principles of infectious disease - bacterial toxins

non protein toxins

• Endotoxin; embedded in the cell surface: Lipopolysaccharide (LPS)

• Other bacterial cell wall components; e.g. Lipoteichoic acid (LTA) & phosphatidylglycerol (PG)

• Mycolactone; polyketide-derived macrolide

    produced & secreted by some mycobacteria

• Both cytotoxic and immunosuppressive properties

protein toxins (exotoxins)

excreted from the cell

• type one (superantigens)

• type II (phospholipases, pore forming toxins)

• type III (A-B) toxins

features of endotoxins

formed from lipopolysaccharides that are part of the outer membrane

they cannot be denatured by boiling

relatively low potency and dont have enzymatic activity

pyrogenicity = fever causing

can cause septic shock - an inflammatory response throughout the body

features of exotoxins

are proteins

extracellular and diffusible

usually denatured by boiling

are antigenic and when denatured form a toxoid with antigenic properties but no longer toxic

relatively high potency and usually act as enzymes

only occasionally pyrogenicity

inflammatory response

4 major events of the inflamatory response

1. Vasodilation

2. Activation of endothelial cells

3. Increased vascular permeability

4. Chemotactic factors

Brought about by a combination of complement activation, cytokine release and monocyte and PMN transmigration/activation

roles of cytokines and complement in the inflammatory response

TNF alpha il-1 and IL-8 - all act on the brain causing - fever, anorexia, chills and wasting

also activate and attract phagocytes and mast cells

complement C3a C4a and C5a all activate and attract phagocytes and mast cells

IL-6 - stiumulates liver to release acute phase proteins = complement proteins (opsonising) and transferrin = drop in Fe in serum

septic shock

Occurs in 4 stages

1. Systemic inflammatory response syndrome (SIRS)

   • Temperature > 38^oC or <36^oC (usually ↑)

   • Higher than normal respiratory rate

   • Usually high neutrophil count (sometimes low)

   • Mild hypotension

2. Sepsis

   • SIRS with proof bacteria in bloodstream

3. Severe sepsis

   • Multiple organ dysfunction

   • Dramatic hypotension

4. Septic shock

   • Hypotension despite fluid administration

   • disseminated intravascular coagulation

   • acute respiratory distress

   • multiple organ failure & death 50% survival + survivors can have amputation and organ damage

bacterial endotoxin structure

there is a relationship between their structure, function and biological activity

Lipid A part responsible for septic shock

Triggering of cytokine release by gram-ve LPS and the role in septic shock

source of septic shock symptoms

bacterial exotoxin names

Cytotoxins: Attack a variety of cell types

Toxins that attack specific cell types include; neurotoxin, leukotoxin, hepatotoxin, cardiotoxin

Cholera toxin: produced by Vibrio cholerae

Shiga toxin: produced by Shigella species (dysentery)

Diphtheria toxin: produced by Corynebacterium diphtheriae

Tetanus toxin: produced by Clostridium tetani

Adenylate cyclase: toxin produced by Bordatella pertussis cause of whooping cough

Phospholipase (lecithenase): toxin produced by Clostridium perfringens cause of gangrene

Enterotoxin: protein toxins that cause diarrhoea or vomiting

toxic shock - identical to septic shock, but you don’t need bacteria in the bloodstream due to the extracellular nature of exotoxins

type I toxin

 (superantigens)

• Bind to the host cell surface, but are not translocated into the host cell

• Antigen-independent activation of T cells

  • Inappropriate activation of IL-2 → TOXIC SHOCK

Exotoxins that act on helper T cells

• Causes a massive release of nonspecific cytokines

• Induce a systemic inflammatory response

type I toxin examples

Toxic shock syndrome toxin-1 (TSST-1)

• Superantigen produced by some strains of Staphylococcus aureus.

• causes toxic shock syndrome (TSS)  - caused by a certain tampon brand

Staphyloccal enterotoxins (SEs)

• Superantigens produced by some strains of Staphylococcus aureus

• Cause food poisoning

Streptococcal pyrogenic exotoxin (Spe)

• produced by group A streptococci (eg Streptococcus pyogenes).

