Bacterial Pathogens and Diseases I (Exotoxins)

Question 1

what is a pathogen?

Answer 1

a microorganism capable of causing disease

Question 2

what is meant by pathogenicity?

Answer 2

the ability of an infectious agent to cause disease

Question 3

what is meant by virulence?

Answer 3

the quantitative ability of an agent to cause disease

Question 4

what is meant by toxigenicity?

Answer 4

the ability of a microorganism to produce a toxin that contributes to the development of disease

Question 5

what are virulence factors?

Answer 5

Factors that bacteria have genetically acquired and encoded in their genome. They are necessary for the bacteria to undergo the process that leads to disease

examples:

adherence factors (to get into tissues)
biofilms
invasion of host cells and tissues
toxins – endotoxins and exotoxins

Question 6

what are exotoxins?

Answer 6

Heterogeneous group of proteins produced and secreted by living bacterial cells into the host tissues, causing disease symptoms

Produced by both gram negative and gram positive bacteria

Question 7

What selective advantages do exotoxins give to the bacteria?

Answer 7

help cause disease

Evade immune response
Enable biofilm formation 
Enable attachment to host cells. 
Escape from phagosomes 
Allowing carriage without disease

Question 8

example of toxins

Answer 8

Haemolytic toxins and Phenol soluble modulins

Question 9

where is s. aureus in our body?

Answer 9

commensal in our nasal cavity – it isn’t doing any damage or releasing toxins

Question 10

how does s. aureus colonise?

Answer 10

in a bio film

PSMs give it gliding properties – useful for when it lives in the nose. Also contains toxins that prevent that phagolyosome fusion and therefore prevent bacteria death. PSM allows bacteria to escape from the early phagosome.

Haemolytic toxins to damage other organisms and prevent them from growing – bacteria are in competition, need to kill off other bacteria so they can grow.

Question 11

Haemolytic toxins

Answer 11

cause cells to lyse by forming pores

Important cause of features of S. aureus disease.

Question 12

Phenol soluble modulins

Answer 12

interfere with and aggregate the lipid bilayer of host cells, leading to lysis

Question 13

bacterial chromosome?

Answer 13

single circular piece of double stranded DNA

Question 14

where are toxin genes encoded?

Answer 14

most toxin genes are encoded on the chromosome, but not all of them - many toxins coded by extrachromosomal genes (plasmids)

Question 15

how do bacteria exchange genetic information?

Answer 15

conjugation, transduction and transfection

plasmids play a role

Question 16

what are plasmids?

Answer 16

plasmids are independently replicating small pieces of circular DNA that exist in the bacteria separate from the chromosome, can be passed onto other bacteria via conjugation, transduction and transfection – this is partly how bacteria exchange genetic information

Question 17

explain transfection

Answer 17

toxins can be transferred from 1 bacteria to another via bacteriophages

a bacteriophage is a virus that infects a bacteria, and as a result it picks up some of the genes in the bacteria, and transfers it to the next bacteria it infects

Question 18

Classification of Exotoxins

Answer 18

1. Membrane Acting Toxins – Type I
2. Membrane Damaging Toxins – Type II
3. Intracellular Toxins – Type III

Question 19

Membrane Acting toxins – Type I

Answer 19

act - on the outside of cells or on the membrane

interfere - host cell signaling by inappropriate activation of host cell receptor

target receptors:

• Guanylyl cyclase, increase intracellular cGMP
• Adenyl cyclase, increase intracellular cAMP
• Rho and Ras proteins (enzymes involved in generating GTP)

Question 20

an example of a type 1 membrane acting toxin

Answer 20

ecoli, produces an STa toxin

once STa engages with the receptor, it causes an increase in cGMP

Question 21

Membrane Damaging toxins – Type II

Answer 21

cause damage to the host cell membrane

• insert channels into host cell membrane (beta sheet or alpha helix toxins)
• enzymatical damage

Receptor mediated or Receptor Independent

Question 22

explain the difference between receptor mediated and receptor independent (type II membrane damaging toxins)

Answer 22

receptor mediated is where a toxin binds to the toxic specific receptor, and defined (hexametric or octametric) pores forms (alpha toxins)

receptor independent is where the toxin attaches itself to the membrane and causes it to disintegrate, and there are formation of short lived pores (many alpha type PSM’s)

Question 23

Intracellular toxins – type III

Answer 23

these toxins go inside cells
2 components: A and B

A part- activity, toxigenic and enzymatic
B part- receptor binding and translocation function

May be single or multiple B units e.g. Cholera toxin AB5. 5 binding subunits: Active domain sitting on the top.

Once the toxin is inside the cell, it releases the A part which has biological activity, usually involves some sort of enzymatic degradation.

The protein is held together by electrostatic attractions, meaning its usually susceptible to heat. On the other hand if it’s a single polypeptide chain, heat will not affect or inactivate it.

Another type of type III toxin squirts effector proteins to disrupt the cell

Question 24

the enzymatic component A of Type III toxins has a wide variety of activities - give some examples:

Answer 24

ADP – ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.

