Bacterial Pathogens and Diseases 1

Question 1

What is a pathogen ?

Answer 1

A micro-organism capable of causing disease

Question 2

What is pathogenicity ?

Answer 2

The ability of an infectious agent to cause disease

Question 3

What is Virulence ?

Answer 3

The quantitive ability of an agent to cause disease. To what extent does it cause disease

Question 4

Whta is Toxigenicity ?

Answer 4

The ability of a micro-organism to produce a toxin that contributes to the development of disease

Question 5

What are the virulence mechanisms ?

Answer 5

The mechanisms through which a pathogen can cause disease :
Adherence factors (molecules and proteins that allow the attachment of bacteria to find a niche and start colonisation )

Biofilms
(Molecules that allow bacteria to form complex structures for macromolecules)

Invasion of Host cells and tissues
To evade phagocytosis , colonise tissues )

Toxins-Endotoxins and exotoxins

Question 6

What are exotoxins ?

Answer 6

These are a diverse heterogeneous group of proteins produces and secreted by living bacterial cells (not byproducts or waste )

Produced by gram negative and positive bacteria

Cause disease symptoms in host during disease

Act via a variety of diverse mechanisms

Question 7

What are gram negative bacteria ?

Answer 7

Gram negative bacteria have a thin peptidoglycan layer and have no outer lipid membrane
They do not retain the violet stain

Question 8

What are gram positive bacteria ?

Answer 8

Gram positive bacteria have a thick peptidoglycan layer and no outer lipid membrane

These stain violet

Question 9

What selective advantages do exotoxins give bacteria ?

Answer 9

They help transmission of disease 
They evade immune response 
They enable biofilm formation 
They enable attachment to host cells 
They escape from phagosomes 

They allow for colonisation ,niche establishment and carriage -Evolutionary advantage

Question 10

What is a example of bacteria found on mucosal surfaces ?

Answer 10

Staphylococcus aureus

Question 11

What exotoxins does Staphylococcus auerus contain ?

Answer 11

Haemolytic toxins

• Cause cells to lyse by forming pores
• Important cause of features of S.aerus disease
• alpha , beta and gamma toxins , Panton Valentine Leukocidin (PVL) , LukAB, LukED, LukMF

Phenol soluble modulins PSM
-Aggregate the lipid bilayer of host cells -lysis

(Majority of S.aureus in humans is asymptomatic carriage in the nose )

Question 12

What are some functions of the toxins found in S.aureus ?

Answer 12

A. When the S.auerus is phagocytosed , the alpha toxins can block the fusion of lysosomes to phagolysosome so the organism can stay.
Modulins can aid the escape of the S.auereus from the phagolysosome

B. Some molecules e.g. phenol soluble modulins can be harmful to other micro-organisms ( competition).This allows it to find a niche within the nasal cavity. It also allows S.aeurus which is a cocci (no flagella) to slide through surfaces to colonise the nasal mucosa.

C.

Question 13

What are Cocci bacteria ?

Answer 13

A coccus (plural cocci) is any bacterium or archaeon that has a spherical, ovoid, or generally round shape.

Question 14

Outline the genetics of exotoxins

Answer 14

Can be encoded by chromosomal genes Shiga toxin in Shigella dysenteriae, TcdA and TcdB in C.difficile

Many toxins coded by extrachromosomal genes
(these can be transferred /exchanged )
-Plasmids -Bacillus anthracis toxin , tetanus toxin

-Lysogenic bacteriophage
(a virus which infects and replicated within bacteria /archaea)
e.g. streptococcal pyrogenic exotoxins in Scarlet Fever , Diphtheria toxins

Question 15

Describe the classification of Exotoxins

Answer 15

1.Membrane Acting toxins -Type 1
(get in contact with cell surface receptors and interfere with processes)

2.Membrane Damaging Toxins -Type 2
(Physically damage )

3.Intracellular Toxins - Type 3
(Interfere with cellular processes)

Issues with this classification =Many toxins have more than one type activity
As mechanisms better understood this classification tend to break down

Question 16

Outline the main characteristics of the membrane acting toxins

Answer 16

Act :
Act from without the cell
(outside the cell)

Interfere:
Interfere with the host cell signalling by inappropriate activation of host cell receptors

Target :
Target receptors include:
Guanylyl cyclase -increases intracellular cGMP

Adenylyl cyclase - Increases intracellular cAMP

Rho proteins

Ras proteins

Question 17

Give an example of a type 1 exotoxin

Answer 17

E.coli Stable heat toxin

It will affect the downstream signalling .

The toxin affects the cystic fibrosis transmembrane receptor causing changes in important ion transporters within tissues.

E.g. Chlorine , bicarbonate ,sodium transporters are affected

Effect is an increase in sodium chloride ions and this can cause diarrhoea.