• causes toxic shock-like syndrome (TSLS)

type II toxins

(pore forming, phospholipases)

• Destroy the integrity of the mammalian cell cytoplasmic membrane

type II toxin examples

pore forming toxins

• Staphylococcal a toxin

  • Produced by Staphylococcus aureus

  • Pore forming cytolysin

• Listeriolysin O (LLO)

  • Produced by Listeria monocytogenes

  • Pore forming membrane damaging toxin, role in mediating escape from phagosome

• Pneumolysin (PLY)

  • Produced by Streptococcus pneumoniae

  • Pore forming cytolysin

    • Induction of inflammation in lung

    • Inhibits cilial beat in respiratory mucosa

phospholipases

• Clostridium perfringens a Toxin

• Very common in the environment

  • Causes gas gangrene

  • Dermonecrotic & haemolytic

  • Is a zinc-metallophospholipase C

 

type III toxins

 (A-B toxins)

• Have a B portion comprising a translocation (T) domain and a receptor binding (R) domain

• Have an enzymatic portion (A) that acts on some intracellular host protein

Binding portion (B) and enzymatic portion (A) connected by disulphide bond

• B portion confers specificity

• A portion elicits effect on target host protein

• Simple or compound

Number of outcomes dependent on target protein

what bacteria use type III AB toxins

Diphtheria

Botulism

Tetinus

AB toxin structure

portions are held together by a disulfide bond the toxin only becomes active when the bond breaks

toxins of tetanus, botulism and diptheria are all simple AB toxins

A is the enzymatic part - targets protein in host cell

mechanism of AB toxin entry to cells

Common mechanism of entry for the toxins

B section mediates entry to cell

Once toxin is in the endosome the PH drop causes a change in the toxin and A portion travels into the cell and A portion is now active

there is a huge diversity of AB toxins due to receptors they bind to = very specific

most common action of AB toxins

A portion remove the ADP-ribosyl group from nicotinamide adenine dinucleotide (NAD and causes ADP-ribosylation of target host protein

resulting in inhibition of protein translation

diphtheria

Caused by Corynebacterium diphtheriae

Gram +ve, non-spore forming, facultative anaerobic rod

Humans are only host (normally childhood)

Transmission usually by inhalation of aerosols

Vaccine is a toxoided (chemically inactivated) version of diphtheria toxin

First symptoms (2-4 days post colonisation): malaise, low grade fever, tonsilitis, sore throat, loss of appetite

typically form a grey white membrane on tonsils and soft palete

complications

If complications: irregular heart beat, difficulty swallowing, stupor, coma & death

Bacteria produsces local toxin but it can go systemic

Toxin encoded onto bacteriophage in the genome 

Only strains infected by the bacteriophage produce the toxin

Toxing gene only made in low iron conditions (similar conditions to mucus in host)

High iron represses transcription allwoing it only to be expressed in the host

structure of diptheria toxin

function of diptheria toxin

Toxin can get cleaved by protease on outside of the cell or cleaved by furine protease in endosome

 

Gets cleaved and A portion stays held to B portion by disulphide bond

B recognises receptor on host cell (HB-EGF receptor)

 

Now translocation domain exposed and due to hydrophobic nature intergrates into cell wall

Puts the A portion in the cell which breaks the disulphide bond

 

Toxin in cytoplasm

Stops protein synthesis by modifying EF-2

Is an enzyme so modifies EF-2 until it is all used up and leads to cell death

therapeutic uses for diptheria toxins

Therapeutic use of diptheria toxin - targeted cancer therapy

Diphtheria toxin derived immunotoxins

Use targeting molecules (receptor specific ligands) conjugated to modified toxin - targets toxin to cancer cells

Cancer cells frequently have specific growth factor receptors and antigens overexpressed on surface which allows selective targeting of immunotoxins

Targeting molecules are typically antibodies, antibody fragments, cytokines or growth factors that recognise a specific cell surface receptor that is either absent on the surface of normal cells or highly up-regulated on cancer cells

strategies based on controlled tumour specific expression of diptheria toxin

Strategies based on the controlled tumour-specific expression of diphtheria toxin

targeted killing of cancer cells by delivering botulinum A chain to cells and limiting its expression to within cancer cells through transcriptional regulation

If you can replace the receptor domain with the targetting molecule to lead the toxin to cancer cells

Issue is most of us are immune to the toxins and react to them

This is changes by introducing the enzyme portion of the toxin (A portion) via genetic engineering and get it to only be expressed in the cancer cells

botulism

Most cases caused by consumption of the toxin not colonisation by the bacteria

Intoxication caused by Clostridium botulinum

relatively large, anaerobic, Gram +ve, rods

forms sub-terminal endospores, strictly fermentative metabolism

Spores often contaminate food

Disease occurs when spores able to germinate and bacteria grow in food

Most cases associated with consumption of unheated canned food

Food heated but spores not destroyed and the bacteria replicates in anearobic environment

