) A has various cellular activities
Figure 1. AB organization of bacterial toxins Diphtheria toxin is an AB toxin where the
N terminal A domain (black) encodes an Enzyme activity, an ADP- ribosyl trasferase
activity, NAD + EF-2 → ADP-r-EF-2 + nicotinamide + H+. The C-terminal B domain
encodes a receptor binding function (grey) that binds to a grwoth factor receptor and
enters cells through receptor-mediated endocytosis and a translocation function (white)
which undergoes a pH-dependent conformation a change where chaafed amino acids
are protonated which allows a pair of hydrophobic alpha helices to insert into the
endosome membrane which is responsible for the delivery of A domain in to the host
cytosol. Structure: PDB 1fol
The plasma membranes of cells contain combinations of glycosphingolipids
and protein receptors organized in glycolipoprotein microdomains termed
lipid rafts. These specialized membrane microdomains compartmentalize
cellular processes by serving as organizing centers for the assembly of
signaling molecules, influencing membrane fluidity and membrane protein
trafficking, and regulating neurotransmission and receptor trafficking. Lipid
rafts are more ordered and tightly packed than the surrounding bilayer, but
float freely in the membrane bilayer. Although more common in plasma
membrane, lipid rafts have also been reported in other parts of the cell, such
as Golgi and lysosomes
Binding of superantigen SEC3 to
TCR β
Staphylococcus aureus superantigen
SEC3 binds to the β chain of TCR
through the hypervariable domain 4
(HV4), acting as a wedge to encourage
TCR and MHCII interaction and
activation lacking foreign peptide
specificity. Arrow indicates HV4 loop
on TCR β. Structure: PDB 1jck
Comparison of antigen and superantigen binding to TCR : MHC class II
complex. Left side: normal antigen presentation. Right side: superantigen
stimulation in absence of antigen recognition. Red = MHC, blue = TCR, green =
antigen, purple = superantigen
MHC class II protein molecule
MHC class I protein molecule
Gram-negative - Type I secretion (ABC secretion)
Properties:
- ATP-binding cassette transporter (also in eukaryotes)
- Single step traversal across CM and OM
- Signal sequence at C-terminus - is not removed
- ABC channel - 6-12 transmembrane helices
- Accessory factor - bridges periplasmic space
Post-translationally
coordinated
synthesistranslocation
OM
accessory
factor
P
CM
ATP
ADP
Genes fused or coordinately expressed on operon:
N
protein - GGXGSD ABC transporter
C
accessory factor (MFP)
Outer membrane transport may not be linked
Type I secreted proteins:
RTX toxin (repeat in toxin)
E. coli hemolysin
bacteriocins
metalloproteases
Type I Secreted Toxins
Proteins secreted via the Type I pathway contain glycine-rich repeats,
usually at the C terminus, which bind calcium
composed of a tripartite protein complex that includes an inner membrane
transporter, a membrane fusion protein within the periplasm, and an outer
membrane pore protein
The adenylcyclase-hemolysin (CyaA) of Bordetella pertussis is a Type I
secreted toxin . Entry into the host cell, via the alpha(M)beta(2) integrin
(CD11b/CD18) cell receptor Upon entry into the cytosol of a host cell, CyaA is
activated by the cofactor, calmodulin, and catalyzes the conversion of ATP to
cAMP. Elevated intracellular cAMP disrupts signaling events within the cell
and inhibits the ability of phagocytes to respond to B. pertussis infections
Specifically, deregulation of cell signaling by CyaA affects protein kinase A,
which modulates neutrophil migration, cytokine synthesis, oxidative bursts
and organization of the actin cytoskeleton
Type I Secreted Toxins
OM
accessory
factor
P
CM
ATP
ADP
N
C
Type II Secreted Toxins
Type II secretion is facilitated by the Sec system and comprises protein
complexes that span the bacterial inner and outer membranes
Sec secretion includes three groups of proteins, a protein complex that spans
the inner membrane, a periplasm-spanning protein complex, and outer
membrane associated proteins. Type II secretion involves recognition of an
N-terminal signal peptide on the nascent protein
The signal sequence consists of N-terminal positive charged amino acids,
internal hydrophobic amino acids, and a C-terminal domain with prolines and
glycine
One group of periplasmic proteins are homologous to pilin-like structures
(known as pseudopilins) and regulatory proteins of the Type IV secretion
system
Type II Secreted Toxins
Gram-negative - Type II secretion
Sec-dependent secretory pathway
signal peptidase
leader peptide
N
++
hydrophobic
1-5
7-15
C
mature protein
3-7
Two step process:
Step 1 - Transfer across cytoplasmic membrane
- Leader (signal) peptide (18-26 aa)
- SecA - binds leader (L), inserts in CM channel
(requires ATP)
- SecB - cytosolic chaperone (keeps unfolded)
- SecYEG - CM channel complex
SecY,E,G
N L
post-translational translocation
SecB
C
(www.genome.ad.jp/kegg/ pathway/map/map03090.html)
Type II Secreted Toxins
Cholera toxin (CT) of Vibrio cholera is a Type II secreted toxin. CT is an AB5
toxin, where the A domain (~27.4 kDa) consists of two components, CT-A1 and
CT-A2 and the B domain (~58 kDa) is a homopentameric protein complex
CT-A1 ADP- ribosylates the Gα- subunit of the heterotrimeric protein, Gs.