Question 18

Outline the Type 2 exotoxins and how they work

Answer 18

These cause damage to host cell membrane
They can interact with the receptor a channel
polymerising a channel causing disruption of ions.
(Action potentials )

1.Insert channels to host cell membrane
-Beta sheet toxins S.aureus –Alpha toxin , gamma toxin , PVL
alpha helix toxins -dipheria toxin
-Can produce pores in membrane by polymerising.

2.Enzymatical damage e.g. S.aureus beta -haemolysin, PSM

Not receptor function but receptor itself
Receptor mediated

Receptor independent

Question 19

How do Membrane damaging toxins function ?

Answer 19

Receptor Mediated interaction

Polymerise a pore , forms a hole in the membrane .
Doesn’t affect function of receptor.

Receptor independent may attach to membrane and alter the phospholipid bilayer structure .

Question 20

Outline the type 3- Intracellular toxins function

Answer 20

These are active within the cell which means they need to gain access.

Usually two components -AB toxins

Receptor binding and translocation function -B
(can bind to host cell receptor) -Internalised through receptor mediated endocytosis

Toxigenic (enzymatic )- A

May be single or multiple B units e.g. Cholera toxin AB(5)- has electrostatic interactions and this makes it heat sensitive.

AB are covalently linked and heat resistance

Question 21

What are some example of the enyzmatic A componenet of intracellular toxins ?

Answer 21

Wide variety of activities
These can modify enzymatic activity

ADP-ribosyl transferases -Exotoxin A of Pseudomonas aeruginosa
pertussis toxin

Glucosyltransferases e.g. TcdA and TcdB of Closetridium difficile.
can affect ribosomal Rna and inhibit protein synthesis

Deamidase - dermo necrotic toxin of Bordetella pertussis

Protease -Clostridial neurotoxins :botulism and tetanus
These can destroy proteins
and affect presynaptic structure

Adenylcyclase -e.g. EF toxin of Bacillus anthracis
This can affect production of cAMP which is a key messenger for many processes

Question 22

Give some further examples of intracellular toxins -type 3 other

Answer 22

Type 3 secretion and toxin injections
e.g. YopE in Yersinia species

This bacteria has managed to inject its toxins into host cells.
Produce multi-protein complexes which anchor to the membrane. Inner membrane and outer membrane + protein complex which forms a needle and injects the toxins.

Type IV secretion and toxin injection

e. g. CagA in Helicobacter pylori
- This is a different secretion process

Question 23

Outline super antigens and inflammatory cytokines

Answer 23

Exotoxins are able to induce inflammatory cytokine response.

Antigen presenting cell

Cytokines which are released:
IL1, IL1 beta , TNF, IL6 , interferon gamma , IL18

Due to hyperactivation of T cells which is not antigen specific

Mechanisms :
Super antigen : non specific bridging of the MHC class 2 and T-cell receptor leading to cytokine production e.g. Staphylococcal exfoliative toxin A , Toxic shock syndrome Toxin 1 (TSST1)

Via activation of the different inflammasome leading to release of IL1 beta and IL18 e.g. S.auereus toxin A, PVL

Activate cell upon detection of pathogen patterns, death by paraptosis

Question 24

What are toxoids?

Answer 24

Toxins can be inactivated by using formaldehyde/glutaraldehyde which creates toxoids

These are inactive proteins but still highly immunogenic - form the basis of vaccines.

E.g.
Tetanus Vaccine
Diphtheria
Pertussis (Acellular)

Treatment of toxin mediated disease can be affected by administering preformed antibodies to the toxin

Diphtheria antitoxin-horse antibodies
Tetanus-Pooled human immunoglobulin (specific /normal)
Botulism-horse antibodies

Experimental and research-Monoclonal antibodies

Question 25

Give an example of a toxin mediated disease

Answer 25

Toxins in Clostridium dificile disease: This is a gram positive bacillus (rod shaped0 Anaerobic Spore forming -highly resistant Toxin-producing Can be carried asymptomatically in the gut -if controlled by microbiota 3 toxins

Question 26

What is the epidemiology of C.dificile ?

Answer 26

Common hospital acquired infection worldwide Spread through ingestion of spores-dormant in environment Coloniser of the human gut up to 5% in adults Risk factors-Antibiotic use, age ,antacids and prolonged hospital stay

Question 27

How can C.difice be caused by antibiotics?

Answer 27

They act to destroy the microbial ecosystem within the gut Antibiotics provide a competitive advantage to spore forming anaerobes over non spore forming anaerobes. Allows C.dificile colonisation +growth All antibiotics have potential for causing disease. Examples: ``` Some antibiotics are worse: 2nd and 3rd generation cephalosporins Quinolones Clindamycin Aminoglycosides Trimethoprim Vancomycin ```

Question 28

Outline the pathogenesis of C.dificle (Toxin-mediated disease)

Answer 28

Mediated by the toxin it contains: Cytotoxin A- TcdA coded by the tcdA gene Cytotoxin B-TcdB coded by tcdb gene Binary toxin-C.diff transferase (CDT)-minor role in disease Tcd A and Tcd B -Type 3 AB toxins The A component of toxins are glycosylating enzymes There is a receptor binding domain which binding domain which interacts with the cell for internalisation. Hydrophobic domain which allows the escape of the active A component into the intracellular space. The GTD and CPD domain (A part of the toxin which mediate disease)

Question 29

What are the components of the TcdA and TcdB ?

