Chronic Infection

Question 1

P.aeruginosa can form biofilms where?

Answer 1

On abiotic surfaces or on biotic surfaces

Question 2

What regulatory switches control biofilm production?

Answer 2

Gac/Rsm cascade

c-di-GMP signalling- high c-di-GMP

Question 3

What are the features of chronic infection?

Answer 3

T6SS highly active
Sessile
T3SS not very active
Exopolysaccharide production
Not many virulence factors being produced

Question 4

What is required for biofilm formation?

Answer 4

Flagella, Type IV pili, cup fimbriae and adhesins

Production of exopolysaccharide

Question 5

What is needed for biofilm attachment to surfaces?

Answer 5

Flagella, type IV pili, cup fimbriae, adhesins

Question 6

What exopolysaccharide is produced in the mucoid biofilm phenotype?

Answer 6

Alginate

Question 7

What exopolysaccharides are produced in the non-mucoid biofilm phenotype?

Answer 7

Pel

Psl

Question 8

What dye binds to exopolysaccharides?

Answer 8

Congo red

Question 9

What is the process of biofilm formation?

Answer 9

1) Transport to the biotic/abiotic surface via flagella
2) Attachment to the biotic/abiotic surface via flagella, type IV pili, adhesins, cup fimbriae
3) Motility on the surface and mushroom formation via type IV pili
4) Biofilm maturation via exopolysaccharide production: Production of alginate, psl, pel, eDNA
4) Dispersal: Breaking down the exopolysaccharides and cleavage of adhesins… glycoside hydrolase and alginate lyase

Question 10

What is needed for mushroom formation?

Answer 10

Type IV pili

Question 11

What is needed for breaking down of the exopolysaccharide?

Answer 11

Enzymes
Alginate lysase
Glycoside hydrolase

Question 12

What is needed to break down alginate?

Answer 12

Alginate lyase

Question 13

What is needed to break down pel/psl?

Answer 13

Glycoside hydrolase

Question 14

How do biofilms protect the bacteria?

Answer 14

Makes them much more antibiotic resistant as it is harder for antibiotics to penetrate the biofilm
Makes it harder for the bacteria to become phagocytosed

Question 15

Exopolysaccharide associated with mucoidy?

Answer 15

Alginate

Question 16

What exopolysaccharide is primarily produced by P.aeruginosa in CF patients?

Answer 16

Mucoidy biofilm phenotype- alginate

Question 17

What is the importance of the T6SS with relation to biofilms?

Answer 17

During biofilm formation is is possible that other bacterial species may enter the biofilm and benefit from its protective environment. The T6SS is therefore beneficial in killing these cells which may compete for nutrients etc

Question 18

How does the T6SS work?

Answer 18

There is a contractile sheath which reveals a nanotube- able to penetrate the target bacterium

Question 19

In acute infection how does the input environmental strain compare to the output strain?

Answer 19

Not many differences
There are hardly any genetic differences
There was no time for differences to be accumulated as there is no persistence

Question 20

In chronic infection how does the input environmental strain compare to the output strain?

Answer 20

There are may genetic differences
The bacteria are persisting in chronic infection and have therefore had time to evolve and develop mutations that have been selected for
They acquire pathoadaptive mutations which help them to survive in the environment

Question 21

Chronic infection is very common in?

Answer 21

CF patients

Question 22

What are the typical types of pathoadaptive mutations acquired in chronic infection?

Answer 22

• Loss of motility
• Increase in mucoidy
• Loss of T3SS
• Increase in T6SS
• Loss of virulence effectors
• Loss of QS
• Increased antibiotic resistance

Question 23

What mutation leads to sessility?

Answer 23

Mutations in rpoN

Question 24

What mutation increases antibiotic resistance?

Answer 24

Mutations in muxZ

Question 25

What is MuxZ?

Answer 25

Negative regulator of an efflux pump

Question 26

How do mutations in muxZ increase antibiotic resistance?

Answer 26

MuxZ is a negative regulator of an efflux pump Therefore, mutation in muxZ will increase the capacity of the bacteria to expel any drugs or antibiotics that enter Increased antibiotic resistance

Question 27

What is mucA

Answer 27

Anti-sigma factor

Question 28

Mutations in mucA lead to?

Answer 28

Increased alginate production and mucoidy

Question 29

Mutations in mucA are typically?

Answer 29

Loss of function mutations

Question 30

How does a mutation in mucA increase alginate production?

Answer 30

AlgU leads to alginate production MucA is able to sequester AlgU and reduce the amount of alginate produced Therefore mutations in mucA increase alginate production

Question 31

What is algU?

Answer 31

Alternative sigma factor

Question 32

What do mutations in retS cause?

Answer 32

Hyperbiofilm phenotype | Increased c-di-GMP levels

Question 33

What is RetS?

Answer 33

Negative regulator of Gac/Rsm signalling

Question 34

What do mutations in lasR result in?

Answer 34

Loss of QS system

Question 35

What is LasR?

Answer 35

Transcriptional regulator | The master quorum sensing regulator

Question 36

What is rpoN?

Answer 36

Alternative sigma factor

Question 37

Summary of all the mutations?

Answer 37

``` mucA loss of function retS loss of function rpoN mutations mexZ mutations lasR mutations ```

Question 38

Why do contingency mutations arise?

Answer 38

Mutations often have a cost involved. Contingency mutations allow the phenotype/effect of a prior mutation to be reverted

Question 39

Why might a contingency mutation for increased alginate production occur?

Answer 39

As increased alginate production is a very energy costly process

Question 40

What is the contingency mutation to the loss of function mucA?

Answer 40

Mutations in algU

Question 41

What contingency mutations are there to the retS mutation?

Answer 41

Mutations in gacA, gacS, rsmA

Question 42

How can strains evolve quickly?

Answer 42

Mutations in mutS and mutL

Question 43

What are mutS and mutL involved in?

Answer 43

They are involved in mismatch repair

Question 44

Mutations in mutL and mutS result in?

Answer 44

The incorperation of more mutations as fewer mutations are fixed/resolved