L2 - THE INFLUENCE OF HOST GENOTYPE ON THE SUSCEPTIBILITY TO INFECTION

Question 1

Define the role of genetics in susceptibility to infection

Answer 1

• In many human infections, only a proportion of exposed individuals develop clinical disease, suggesting a role for heritable factors.
• Example, twin studies revealed that host genetics is implicated in infectious disease risk
• Adopted children have a markedly increased risk of death from infectious disease if one of their biological parents had died prematurely from infection
• Mouse studies further support role for host genetics
  Different inbred strains show differences in bacterial loads, cytokine responses and outcomes following bacterial infection

Question 2

Sickle cell carriers & malaria

Answer 2

Sickle cell anaemia caused by mutation in haemoglobin-encoding gene; particularly common in sub-Saharan Africa where the carrier rate can be 10-40%.

PROTECTION AFFORDED BY SICKLE-CELL TRAIT AGAINST SUBTERTIAN MALARIAL INFECTION

Question 3

Individuals vary in their genetic susceptibility to infection: EVOLUTION
Streptococcus pneumoniae

Answer 3

Individual may have susceptibility to invasive disease due to a probable impairment of the TLRIL1R signalling

Question 4

Individuals vary in their genetic susceptibility to infection: EVOLUTION
Encapsulated bacteria

Answer 4

Idividual may have susceptibility to invasive disease due to a probable deficiency in the compliment pathway

Question 5

Individuals vary in their genetic susceptibility to infection: EVOLUTION
Mycobacteria

Answer 5

Idividual may have susceptibility due to an impairment in the IFNy response causing impaired macrophage respiratory burst

Question 6

Individuals vary in their genetic susceptibility to infection: EVOLUTION
HIV-1, norovirus, plasmodium vivax

Answer 6

Individual may have resistance due to the absence of (co)receptors for pathogen

Question 7

What is the Cystic Fibrosis transmembrane conductance Regulator (CFTR)?

Answer 7

A chloride channel on the apical surface of epithelial cells. It regulates the flow of ions, and consequently fluid

Question 8

How many different cftr mutations have been identified?

Answer 8

> 1,600

Question 9

CF is the most common life-threatening inherited disease in the UK. What are the statistics?

Answer 9

Over 10,000 sufferers, and a 1 in 25 carrier rate in the UK

Question 10

What is the most common cftr mutation in the UK?

Answer 10

ΔF508 (~ 75% in UK)

Question 11

What is the leading cause of morbidity & mortality in CF patients?

Answer 11

Chronic lung infection and ultimately respiratory failure

Question 12

What is the median age of survival in CF patients?

Answer 12

Approx. 40 years, although is heavily influenced by patient’s microbiology status

Question 13

Why is the CF lung susceptible to infection?

Answer 13

Due to loss of (or impaired) CFTR function, the composition of the airway surface liquid (ASL) in the CF lung is altered.

Dehydrated ASL impairs mucociliary clearance - Airways are lined by ciliated epithelial cells & mucus
Bacteria get trapped in mucus, and the beating action of cilia expel mucus & bacteria
With dehydrated ASL, this cannot function properly.

Increased salt concentration in ASL inhibits antimicrobial peptides - epithelial cells produce cationic antimicrobial peptides
Small (12-50 amino acids) peptides with potent antimicrobial activity
Positively-charged and hydrophobic
Interact & disrupt bacterial membrane
Typically, their activity is salt-sensitive

Question 14

What is the Pseudomonas paradox in CF?

Answer 14

The profile of respiratory pathogens in CF patients is very different from that observed in non-CF patients
Respiratory infections in non-CF patients are dominated by Streptococcus pneumoniae.
In contrast, by adulthood, approx. 80% of CF patients are chronically-infected with Pseudomonas aeruginosa

Defects in non-specific immune defences like mucociliary clearance and antimicrobial peptides cannot explain this bias for Pseudomonas

Question 15

What % of adult CF sufferers are chronically infected with P.aeruginosa?

Answer 15

80% of CF patients are chronically-infected with Pseudomonas aeruginosa

Question 16

What is Pseudomonas aeruginosa?