Answer 29

GTD_glucosyltransferase domain CPD-Cycteine protease domain DD-delivery hydrophobic domain RBD-receptor binding domain There is a receptor binding domain which binding domain which interacts with the cell for internalisation. Hydrophobic domain which allows the escape of the active A component into the intracellular space. The GTD and CPD domain (A part of the toxin which mediate disease)

Question 30

How does the toxin interact with the membrane and get internalised ?

Answer 30

1. The toxin will bind to specific host cell receptors 2. Receptor mediated endocytosis =internalised and cause damage 3. Endosome will acidify which leads to creation of a pore-mediated by hydrophobic domain 4. This allows release of active component A of toxin 5. GTD will interact with Rho GTPases 6. Deactivation effects within the host cell Consequences: Cytopathic effects changing structure of cell cytoskeleton breakdown, epithelial cell permeability increase ,epithelial to epithelial cell contact loss Cytotoxic Activation of production of reactive oxygen species which are very toxic and damaging -can induce apoptosis

Question 31

Outline cytopathic and cytotoxic

Answer 31

Patchy necrosis with neutrophil infiltration Epithelial ulcers Pseudomembranes,leuokocyes fibrin mucous cell debris May be asymptotic Watery diarrhoea dysentery Pseudomembranous colitis

Question 32

How can C.dificiel be diagnosed ?c

Answer 32

Clinical signs and symptoms Raised white cell count in the blood Detection of organisms and toxins in stool 2 phase test : Glutamamte dehydrogenase -detects if C.dificile organism present 2.Toxin enzyme linked immunosorbent assay (ELISA) for TcdA and TcdB toxins) Detection of tcdA/tcdB gene -PCR Colonoscopy-pseudomembranous colitis

Question 33

How can c.dificiel be treated ?

Answer 33

Treatment is dependent on severity and presence of surgical complications Ideally removal of offending antibiotic -not always possible Antibiotics : fidaxomicin or metronidazole or vancomycin. Surgery-Partial , total colectomy recurrent-Faecal transplant (recover microbiota from a patient )

Question 34

What is the Veroocytotoxin Escheria coli (VTEC) disease ?

Answer 34

VTEC or Shiga-toxin (Stx) producing E.coli (STEC) can cause disease mild to life threatening disease Stx carried by some E.coli -most commonly 0157:H7 - Identified usually by growth on sorbitol MacConkey agar (SMac) -does not feremtn sorbitol and hence is clear - other less common types n

Question 35

What is the epidemiology of VTEC disease

Answer 35

E.coli 0157:H7 naturally colonises the gastrointestinal tracts of cattle who are generally asymptomatic Transmission : Predominantly via consumption of contaminated food/water Person to person Animal to person Low infectious dose

Question 36

What is the pathogenesis of VTEC diseases?

Answer 36

Toxin -Shiga like toxin (SLT)= shigatoxin (STx) = Verocytotroxin Stx,Stx1,Stx1a,1c,1d Stx2a,2c,2d -variations in a.a sequence gene carried on lysogenic bacteria Type 3 exotoxin -AB 5 Enzymatic component A= N-Glycosidase Bound to 5 B subunits

Question 37

What is the Mechanism of VTEC disease?

Answer 37

Bind to receptor globotriaosylceramide Gb3 or glovbotetraosylceramide (Gb4) on host cell membrane Bound toxin internalised by receptor mediated endocytosis Carried by retrograde trafficking via the Golgi apparatus to the endoplasmic reticulum The A subunit is cleaved off by membrane bound proteases Once in the cytoplasm A1 and A2 dissociate A1 binds to 28S RNA subunit -blocks protein synthesis This can also cause a haemolytic uermic syndrome ( condition that affects blood /blood vessels )

Question 38

Outline STEC pathogenesis

Answer 38

STEC closely adheres to the epithelial cells of the gut mucosa. Stx is transported from the intestine to the kidney and other tissues is possibly through polymorphonuclear neutrophils (PMNs). Bind to glomerular endothelial cells of kidney,cardiovascular and central nervous system. very high levels of Gb3 in kidey so kidneys most affected. Thought that Stx favours inflammation resulting in microvascular thrombosis and inhibition of fibrinolysis.

Question 39

What can STEC disease lead to ?Symptoms

Answer 39

``` Abdominal cramps Watery diarrhoea Haemolytic uraemic syndrome Anaemia Renal failure Thrombocytopenia Lethargy Severe headache Convulsions Encephalopathy ```

Question 40

How can STEC be diagnosed and treated ?

Answer 40

Clinical signs and symptoms Haemotological and biochemical evidence Stool culture -Grow on SMAc PCR for Stx genes Treatment : Supportive including renal dialysis and blood product transfusion Antibiotics have little to no role