Bacterial Pathogens and Diseases I Flashcards
What is a pathogen?
A microorganism capable of causing disease
Pathogenecity
the ability of an infectious agent/microorganism to cause disease
Virulence
quantitative ability of an agent to cause disease
-degree of pathogenecity
Toxigenicity
the ability of a microorganism to produce a toxin that contributes to the development of the disease
What are virulence mechanisms?
Mechanisms that allow microorganisms to attach/proliferate:
- Adherence factors
- Biofilms
- Invasion of host cells and tissues
- Toxins (endotoxins & exotoxins)
What are exotoxins?
Exotoxins are a heterogenous group of proteins produced and secreted by living bacterial cells
What kind of bacteria are exotoxins produced by?
BOTH gram-negative and gram-positive
What is the effect of exotoxins?
They act via a variety of diverse mechanisms to cause tissue damage and result in disease symptoms in the host
Why do bacteria produce exotoxins?
Gives them an evolutionary advantage by allowing for colonisation, niche establishment and carriage:
· Exotoxins may help transmission of disease, however in severe disease host may be a literal and evolutionary dead end. However, with many toxins the disease-causing activity may not be the primary function
· Evade immune response
· Enable biofilm formation
· Enable attachment to host cells
· Escape from phagosomes
What kind of bacteria is Staphylococcus?
Gram positive
Exotoxins produced by Staphylococcus aureus
Haemolytic Toxins
- Cause cells to lyse by forming pores → causing tissue damage
- ⍺,β,ẟ toxins, Panton Valentine Leukocidin (PVL), LukAB, LukED, LukMF
Phenol Soluble Modulins (PSMs)
- cause damage to cells by interfering with the membrane structure- cause the lipid membrane of cells to become disaggregated→lysis
S.aureus in humans
Majority of S.aureus in humans is asymptomatic carriage in the nose existing as a commensal organism.
In what form does S.aureus colonise the nose?
it colonises in a biofilm
What is a biofilm?
communities of bacteria that exist as colonies
How is S.aureus able to survive as a commensal organism in humans?
Secretes exotoxins useful for colonisation:
- e.g. haemolytic toxins to stop other organisms from growing (competition to establish itself in an environmental niche)
- e.g. PSMs and S.aureus ⍺ which inhibit phagolysosome fusion, enabling bacteria to escape phagosome into the cytoplasm for intracellular niche establishment and replication
- e.g. PSMs target cohabiting bacterial species, competing for resources and exclusion of non-kin isolates
How is a S.aureus biofilm formed?
1) ⍺-toxin establishes cell-cell contacts, enabling the formation of secondary biofilm structures
2) Extracellular matrices develop, surrounding the cells within the biofilm. In the presence of extracellular DNA (eDNA), β-toxin covalently cross-links with itself, adding to this extracellular nucleoprotein biofilm matrix and contributing to the formation of the complex biofilm secondary structuring
3) PSMs detach from the mature biofilm, aiding in the release of cell clusters from the main body of the biofilm which allows for dispersal to new sites of infection
What are exotoxins encoded by?
Some by chromosomal genes:
- Shiga toxin in Shigella dysenteriae
- TcdA & TcdB in C.difficile
Many by extrachromosomal genes:
· Plasmids- bacillus anthracis toxin, tetanus toxin
· Lysogenic bacteriophage- e.g. streptococcal pyrogenic exotoxins in Scarlet Fever, Diphtheria toxin
Classification of exotoxins
Exotoxins are classified by their activity:
Membrane Acting Toxins (Type I)
Membrane Damaging Toxins (Type II)
Intracellular Toxins (Type III)
Why is classifying exotoxins by their activity problematic?
many toxins may have more than one type of activity
as mechanisms are better understood this classification tends to break down
Membrane Acting Toxins- Type I
Toxins which act from the outside of the cell or on the membrane and interfere with intracellular host cell signalling by inappropriate activation of host cell receptors
-generates changes in the activity of the pores on the membrane, which can lead to disease
Target receptors of membrane acting toxins (Type I)
Guanyly cyclase →↑cGMP activity
Adenylyl cyclase →↑cAMP activity
Rho proteins
Ras proteins
MOA of Type I-E.coli Stable Heat Toxin
1) Exotoxin binds guanylyl cyclase C membrane receptor and causes an increase in cGMP production
2) cGMP then promotes further downstream signalling. It acts via protein kinases and a CFTR pump. This CFTR channel pumps out chloride and bicarbonate ions out of the cell.
3) The cGMP, via another mechanism, produces cAMP which then inhibits the pumps which pump out hydrogen and bring in sodium.
4) Net effect is you get chloride ions, bicarbonate ions and sodium chloride ions being pumped out of the cell. Where salt goes (sodium chloride), water follows, hence why you get diarrhoea from this infection.