CT-A1 associates with the CT-A2 via a disulfide bond where CT-A2 inserts into the
channel within the center of B5.
The B5 domain binds specifically to the ganglioside, GM1a on the surface of
intestinal epithelial cells [84]. Once bound, CT enters the cell through both clathrin
-dependent and non- clathrin-dependent vesicle mechanisms involving lipid rafts
Cytosolic CT-A1 binds A DP- ribosylation factor (ARF) and the activated CTA1
mono-ADP-ribosylates Arg 201 of Gsα, which blocks intrinsic GTPase activity and
constitutively activates Gsα
Gsα is a positive regulator of adenylate cyclase. Increased intracellular cAMP
activates protein kinase A (PKA increasing active secretion of chloride ions
Inhibition of the Na/K/2Cl co-transporter at the same time increases the
unidirectional flow of chloride into the gut lumen, causing osmotic H20 flow into
the gut lumen, the pathological outcome of cholera
Type III Secreted Toxins
The Type III secretion system (TTSS) is a bacterial virulence factor which
injects cytotoxins (also termed effectors) in an unfolded or semi-folded state
into a host cell.
TTSS comprises inner and outer membrane protein complexes, but includes
a hollow, pilin-like structure that extends beyond the outer membrane ring
complex.
The outer membrane structure is also similar to the Type II secretion system
outer membrane ring and the Type IV pilus.
Extracellular components of the TTSS include a needle, needle extension,
and translocation pore, which deliver cytotoxins into the cytoplasm of host
cells.
The needle tip proteins appear to be adaptors that link the translocator
proteins within the host membrane to the needle body for efficient transfer
of cytotoxins
The translocon protein complex prevents cytotoxin secretion before contact
with the host cell by physically blocking the hollow channel of the needle
complex
Type III Secreted Toxins
ExoS is a Type III secreted cytotoxin. ExoS is a 453 amino acid protein produced
by Pseudomonas aeruginosa and is a dual function toxin, containing a Rho
GTPase Activating Protein (Rho GAP) activity in the N terminus (96–219)
and an ADP-ribosylation domain in the C terminus (234–453)
ExoS Rho GAP domain increases γ-phosphate hydrolysis of GTP bound to Rho
GTPases and in actives RhoA, Rac1, and Cdc42
Functional orgainzation of the type III cytotoxin Pseudomaons aeruginosa
ExoS is a bi-functional toxins and is orgainzed inot discret functional doamisn
(amino acids): secretion domain (1–15), chaperone binding domain (16–51),
membrane localization domain (51–77), Rho GAP domain, active site residue
R146 (96–243), and ADPribosyl transferase domain, active site residues E379,
E381 (233–453)
ADP-ribosylation is the addition of one or more ADP-ribose moieties
to a protein . ADP-ribosylation is also responsible for the actions of
some bacterial toxins, such as cholera toxin, diphtheria toxin, pertussis
toxin, and heat-labile enterotoxin. These toxin proteins are ADP- ribosyl
transferases that modify target proteins in human cells. For example,
cholera toxin ADP- ribosylates G proteins, causing massive fluid
secretion from the lining of the small intestine, resulting in lifethreatening diarrhea. P. aeruginosa ADP- ribosylates cytoskeleton and
GTP-binding proteins
ADP Ribosylation Factors (ARFs) are members of the ARF family of
GTP-binding proteins of the Ras superfamily. ARF family proteins are
ubiquitous in eukaryotic cells, and six highly conserved members of the
family have been identified in mammalian cells.
