Bacterial Pathogenesis II (Toxins) - L8 Flashcards
Superantigens
proteins that produce a massive cellular immune response that can lead to fatal toxic shock
What to superantigens lead to?
a continuum of worsening inflammation:
systemic inflammatory response syndrome -> sepsis -> severe sepsis (sepsis+organ dysfunction) -> septic shock
Sepsis
documented, or suspected infection, with other criteria that would suggest an inflammatory response
Why is sepsis important?
very common, expensive, and a high mortality:
- 750K cases: 52% in IC, 17% life support
- 28% mortality overally
- most common cause of death of IC patients (excluding coronary IC patients)
- $14 billion in US
- 25% fatality sever sepsis
- 50% fatality septic shock
Sepsis causative agents
47% gram-pos, 62% gram-neg
gram-pos: staphylococcus aureus, MRSA, s. epidermidis, s. pneumoniae, GAS
gram-neg: pseudomonas sp., e. coli, klebsiella sp., acinetobacter sp, enterobacter
also fungi
sites of infection for sepsis
- respiratory system
- urogenital tract (female > male)
- abdomen
- medical device (catheters, ventilators)
- wound/soft tissue
- CNS
superantigen mechanism
- DIRECT stimulation of T cells
- bypasses antigen processing and presentation
- bind MHC-II and variable portion of beat chain or T cell receptor
- may also engage CD28, a costimulary molecule on T cells
What do SAgs activate?
- far more T cells than normal pathway (20% vs
endotoxin lipid A
may also cause toxic shock, but through a pathway mediated by TLR4
GAS (group A streptococcus) key facts
- wide ranging disease: impetigo, necrotizing fascilititis (flesh-eating bacteria, to toxic shock)
What are important virulence factors for GAS?
Superantigens, including:
- SpeA
- SpeC
- exotoxin Z
- streptococcal superantigen (SSA)
most are encoded within bacteriophage genomes
What is the generalized mechanism of ADP-ribosylating toxins?
- catalytic glutamate (Glu, E) residue of toxin forms hydrogen bond with NAD
- nucleophilic atack of NAD
- transfer of ADP ribose to substrate
What kind of targeting do ADP-ribosylating toxins have?
- some very precise (diptheria toxin)
- some modify numerous (pertussis toxin, ExoS)
What kind of toxin are Cholera and Heat-Labile (LT) toxin?
ADP-ribosylating
What is the structure of cholera/LT toxins?
AB5 toxin pentamer of B bununits surrounding the catalytic A subunit “lunar landers”
Where are the cholera/LT toxins located?
- cholera within prophage genome
- LT on virulence plasmid
How are cholera/LT secreted?
Type II systems, with additional outer membrane vesicles (OMV’s) for the LT
What do cholera/LT toxins target, and what is the result
- target Gs, which regulated the activity of adenylate cyclase
- results in the inhibitio of GTPase activity of Gs, massive overproduction of cAMP, dysregulation of chloride and other ion transporters, diarrheal disease
What are visible signs of cholera?
severe dehydration, decreased skin turgor which produces “washer woman’s hand”
How do CT and LT differ after secretion?
- CT diffuses away from the bacterium after secretion
- LT binds to LPS via a binding site on B subunits that the CT-B subunits lack, then is released from the pathogen as OMVs form
Where does diptheria toxin (DT) originate from?
Corynebacterium diptheriae, a gram-pos faculative anaerobe that causes respiratory and cutaneous diptheria
What is the structure of DT?
an AB toxin that is encoded by a single gene within a prophage;
in order A, T, B
How is DT secreted?
secreted as a single polypeptide via an amino terminal signal peptide (GSP), cleavage of signal peptide (signal peptidase)
What happens to DT after secretion?
DT is cleaved b host furin (a protease) into A and B fragments, but the disulfide bond between cys residues keeps proteolytic fragments covalently bound to each other
What does DT target, and what does that result in?
- targets elongation factor-2 (EF-2)
- results in inhibition of protein synthesis
How toxic is DT?
DT is EXTREMELY POTENT:
- a single molecule is enough to kill a cell
- lethal dose is ~100 ng/kg in humans
- 1/3 most deadly toxins: diptheria, tetanus, botulism
A detailed pathway of DT
- B domain recognizes the EGF-like growth factor (HB-EGF) in the plasma membrane
- receptor-meditated endocytosis
- low pH of the endosome induces T to begin producing pores, reduction of the disulfide bond, and escape of catalytic A fragment into the cytosol
- a fragment catalyzes the transfer of ADP-ribose from NAD to EF-2, which inhibits the function of EF-2 in protein synthesis
- ADP-ribosylation of EF-2 inhibits translation (EF-2 is needed for moving of tRN)
- no translation = cellular death
Do all living cells require iron?
Yes
Where is Fe++ in the human body?
it’s tightly sequestered by iron binding proteins (ferritn, etc), hemoglobin, and within enzymatic reaction centers
What is the relationship between DT and iron?
in the absence of Fe++, the DT gene is expressed, and in its presence DT is repressed by DtxR;
the death of host cells makes Fe++ bioavailable to C. diptheriae
Where does Exotoxin A originate from?