Membrane Damaging Toxins- Type II
cause damage to the host cell membrane
How do membrane damaging toxins cause damage to the host cell membrane?
Receptor Mediated:
-Insert channels/pores into host cell membrane, disrupting homeostatic mechanisms (E.g. ion flux across membrane)
·β sheet toxins e.g. S.aureus ⍺-toxin, 𝛾-toxin, PVL
·⍺ helix toxins- e.g. diphtheria toxin
Receptor Independent:
- Causes enzymatic damage
- e.g. S.aureus β-haemolysin, PSMs
Intracellular Toxins- Type III
Toxins which are active within the cell (must gain access to the cell)
Structure of intracellular toxins (Type III)
Most of these toxins are proteins known as AB toxins because they usually have 2 components:
- receptor binding and translocation function (B)
- toxigenic/enzymatic (A)
*may be single or multiple B units e.g. Cholera toxin AB5
How do intracellular AB toxins (type III) work?
1) B component of the toxin binds to the receptor and toxin is internalised inside the cell.
2) Once the toxin is internalised, it is cleaved by proteolytic degradation, releasing the active component of the toxin (A).
3) Active enzymatic component has a wide variety of activities, acting at multiple sites within the cytoplasm and causing damage
what are some activities of enzymatic component A
ADP – ribosyl transferases - e.g. Exotoxin A of Pseudomonas aeruginosa, pertussis toxin.
Glucosyltransferases – e.g. TcdA and TcdB of Clostridium difficile
Deamidase – e.g. dermonecrotic toxin of Bordetella pertussis.
Protease – e.g. Clostridial neurotoxins: botulism & tetanus
Adenylcyclase - e.g. EF (Edema factor) toxin of Bacillus anthracis
Type III toxins other than AB toxins
Type III secretion and toxin injection
-bacteria adheres to surface and injects effector proteins inside cells through a needle structure which penetrates the membrane of the host cell (e.g. YopE in Yersinia species)
Type IV secretion and toxin injection
-pumps in larger proteins (e.g. CagA in Helicobacter pylori)
Effect of exotoxins on immunity
Exotoxins are able to induce inflammatory cytokine release, which causes damage to tissues.
- IL1, IL1β, TNF, IL6, ẟ interferon, IL18
How do exotoxins induce inflammatory cytokine release?
Superantigen (bacterial toxin)
- cause non-specific bridging of the MHC Class II and T-cell receptor without any antigen presentation
- clonal activation of T-cells, producing many different cytokines which drive inflammation and tissue damage
- e.g. Staphylococcal Exfoliative Toxin A, Toxic Shock Syndrome Toxin 1 (TSST1)
Inflammasome
-exotoxin activates different inflammasomes leading to release of IL1β and IL18 e.g. S.aureas toxin A, PVL
Toxoid
inactivated toxin
How can toxins be inactivated to produce toxoids?
Toxins can be inactivated using formaldehyde or glutaraldehyde
What are toxoids used for?
Toxoids are inactive proteins but still highly immunogenic- form the basis for vaccines:
· Tetanus vaccine
· Diphtheria vaccine
· Pertussis (acellular) vaccine
Treatment of Toxin-Mediated Disease
Administering antibodies to the toxin:
- Diphtheria antitoxin- horse antibodies
- Tetanus antitoxin- pooled human immunoglobulin. Specific or normal
- Botulism antitoxin- horse antibodies
Monoclonal antibodies are also used in experiments and research in the treatment of toxin mediated disease
Bacteria which can cause toxin mediated disease include…
Clostridium difficile
Verocytotoxin/Shiga-toxin (Stx) producing Escherichia Coli (VTEC)(STEC)
Microbiology of C.difficile
· Gram-positive bacillus (rod) · Anaerobic · Spore-forming · Toxin-producing · Can be carried asymptomatically in the gut · Produces 3 toxins
Epidemiology of C.difficile
· Common hospital acquired infection worldwide
· Spread by ingestion of spores (remain dormant in the environment) or spread from patient to patient
· Coloniser of the human gut up to 5% in adults
· Risk factors- antibiotic use, age (>60 years), antacids & prolonged hospital stay
How do antibiotics cause C.difficile infection?
Antibiotics disrupt the microbial system within the gut, providing a competitive advantage to spore forming anaerobes over non-spore forming anaerobes
-allows C.difficile colonisation and growth
Which antibiotics are likely to cause C.difficile infection?
Some antibiotics are worse than others
- 2nd and 3rd generation cephalosporins
- quinolones
- clindamycin
Others less likely to cause C.difficile infection:
- Aminoglycosides
- Trimethoprim
- Vancomycin
What are the toxins produced by C.difficile?