Glucosyltransferases – e.g. TcdA from Clostridium difficile, modifies ribosomal RNA so protein synthesis doesn’t happen efficiently

Deamidase – e.g. dermonecrotic toxin of Bordetella pertussis.

Protease – e.g. Clostridial neurotoxins: botulism & tetanus, damage to presynaptic vesicles on neuromuscular junction or in the brain

Adenylcyclase - e.g. EF toxin of Bacillus anthracis

Question 25

Toxins can have an effect via dysregulation of immunity- what does this mean?

Answer 25

• they can induce inflammatory cytokine release
• they can act as super antigens, covalently cross links MHC class II to the t-cell receptor. Activates huge numbers of T-cell populations, which will all produce inflammatory cytokines. The super-antigens causes clonal activation of T-cells, leading to sepsis and septic shock.

Question 26

what are toxoids?

Answer 26

Toxins, which are inactivated using formaldehyde or glutaraldehyde
they are inactive proteins but still highly immunogenic – form the basis for vaccines

examples: Tetanus Vaccine, Diphtheria or Pertussi
(also, we can use antibodies against toxins as part of the treatment, so tetanus is treated by cooled human Ig that contains antibodies against the toxin)

Question 27

microbiology of Clostridium difficile

Answer 27

gram-positive bacillus.
anaerobic.
toxin-producing.
can be carried asymptomatically in the gut.
3 toxins.
makes spores that can survive in the environment for months, difficult to kill

Question 28

is clostridium difficile common?

Answer 28

yes it is a common hospital acquired infection worldwide

Question 29

how does Clostridium difficile act and spread?

Answer 29

disruption of normal gut flora meaning c diff can grow and start to produce toxins that damage the epithelial surface of the gut

spread by ingestion of spores – remain dormant in environment

Question 30

risk factors of c. diff?

Answer 30

age

prolonged hospital stay

antacids – for people with acid reflux problems. If there is a spore, usually the stomach acid can kill it off, but if you’re on antacids the spore can get into your cut and cause acute watery diarrhoea

antibiotics - provide a competitive advantage to spore forming anaerobes over non spore forming anaerobes, allowing c diff colonisation and growth

Question 31

pathogenesis of c. diff?

Answer 31

Receptor binding domain
Hydrophobic domain that allows it to get across the membrane
Glucosyltransferase domain and Protease domain

• toxins get into the cell via receptor mediated endocytosis
• acidification of the endosome
• pore formation, releasing the toxic component
• disruption of cAMP and gCMP and disruption to ros and ras proteins.

Question 32

what effects does c. diff have on the body?

Answer 32

watery diarrhoea
dysentery
pseudomembranous colitis (necrotic damage to the mucous membrane inside the colon, look via a colonoscopy)
toxic megacolon
raised white cell count in blood
detection of toxins in stool (Toxin ELISA)

Question 33

C. diff treatment

Answer 33

depends on severity and presence of surgical complications

removal of offending antibiotic

surgery – partial, total colectomy

Recurrent – faecal transplant

Question 34

what is verocytotoxcin?

Answer 34

some e.coli that has acquired a gene that encodes verocytotoxcin – a very damaging toxin, causes haemorrhagic acute watery diarrhoea

it is also called STEC

Question 35

transmission of e coli?

Answer 35

consuming contaminated food and water

person to person, particularly in child day-care facilities

animal to person (petting zoos, dairy farms, camp grounds)

very low infectious dose

Question 36

what type of toxin is verocytotoxcin/shiga toxin?

Answer 36

Stx toxin is a type II exotoxin with an AB5 structure

A is the enzymatic component, which is N-Glycosidase. This damages ribosomal RNA, inhibiting protein synthesis and causing cell damage. The A is bound to 5 B subunits

The gene is carried on lysogenic bacteria.

Question 37

mechanism of action of the Stx (shiga) toxin?

Answer 37

Binds to Gb3 receptor on host cell membrane, and internalised by endocytosis

transported through the golgi apparatus into the ER where the ribosomes are sitting

Active subunit cleaved off, it will N-glycosylate the 28s ribosomal RNA in eukaryotic ribosomes and prevents protein synthesis

Question 38

Pathogenesis of STEC?

Answer 38

b-binding domain of the toxins allows it to bind to endothelial cells as well as epithelial cells in the mucosa

Stx binds to glomerular endothelial cells of kidney, cardiovascular and CNS - v high levels of Gb3 in kidney so kidneys most affected

inflammatory response, causes thrombotic clotting of the bacteria, blocking the glomeruli – gives you haemolytic urine syndrome

Question 39

STEC Disease

Shiga toxin-producing Escherichia coli, or Verotoxigenic E. coli/VTEC

Answer 39

severe and life threatening

children < 5 years are at the greatest risk

abdominal cramps, watery or bloody diarrhoea

haemolytic uraemic syndrome (anaemia, renal failure and thrombocytopaenia)

neurological symptoms are less common (lethargy, severe headache, convulsions)

Question 40

STEC Diagnosis

Answer 40

Clinical signs and symptoms
Haematological and biochemical evidence.
Stool culture – Growth on SMac
PCR for Stx genes

Question 41

STEC Treatment

Answer 41

Supportive including renal dialysis and blood product transfusion
Antibiotics have little to no role