Pseudomonas aeruginosa, a gram-neg aerobe that causes mild skin, ear infections to severe pneumonia (in immune compromised, cystic fibrosis patients)
What is the structure of exotoxin A?
an AB toxin with domains in the reverse order of DT, without proteolytic cleavage;
in order, B, T, A
How is exotoxin A secreted?
as a single polypeptide via an amino terminal signal peptide (GSP)
What happens to exotoxin A after it is secreted?
a signal peptidase cleaves signal peptide in periplasm type II secretion across the outer membrane
What does exotoxin A target, and what does this result in?
- targets EF-2
- this results in the inhibition of protein synthesis, which leads to cell death
What is the overall pathway of exotoxin A?
- receptor meditated endocytosis (LDL receptor related protein 1)
- proteolytic cleavage
- retrograde traffic to endoplasmic reticulum
- translocation of A domain into cytoplasm
What do bacteria producing shiga-like toxins commonly cause?
hemolytic uremic syndrome (HUS)
What is hemolytic uremic syndrome (HUS)?
the most common cause of acute kidney failure in children; causes destruction of red blood cells and kidney failure;
Gb3 is present in greater amounts in renal epithelial tissues
Where do shiga-toxins orignitate?
shigella dysenteriae, and some strains of E. coli (EHEC)
What is the structure of a shiga-toxin?
an AB5 toxin, encoded by two genes on bicistronic mRNA (fat star)
How is shiga-toxin secreted? (detailed)
- amino-terminal signal peptides, cleavage by ignal peptidase and assembly in periplasm
- Stx type 1 is retained in the periplasm and only released upon death of bacterium
- Stx2 may be released like Stx1, but could also have an alternative pathway across OM
What does Stx target, and what does that result in?
- A subunit is an N-glycosidase that enters the cytosol and cleaves off a single adenine residue from the 28S rRNA of the 60S ribosomal subunit;
this ultimately inhibits protein synthesis - the B pentamer mediates holotoxin binding to the well-characterized glycolipid receptor Gb3
- the overall result is inhibition of translation, which leads to cell death
What is the detailed mechanism of Stx toxicity?
- A1:B5 toxin binds to Gb3 receptor
- binding mediated by subunits
- entire toxin-Gb3 complex is endocytosed
- retrograde transport to gogli then ER
- A1 fragment released into cytoplasm
- loop b/w A1 and A2 cleaved by trypsin and/or furin
- A1 fragment has N-glycosidase activity that depurinates a critical residue in the 28S rRNA of 60S ribosomes
Which major toxin is a protease?
Tetanus toxin
Where does tetanus toxin originate?
Clostridium tetani, a gram-pos, obligate anaerobe that causes tetanus: painful tightening, spams, rigidity of the muscles, lockjaw with a 10% fatality rate
What is the structure of tetanus toxin?
a single gene, plasmid encoded that gets cleaved into two fragments linked by a disulfide bond
What does tetanus toxin target?
it’s a neurotoxin, so the CNS
What is the tetanus toxin pathway (detailed)?
- toxin binds peripheral nerve terminals
- gets internalized
- retrograde transport within axon to reach CNS
- delivered into intersynaptic space b/w motor neuron and inhibitory interneuron
- attaches to gangliosides at the presynaptic inhibitory motor nerve endings
- taken into axon by endocytosis
- proteolytic cleavage of synaptobrevin/VAMP prevents neuroexocytosis
- release of inhibitory neurotransmitters across synaptic cleft
Which major toxin is a proteolytic toxin?
botulinum toxin
Where does BoTox originate?
clostridium botulinum, a gram-pos obligate anaerobe; which causes botulism (aka flaccid paralysis)
What is the structure of botox?
a single gene, may be located on the chromosome, a plasmid, or a bacteriophage cleaved into two fragments linked by a disulfide bond
How is botox secreted?
- binds to presynaptic stimulatory terminals
- proteolytic cleavage of VMAP/synaptobrevin
- release of stimulating neurotransmitter (acetycholine) across synaptic cleft
What does botox target, and what is the end result?
- it is a neurotoxin that targets peripheral neuromuscular synapses; permanently damages nerve endings
- also causes food poisoning; gets absorbed by the GI and passes into the blood stream to reach synapses
What is heat-stable toxin (ST)?
a hormone (guanylin and urogunalyin) mimic
How is ST secreted?
through a TolC system
What is the largest class of bacterial toxins?
pore forming toxins (~30% of known toxins)
What are pore forming toxins?
toxins that are secreted as soluble proteins, but still have the capability to form transmembrane channels (pores)
What is a common pore forming toxin?
Staphylococcal a-toxin (a-hemolysin)
where does staphylococcal a-toxin (a-hemolysin) originate?
from staphylicoccus aureus, a gram-pos facultative anaerobe, that causes impetigo, pneumonia, food poisoning, bacteremia, and toxic shock
What is the structure of staphylococcal a-toxin (a-hemolysin)?
a single gene encoded on chromosome, expressed as a single polypeptide with N-term signal peptide for secretion via GSP