Cytotoxin A
-TcdA coded by tcdA gene
Cytotoxin B
-TcdB coded by tcdB gene
Binary Toxin
-C.difficile transferase (minor role in disease)
What kind of toxins are TcdA and TcdB?
Type III AB toxins
-A component are gycosylating enzymes
MOA of TcdA and TcdB toxins
1) Toxins binding to specific host cell receptors and get into the cell through receptor mediated endocytosis
2) Toxins are internalised and packaged within an endosome
3) Endosome is acidified
4) Pore formation in the endosome
5) Pore allows for GTD release from the endosome to the host cell cytoplasm
6) There is Rho GTPase inactivation by glucosylation
7) This results in downstream effects within the host cell: Cytopathic effects >cytoskeleton breakdown >loss of cell-cell contacts >increased epithelial permeability
Cytotoxic effects
>activation of the inflammasome (more cytokine release causing damage)
>increase in ROS levels
>induction of programmed cell death
What is the result of cytopathic & cytotoxic effect of C.difficile toxins?
· Patchy necrosis with neutrophil infiltration
· Epithelial ulcers
· Pseudomembranes over the ulcers- combination of leucocytes, fibrin, mucous, cell debris
How can C.difficile disease present?
The C-difficile disease can range from:
- Asymptomatic
- water diarrhoea
- dysentery (diarrhoea with blood and straining)
- pseudomembranous colitis
- toxic megacolon and peritonitis
Diagnosis of C.difficile
Clinical signs and symptoms
Raised white cell count in blood
Detection of organisms and toxins in stool via 2 phase test:
1) Glutamate dehydrogenase- detects if C.difficile organism is present
2) Toxin enzyme linked immunosorbent assay (ELISA) for TcdA and TcdB toxins
Detection of tcdA and tcdB genes via PCR
Colonoscopy- pseudomembranous colitis
Treatment of C.difficile
Treatment is dependent on severity and presence of surgical complications.
· Ideally, removal of offending antibiotic- not always possible
· Antibiotics fidaxomicin, metronidazole or vancomycin work against C.difficile
· Surgery- partial or total colectomy
· Recurrent C.difficile - treat with faecal transplant to re-establish the normal gut flora
Shiga toxin-producing E. coli (STEC)
Stx is carried by some E.coli- most commonly O157:H7 (particular type of bacteria)
Epidemiology of E.coli O157:H7
E.coli O157:H7 naturally colonises the gastrointestinal tracts of cattle who are generally asymptomatic
· Transmission of E.coli:
- predominantly via consumption of contaminated food and water
- person to person, particularly in child day-care facilities
- animal to person e.g. in petting zoos, dairy farms or camp grounds
· Very low infectious dose needed to cause infection (meaning it is quite infectious)
Toxin produced by E.coli O157:H7
- Toxin: Shiga like toxin (SLT)= shigatoxin (Stx)= verocytotoxin (VTEC)
- Stx, Stx1, Stx1a, 1c, 1d, Stx2a, 2c, 2d- variations in amino acid sequence
- Gene carried on lysogenic phage
Structure of Shiga toxin
- Type III exotoxin- AB5
- Enzymatic component A= N-Glycosidase
- Bound to 5 B subunits
MOA of Shiga toxin (Stx)
· Toxin binds to receptor Gb3 or 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 disassociated
· A1 binds to 28S RNA subunit→blocks protein synthesis
Pathogenesis of STEC
- STEC closely adheres to the epithelial cells of the gut mucosa
- Stx is transported from the intestine to the kidney and other tissues via polymorphonuclear neutrophils (PMNs); but this is debated
- Bind to glomerular endothelial cells of kidney, cardiovascular and central nervous system
- Very high levels of Gb3 in kidney so kidneys most affected
- Thought that Stx favours inflammation resulting in microvascular thrombosis and inhibition of fibrinolysis (damaging the kidney)
Presentation of STEC Disease
· Abdominal cramps, watery or bloody diarrhoea- may not be present
· Haemolytic uraemic syndrome presentation
>anaemia
>renal failure
>thrombocytopaenia
· Less common are neurological symptoms >lethargy >severe headache >convulsions >encephalopathy
Severity of STEC Disease
· Can be severe and life threatening
Often in children <5 years are at greatest risk
Diagnosis of STEC Disease
- Clinical signs and symptoms
- Haematological and biochemical evidence
- Stool culture- growth on SMac
- PCR for Stx genes
How can we identify STEC on a culture?
Stx is identified usually by growth on sorbital MacConkey agar (SMac)
O157:H7 does not ferment sorbitol and hence is red
Treatment of STEC Disease
- Supportive including renal dialysis and blood product transfusion
- Antibiotics have little to no role