Answer 16

• Gram-negative opportunistic pathogen
• Commonly found in soil & water
• Increasing global importance as a cause of healthcare- acquired infections
• Urinary tract infections, respiratory infections, dermatitis, soft tissue infections, bacteraemia, bone & joint infections, GI infections and a variety of systemic infections (burns/AIDS/cancer)
• Primary pathogen in CF

Question 17

During infection of the CF lung, P. aeruginosa typically converts to a what?

Answer 17

A “mucoid phenotype” associated with the overproduction of the polysaccharide, alginate

Question 18

How do Pseudomonas factors contribute to CF infection?

Answer 18

Alginate overproduction, leading to mucoidy

It converts to a “mucoid phenotype” associated with the overproduction of the polysaccharide, alginate. Influences biofilm architecture, antibiotic tolerance and interaction with immune cells.

Pyocyanin production impairs mucociliary clearance
- Pyocyanin is present in CF sputum up to 27 μg/mL

Question 19

How does P. aeruginosa inhibit mucociliary clearance in CF infection?

Answer 19

By reducing the beating frequency of the cilia. Pyocyanin achieves this by depleting cellular ATP levels.

Question 20

What is a receptor for P. aeruginosa?

Answer 20

CFTR appears to act as a receptor for P. aeruginosa. The P. aeruginosa-infected epithelium shows the organisms entering the tracheal epithelial cells, usually with one of the polar ends being taken into a membrane invagination. Expression of CFTR on tracheal epithelial cells is required for the binding and internalisation of P. aeruginosa.

Question 21

How does the binding (to CFTR) of P. aeruginosa occur?

Observations from a transgenic mouse model and the Zebrafish embryo model.

Answer 21

Wildtype and CF mice were challenged with P. aeruginosa. To determine the % internalised, lungs were extracted from infected mice and homogenised to obtain single cell suspensions of lung epithelial cells. This suspension was treated with antibiotic to kill any Pseudomonas that were NOT inside epithelial cells. The only Pseudomonas surviving would need to be INSIDE epithelial cells (and thus protected from the antibiotic). After the antibiotic treatment, epithelial cells are simply lysed and the lysate is plated on agar plates to allow growth of surviving Pseudomonas. Bacterial counts are determined and compared to the original inoculum to determine the & bacteria internalised.
To quantify the bacteria in the lung (right-hand graph), lung tissue is simply homogenised and plated to enable viable counts of bacteria.

Very elegant study examined the response of zebrafish embryos to Pseudomonas aeruginosa, and how the response differed between control zebrafish and those with reduced expression of CFTR (termed, CFTR morphants). Data very clearly showed that the clearance of P. aeruginosa was significantly impaired when CFTR expression was reduced. This is shown clearly in the top images that show fluorescently-labelled P. aeruginosa within zebrafish embryos – there is a much higher bacterial burden in the CFTR morphants. The graph below shows that this is a Pseudomonas aeruginosa-specific phenomenon that is not observed with other bacterial species. The graph plots the ratio of bacterial burden in CFTR morphants compared to controls – a number above 1 indicates increased burden in morphants compared to control. You can see that this is only the case for P. aeruginoa infection. Infections with other bacterial species (Edwardsiella tarda, Escherichia coli, Burkholderia cenocepacia, Staphylococcus aureus and Haemophilus influenzae) results in a comparable bacterial burden in both control and CFTR morphants.

Question 22

What type of receptor does CFTR is act as in P.aeriginosa ?

Answer 22

A pattern recognition receptor.

P. aeruginosa binding to CFTR is NOT being used by the bacteria to establish infection

Rather, CFTR is acting as a pattern recognition receptor specifically for P. aeruginosa, triggering an immune response

In the absence of CFTR, that immune response is not triggered

CFTR acts as an immunomodulator:
See picture in notes

Question 23

What part of P. aeruginosa does the CFTR bind to?

Answer 23

The LPS.
The Pseudomonas-CFTR interaction is between the core region of the Pseudomonas LPS and the first extracellular domain of CFTR (amino acids 108-117)
Mutants of P. aeruginosa with truncated LPS core are not internalised
Presence of exogenous LPS inhibits P. aeruginosa internalisation, as does a synthetic peptide corresponding to CFTR108-117

Question 24

Pseudomonas & CFTR – conclusions thus far

Answer 24

The predisposition of CF patients to P. aeruginosa and the subsequent success of the organisms as a CF pathogen is due to a combination of host and bacterial factors

CFTR genotype influences the epithelial innate immune function within the airways, specifically in relation to the response to P. aeruginosa

CFTR deficiency specifically predisposes to P. aeruginosa infection

Question 25

What’s the benefit of being a cftr heterozygote?