Disease symptoms proportional to amount ingested

botulism toxin

Botulinum toxins: potent neurotoxins - picograms can be deadly

Initial symptoms (4-36 hours after ingestion): nausea & vomiting, headache, double vision, slurred speech & other neurological symptoms

Generalised flaccid paralysis occurs due to blocking neurotransmitter release preventing muscle stimulation

Death if sufficient to interfere with breathing & heart function

Named after the latin for sausage as report comes from a fair in germany where people got ill from the sausage

Bacteria survives in farm animals intestine and spores get into fertiliser where food grows

three types of botulism

Other types of botulism are very rare

Most cases botulism outcompeted by microbiota but not in babies

Hence why you don’t feed honey to babies under one

 

Wound botulism only occurs in anearobic wounds (seen in war time)

structure of botulism toxin

Multiprotein complex called progenitor toxin other proteins protect the toxin from low PH in stomach

If derivative toxin ingested its much less active as its not protected

If injected the derivative toxin is more deadly

Then its cleaved by protease and end up with A (light chain)  and B chain (heavy chain)

how the toxin gets in cells

Action of botulinum toxins

Uses 2 receptors to get in

Ganglioside to make lose connection the strong connection by a protein receptor

botulism toxin actions

Different serotypes cleaves different snare proteins

Block acetyl choline vesicle form fusing with cell membrane - no neurotransmission

results in flaccid paralysis

actions of diferent botulism serotypes

Tetanus

Intoxication caused by Clostridium tetani

relatively large, anaerobic, Gram +ve, rods

Forms terminal endospores, strictly fermentative metabolism

Spores found in soil

Disease occurs when spores germinate in wounds

Has to be a tight wound so its anaerobic

Vaccine is toxoided version of tetanus toxin

Tetanus toxin: potent neurotoxin

Highly fatal (40-80% mortality)

Early symptoms (4 days to several weeks): painful spasms and rigidity of the voluntary muscles (‘lockjaw’: spasms of the masseter muscle) - lock jaw

Progressive rigidity and violent spasms

Death usually from exhaustion & respiratory failure & occlusion of carotid in neck

action of tetanus toxin

Very similar to botulinam toxin and cleaves snare proteins

Targets inhibitory neurotransmitters

key differences between tetanus toxin and botulism

When botulism toxin goes in there is a PH drop in the endosome and the A chain comes out of it

For tetanus it associated with lipid rafts - no PH drop

Toxin goes through  retrograde trafficking and goes into inhibitory neurons by trancytosis

Now PH drops in endosome and A chain comes out

In inhibitory neuron there is gamma and glycine

Does the same thing as botulism

therapeutic uses of botulism toxins

Native toxin administered into peripheral tissue resulting in reversible blockage of the neuromuscular junction:

Dystonias and other involuntary movements, eg:

Face and neck dystonias

Limb, head, voice, chin tremor

Inappropriate muscle contractions, eg:

Spasticity

Chronic tension (muscle contraction) headaches

Neuropathic pain

Neurogenic bladder

Hyperhidrosis - excessive sweating

Sialorrhea - excess saliva within the oral cavity

May result from stroke, cerebral palsy, head injury, multiple sclerosis etc

Other cosmetic applications include:

wrinkles, brow furrows, frown lines, "crow’s feet", platysma lines, facial asymmetry

Commercial preparations include:

From BoNT/A: Dysport®, Botox®, Xeomin® & CBTXA®

From BoNT/B: Myobloc® (also known as NeuroBloc ®)

cholera

Caused by water/ food contaminated with human feaces

Causative agent: Vibrio cholerae

Gram –ve, curved rod, motile (single polar flagelllum)

capable of respiratory & fermentative metabolism

Transmission to humans is by water or food

Reservoirs: Infected humans esp. asymptomatic carriers (marine invertebrates)

Can survive in water but need to be in human to replicate in intestines

Disease due to secretion of enterotoxin: cholera toxin

Highly fatal (50-60% mortality) and very rapidly fatal death within hours potentially

Symptoms (18h to 5 days): sudden onset of diarrhoea (‘rice water’ stool)