Although ARFs are soluble, they generally associate with
membranes. They function as regulators of vesicular traffic and
actin remodelling
Pseudomonas aeruginosa produces exotoxin A (ETA) and four type III cytotoxins: ExoS,
ExoT, ExoU and ExoY. Different clinical isolates of P. aeruginosa can express one or more of
these four cytotoxins. The catalytic activity of each type III cytotoxin is activated by a host
protein. ExoU is a recently described lipase that disrupts membrane function in mammalian
cells. ExoY is an adenylate cyclase that elevates intracellular cyclic AMP (cAMP) to supraphysiological levels, which indirectly disrupts the actin cytoskeleton. ETA is the most potent
protein toxin that P. aeruginosa secretes, and it inhibits mammalian protein synthesis by
ADP- ribosylation of elongation factor 2 (EF2). One role of ExoS and ExoT is to disrupt the
actin cytoskeleton through two independent enzymatic activities: Rho GTPase-activating
protein (GAP) activity and ADP- ribosylation
Gram-negative - Type IV secretion
Properties
- Used in export of protein complexes / DNA
- Can translocate directly into host cell
- Show homology to pilus-mediated conjugal
transfer systems
- Sec-like dependent translocation into
periplasm
- B11 - related to ATP-ases of type II system
- D4 - DNA binding - may function in DNA
transfer
- B6, B7, B8 B9, B10 - core periplasmic
components
- B2, B5 - pilus components
Bacteria that use type IV secretion:
(H-J. Yeo, G. Waksman, J. Bacteriol. 2004)
A. tumefaciens
Agrobacterium tumefaciens - VirB-VirD
Bordetella pertussis - pertussis toxin
Helicobacter pylori - CagA
Legionella pneumophila
Type IV Secreted toxins
The Type IV secretion system is a multi-functional protein complex,
which transfers DNA between bacteria through conjugation (Type
IVA), and transports effector proteins into host cells to regulate host
responses to bacterial infection (Type IVB).
The VirT IVA secretion system in Agrobacterium tumefaciens is the
best characterized Type IV secretion system, which transfers DNA
and proteins into plants to cause disease.
DNA transfer requires a protein relaxase, which binds covalently to
the 5′ end of ssDNA and causes secretion specifically in a 5′ to 3′
direction
Type IV Secreted toxins
Type V secretion system (T5SS)
The Type V secretion system, also termed the
autotransporter secretion group, includes 3 transport
secretion mechanisms: termed Va, Vb, and Vc.
Va group autotransporters are translated as a single protein
composed of a N-terminal signal peptide sequence for
transport by the Sec system, the effector domain, and a Cterminal outer membrane translocation domain
Type V secretion system (T5SS)
Type VI and Type VII Secretion Systems
A type VI secretion system has been proposed in V. cholerae
and P. aeruginosa .
Type VI effectors do not possess N-terminal signal peptides and
are Sec secretion independent, implicating a unique mechanism
for effector transport relative to the Types I–V secretion
systems.
Recently, a secretion system was identified in the mycobacteria
that was classified as the Type VII secretion for Gram-Positive
bacteria
Type I - ATP-binding cassette (ABC) transporter
Type II - general pathway (Sec-dependent) - major secretory pathway
Type III - contact-dependent translocation into eukaryotic cells
Type IV - (Sec-like dependent) - translocation of DNA / protein complex
Type V - auto-transporter (Sec-dependent) - includes b-pore forming domain
Gram-negative secretion
Sec-(or Sec-like) dependent
Sec-independent
Type I
Type II
Type III
Type IV
Type V
host
cell
host
cell
OM
P
Sec B11
CM
ATP
ADP
ATP
ADP
N
C
ATP
ATP
ADP
N
C
Sec
ADP
N
C
(Adapted from Stathopoulus et al. (2000); provided by E Rucks)