Answer 25

Until relatively recently, CF children died in infancy

Why has the cftr mutation been maintained at such high levels in the population?
Carriers of CF have less functional CFTR on the surface of epithelial cells
The high carrier rate implies a selective advantage of being heterozygous (the “heterozygote advantage”)

Question 26

Investigating selective pressures for cftr mutations Molecular evidence

Answer 26

Molecular & cellular studies should identify a mechanism through which heterozygosity provides resistance

Question 27

Investigating selective pressures for cftr mutations

Clinical evidence

Answer 27

Clinical studies should indicate correlations between host genotype and disease morbidity/mortality

Question 28

Investigating selective pressures for cftr mutations

Historical-geographical evidence

Answer 28

Temporal & spatial distribution of the selective agent must be consistent with:
the observed allele frequency
the incidence of the genetic disease in different geographical regions

CF incidence is approx 1/2,500 in newborns of European descent, 1/15,000 in Middle Eastern descent, and 1/40,000 in Indian descent

Question 29

CFTR and susceptibility to typhoid fever?

Answer 29

Salmonella enterica Serotype Typhi (“Salmonella typhi”) causes typhoid

The type IV pili of S. typhi, composed of the protein PilS, enable the attachment to epithelial cells by binding to CFTR
PilS binds to the 1st extracellular domain of CFTR, the same domain that binds P. aeruginosa
Type iV pilus mediates attachment of Salmonella to epithelial cells, via binding of PilS to CFTR

Molecular evidence:
The PilS-CFTR interaction has been reported to enable translocation of S. typhi across the gut epithelium; translocation is reduced in cftr heterozygotes
Clinical evidence:
Numerous population studies have confirmed a correlation between cftr polymorphisms & protection from enteric fever

Question 30

CFTR and susceptibility to cholera?

Answer 30

Vibrio cholerae causes cholera via the production of cholera toxin (CTX)responsible for the symptoms of cholera – namely, profound diarrhoea. This is largely brought about by CTX causing activation of CFTR. The resulting efflux of chloride ions is associated with a simultaneous efflux of fluid (osmotic gradient) into the lumen of the gut – hence the diarrhoea.
Molecular evidence:
CTX activates host cell adenylate cyclase, leading to excessive cAMP production.
High cAMP levels activate CFTR, causing efflux of ions & water from infected cells, leading to the characteristic watery diarrhoea
cftr heterozygotes show reduced response to CTX (reduced fluid accumulation)

However, some lab-based studies have been contradictory, and clinical studies are lacking

Question 31

CFTR and susceptibility to tuberculosis?

Answer 31

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). Population genetics models support Tb as the selective pressure. Neither typhoid nor cholera provide enough historical selective pressure 
In contrast, the TB epidemic is predicted to have provided adequate selective pressure to produce the modern CF incidence

Molecular evidence:
Sulfated glycosaminoglycans (sGAGs) accumulate in the CF lung due to diminished activity of the enzyme, arylsulfatase B (ARSB)
Ordinarily, ARSB degrades sGAGs, liberating sulfate

cftr mutation → Possible disruption to Cl- homeostasis → Reduced ARSB activity → Reduced hydrolysis of sGAGs, thus reduced liberation of sulfate.

Mtb incorporates sulfate into its cell wall
Sulfated cell wall components contribute to virulence
The host is the main sulfate source, as Mtb lacks its own sulfatase
Impaired ARSB activity in CF patients & carriers therefore deprives Mtb of a necessary nutrient, leading to reduced virulence.

Clinical evidence:
A lower tuberculosis mortality rate has been observed among cftr heterozygotes versus controls
Several studies report low incidence of TB amongst CF patients

Historical & geographical evidence:
TB has been globally endemic for ≥15,000 years
However, a TB epidemic associated with record mortality broke out in Europe in the early 17th century
TB linked to 20-25% of all deaths from 16th to early 20th century
Epidemic did not spread to India & Asia until late 19th century

Question 32

Conclusions

Answer 32

The ability to establish infection is complex, involving a multitude of host and microbial factors

Polymorphisms in host proteins can positively and negatively influence the susceptibility to diverse infectious agents through a variety of mechanisms