micro 3

Question 1

how many different mutation has been found of the CFTR protein?

Answer 1

over 1800

Question 2

what is the most common CFTR mutation?

Answer 2

Phe508del most common (>80% of cases)

Question 3

what is the mucocillary elevator?

Answer 3

• Cilia in the lung beat which then moves mucus up to the throat and swallowed
• Cl-secreted into ASL via CFTR

Question 4

what happens in the mucocillary elevator in someone with CF?

Answer 4

In Phe508del CF, CFTR protein absent
•Cl-cant be pumped out
•ASL dehydrates, mucus attracted to cell surface and cilia unable to beat properly
•Mucus becomes stuck

Question 5

what are the comvectional CF pathogens?

Answer 5

P. aeruginosa
S. aureus
H. influenzae
B. cepaciacomplex

Question 6

how does the prevalence of microorganisms differ?

Answer 6

varies by country-•MRSA in adults: UK ~3%; USA >30%

Question 7

what did Muhlebachet al 2018 find in his study?

Answer 7

• Took BAL samples and looked at different types of microbes (particularly bacteria in lung) using 16s rRNA sequencing
• Initial microbial load is similar to that found in oral cavity
• Transitions to pathogen dominated by age 4

Question 8

how do bacteria change in infections in CF patients over time?

Answer 8

• Initially with H. influenza and S. aureus

* Progresses to intermittent and chronic P. aeruginosa

Question 9

what are the two main types of Staphylococcus aureus that are in CF infections in the lung?

Answer 9

• MRSA

* Small colony variants (SCV)

Question 10

how big are small colony variants?

Answer 10

<1mm, nonpigmented, non haemolytic

Question 11

what happens if the growth rate decreases in small colony variants?

Answer 11

increased resistance

Question 12

what are the clinical implications of scv?

Answer 12

Children with SA SCV had lower mean % of predicted FEV1(85.5% Vs 92.4%)
•SCVs which were thymidine dependent →strongest association with lung function

Question 13

what is Pseudomonas aeruginosa?

Answer 13

Gram negative facultatively anaerobic bacillus •Environmentally ubiquitous, generally an opportunistic pathogen
•Found in UTIs, sepsis, non-CF lung infections, GI etc

Question 14

what is the main priority on WHO list of antibiotics to make?

Answer 14

Pseudomonas aeruginosa
Intrinsically resistant to multiple antimicrobials;
•Multiple efflux systems
•Multiple secreted and cell based virulence factors

Question 15

what is Pseudomonas aeruginosa’s role in CF?

Answer 15

Generally thought of as an opportunistic pathogen;•Several groups of strains have become more specialised pathogens and are associated with CF infection

Question 16

how can New infections with PA can be eradicated?

Answer 16

• Combination of oral/IV antibiotics AND inhaled antibiotic
• Non consensus on what is the best combination
• Can be successful in approx. 80% of cases

Question 17

what is Chronic colonisation with PA associated with?

Answer 17

• Worse lung function outcome

* Higher inflammation

Question 18

how does Pseudomonas aeruginosa adapt in the CF lung?

Answer 18

• Has to overcome challenges in the CF lung•Protection from immune system•Less oxygen in mucus layer
• Switches gene expression•Begins to adapt•Numerous mechanisms

Question 19

how are the PA adaptations in the lung regulated?

Answer 19

Many of the changes are regulated by quorum sensing system

Question 20

what is Quorum sensing?

Answer 20

Ability to detect and respond to cell population density by gene regulation
•Expression or repression of specific genes which produce phenotypes which will be beneficial

Question 21

what are the 3 characteristics bacteria must have to be able to us QS?

Answer 21

• Ability to produce a signalling molecule (an autoinducer)
• Be able to detect the change in [signalling molecules]
• Regulate gene expression as a result

Question 22

how can PA use QS?

Answer 22

• Biofilm formation

* EPS production

Question 23

what is type 3 secretion system?

Answer 23

• T3SS is needle like machinery that lets bacterial effectors translocate directly into the cell
• Causes tissue injury and cytotoxicity
• Can also activate innate immune response via IL-1β
• Chronic PA infections select against T3SS expressing PA

Question 24

what are the 4 T3SS dependent effectors PA has?

Answer 24

• ExoS, ExoT, ExoY, and ExoU(reviewed in Hauser, 2009)
• ExoS, T and U disrupt host cell cytoskeleton
• ExoUcan cleave phospholipases (leads to cell death)

Question 25

what happens to PA’s LPS?

Answer 25

O antigen lost during early CF infections

•Lipid A structure in CF clinical isolates changes

Question 26

what effect can LPS modifications have?

Answer 26

LPS modifications may make PA less visible to immune system

Question 27

what are free floating bacterica called?

Answer 27

platonic

Question 28

what happens in chronic cf - biofilm?

Answer 28

• In chronic CF bacteria attach to a surface and become embedded in an acellular matrix
• Surface is damaged epithelia of airway
• Matrix varies by bacteria (also includes mucus)
• PA has Alginate, PEL, PSL, extracellular DNA etc

Question 29

what are biofilms?

Answer 29

• Biofilms are intricate structured bacterial communities
• Generally polymicrobial
• Different micro-environments present within them

Question 30

what are the 5 steps in biofilm formation?

Answer 30

1. Initial attachment
2. Irreversible attachment•Needs various factors (flagella, type IV pili)
3. Microcolonyformation•Starts to produce EPS
4. Maturation•Formation of complex 3D structures•Creates oxygen gradient (more anaerobic nearer centre of the biofilm mass
5. Dispersion

Question 31

what can PA do in chronic infections?

Answer 31

n chronic infections PA can switch from non-mucoid to mucoid phenotype
•Generally due to large amounts of alginateproduced

Question 32

what is alginate? what are its properties?

Answer 32

•Linear polysaccharide
•Initially produced as homopolymer of D-mannuronic acid residues
Information from Franklin et al 2011, Colvin et al 2011, Jennings et al 2015•Modified by different enzymes (AlgI, AlgJ, AlgF)
•Epimerized by AlgG(converts D-mannuronic acid to L-guluronic acid
•Best characterised of the PA EPS
•Negatively charged

Question 33

what is PSL and what does it do?

Answer 33

•Neutral pentasacchariderepeat •contains glucose, mannose, and rhamnose

Question 34

what is pel?

Answer 34

• Suggested to be cationic amino sugars

* 1→4 linked partially acetylated galactosamine and glucosamine sugars

Question 35

what is PA invasion of the hosts response?

Answer 35

• PAiscapable of evading the immune response;
• Complement evasion important during early stages of infection
• Produces enzymes (alkaline protease and elastase) degrade C3b
• Neutrophils produce reactive oxygen species and epithelial cells produce hypothiocyanite •P. aeruginosa produces pyocyanin
• PA biofilms can both stimulate and supress the immune system
• Expression of flagellin and T3SS down regulated•↓ inflammation and cytotoxicity
• PA in biofilms can trigger neutrophil cell death•↑ inflammation and tissue damage

Question 36

what is Burkholderiacepaciacomplex (BCC) ?

Answer 36

•Comprised of at least 18 different species of Burkholderia•Most common are; B. cenocepacia(most virulent), B. multivorans, B. dolosa•Associated with;•More severe lung disease•Transmissible between CF patients•Unpredictable clinical outcomes

Question 37

what is Non tuberculosis mycobacteria?

Answer 37

Over 150 NTM species•Majority (95%) of NTM isolates in CF patients are from 2 “complexes”•Mycobacterium avium•Most common•Includes 4 M. avium species and M. intracellulare•Mycobacterium abscessuscomplex•Impact on health outcome is variable