Leads to dehydration, anuria (kidneys stop urine production), acidosis(low PH in blood) & shock

Loss of potassium ions leads to cardiac complications & circulatory failure→death

Prevention: clean water supply, effective sewage treatment

Treatment: Fluid replacement therapy

Vaccine recently became available

identification of cholera

start largely from indian sub continent

Identified by koch (kochs postulates)

Became more virulent with the el tor strain which took over in 7th pandemic

In 8th pandemic it went through mutation in the el tor strain and could infect people who were immune to previous strains

Still kills millions and is still endemic in many areas

Every so often you end up with a pandemic

virulence factors of cholera

Motility

• Flagellum

• Acf (accessory colonisation factor) gene products regulate motility

Neuraminidase

• penetration of mucin layer

Adherence

• Toxin coregulated pili (Tcp pili)

  • clumped at one pole of the cell

  • Tcp genes (except tcpG) are clustered on a pathogenicity island (VPI)

• Non-fimbrial adhesins: Haemagglutinins

• The genes responcible for the TCP pilli clustered on a gene island that has other adhesion but most important adhesion is the TCP pili

Cholera toxin

Other toxins produced by some strains

• Zot (zonula occludens toxin)

• Ace (accessory cholera enterotoxin)

ChxA: not in O1 & V. cholerae O139 strains

Other toxins can cause symptoms in absence of the cholerae toxin but it is still the main toxin

acquisition of virulence

Two key events allowing the bacteria to cause toxin that results in pandemics

Once the pathogenicity island thought to be acquired by a phage but that is now controversal

Pilli acts as a receptor for the phage to intergrate the toxin

pathogenesis of cholera

Pathogenesis of cholera

• V. Cholerae ingested

Virulence genes expressed

(pH shock in stomach, t temp)

• Adheres to & colonises  small-intestinal mucosa (flagella, TCP pili, other adhesins?, neuraminidasg?)

• Produce toxins (cholera toxin, Zot?, Ace?)

• Cholera toxin acts on mucosal cells

• Extensive fluid & ion loss from tissues leading to hypotension, electrolyte imbalance & death

action of cholera toxin

resulrs in changes in the movement of ions and water across intestinal mucosa - cl- out and NA+ blocked from entering

results in massive loss of water from cells

gene structure of cholera toxin

Symptoms caused by cholera toxin

It is a complex AB toxin

Coded by two genes

Co-transcribed in a operon

ctxA and ctxB

Need more ctxB that ctxA

5-1 ctxB ratio to ctxA

More translation of B than a due to stronger ribosomal binding site

assembly of the cholera toxin

interaction of B to A

A1 is the catalytic part, A2 is responsible to tethering A to B

cholera toxin cell entry and action

Toxin is not endocytosed and the breaks out of endosome like normal AB toxins

Gets taken into a apical endosome by ganglioside

Gets taken to endoplasmic reticulum

B subunit still attached at this point

 

Then a isomerase breaks the disulphid bond that releases the A portion releasing the toxin from the endoplamic reticulum

 

Once in the cytosol acts on adenelate cyclase that is responsible for synthesisina cyclic AMP levels

• A subunit nicked for activity; A1 translocated into host cell cytoplasm

  • A1 ADP ribosylates Gs (membrane protein) causing continual activation of adenylate cyclase

These are responsible for cell chloride secretion

Cholera toxin upregulates camp production resulting in more chloride loss and Na+ absorbtion

(via ADP-ribosolation)

whooping cough

Causative agent: Bordetella pertussis

 Very small Gram –ve, aerobic, cocco-bacillus

respiratory metabolism, nutritionally fastidious

Transmission by aerosols or direct contact

Reservoirs: Infected humans esp. asymptomatic carriers

1st stage: resembles common cold

2^nd stage: Dry cough which becomes paroxysmal,

excess mucus production & vomiting

Convulsions & cyanosis may occur

Virulence factors include pertussis toxin (Ptx) and ACase toxin

Prevention: antibiotics (don’t lessen duration of symptoms)

effective vaccine

resurgence of whooping cough

Rare thanks to vaccine but starting to make a come back particularly in Manchester

People stopped taking vaccine and started getting cough

Older people with the vaccine began to get a drop in immunity and caught it of unvaccinated people

whooping cough pertussis toxin

Inactivates GI so it cant switch of active form of the enzyme results in exactly the same effect of cholera toxin

but acts in the airways

results in to much fluid being produced in the airways

toxins summary