C4- Fungi-immunity And Genetics

Question 1

What do most prr use as adaptors for nfkb txn

Answer 1

Myd88

Question 2

What is the importance of prr in fungi (opportunists depend on defects in these responses)

Answer 2

Th1 diff, th17, treg(if tlr2)

Also induce respiratory burst

Question 3

What does tlr4 recognise and in heterodimer with what

Answer 3

Glucoronoxylomannan gxm
particularly hyphae responsive
Also outer cell mannans from cell wall (o linked vs MR n linked)

In the tlr4-md2 complex

Question 4

What does this signalling help differentiate important to stop cryptococcal meningitis

Answer 4

Th1 through ifny and il12 (via nfkb)

Question 5

What recognises n linked mannans

Answer 5

MR

Question 6

What are the 2 major co receptors of tlr which work together

Answer 6

Decrin 1 ctl for b1,3 glucan and mr

Question 7

What’s the diff between tlr ,7 and 9

Answer 7

Tlr7 recognises ssrna of candida

Tlr9 recognises all dna e

Eg during phagocytosis

Question 8

Phagosome maturation and acidification stopped by ca and cf means what

Answer 8

Immune evasion through tlr7 and 9

Question 9

Why is tlr2 useful

Answer 9

Recognition of phosholipomannans

It helps to dampen responses through treg differentiation

Does this through mapk signalling (rather than nkfb) tgfb / il10 induction

Question 10

Which th is required for fungal clearance eg during allergic aspergillosis

Answer 10

Th17 through il23 induction by prr

Question 11

Which damps proteins released during inflammation or tissue damage and what sensed by to promote clearance

Answer 11

S100b protein

Tlr2

Question 12

What is the evidence that over stimulation of tlr9 or any is bad

Answer 12

Snp in tlr9 sensing dna can increase nfkb binding affinity and is linked to ABPA

Question 13

Snp in what inflammasome associated with recurrent vaginal candidiasis

Answer 13

Nlrp3

Question 14

What changes happen during Ibd

Answer 14

Increased candida and sach in gut

colonisation in gut and asca antifungal ab

Question 15

What sort of diet alterations have impact

Answer 15

and plant based increase candida vs penecilum in meat diet

High carb diets cause overgrowth

Question 16

What complex is recruited by syk downstream of decrin 1 through signalling

Answer 16

Card9 adaptor protein, bcl10, malt1

Question 17

What tf is induced by this and what differentiation primarily does this cause

Answer 17

Nfkb

Il23/6 for th17 differentiation
For NETs, phagocytosis and b defensin via il22

Question 18

What does syk induce directly for clearance

Answer 18

Respiratory burst and phagocystosis

Question 19

What does resp burst link to

Answer 19

Inflammasome nlrp3 activation and binding to Asc and caspases 1 for Il1b

Question 20

Dysbiosis linked to antibiotics causes what which can trigger Ibd aswell as genetics

Answer 20

Candida outgrowth due to loss of commensal control

Question 21

Give 2 genetic Ibd examples of poor fungal control

Answer 21

Decrin 1 snp linked to severe form of UC
Card9 linked to uc and crohns

Question 22

Which immune response impaired by the card9 response

Answer 22

Th17 also phagocytosis in decrin snp

Question 23

Fungal virulence determinants

Question 24

How can comparative genomics be used to determine virulence traits

Answer 24

Identify species specific genes in the genomes of the 3 fungal opportunists

Question 25

How many genes with no known function or orthologue in sacharomyces found in c albicans which could be for virulence

Answer 25

1000

Question 26

How many af specific genes with pathogenic function are there not in benign yeast like neosartorya fischeri

Answer 26

Around 700

Question 27

Do they have shared genes for virulence like the spi in diff bacteria

Answer 27

Non are identified yet

Question 28

What is it suggested they do instead to cause virulence

Answer 28

Adapt conserved parhways eg carbohydrate metabolism pathways

Question 29

What 2 other approaches can be done to find virulence traits

Answer 29

In vivo indetificatikn of genes Or study of mutant libraries from insertational mutagenesis

Question 30

How did thewes 2007 examine transcriptomic profiles of a highly invasive candida strain vs less invasive

Answer 30

Inject mice models and study liver invasion ana take rna samples at diff time points Because quite small tissue you can’t get replication in tranxriptomic data so also used ex vivo pig liver Or remove liver from pig and topically add both candida Also looked at transcriptimic profiles at diff time points

Question 31

What did they find comparing virulent strain with non virulent

Question 32

How could you identify genes expressed at diff time points of the sc5314 strain

Answer 32

Take rna samples at diff time points and sequence them

Question 33

How many genes found expressed during time points of infection in ex and in vivo both models and how many were sc5314 specific

Answer 33

63 5 specific

Question 34

Give example of one gene and how they tested this further. Also give example of a protein downregulated in these mutant types

Answer 34

Dfg16 plasma membrane protein required for filamentation in response to change in ph alkaline First confirmed it’s expression on the plasma membrane To analyse the function deleted Dfg16 and found mutants were not able to form hyphae in alkaline conditions anymore Looked at effect of Dfg16 deletion and found that iron permease ftr1 was downregulated. This would usually be important in alkaline conditions as these conditions cause iron starvation Suggests as well as filamentation process, Dfg16 induced exp of these survivial proteins

Question 35

Does transcriptional profiling prove it is involved in virulence

Answer 35

No only looks at expression

Question 36

Why is insertational mutagenesis easier with glabrata than albicans

Answer 36

Haploid so only one copy of interest gene vs diploid need to ko 2

Question 37

What sort of marker gene from yeast doen for IM

Answer 37

Ura3

Question 38

A library of how many homozygous mutants of c albicans used for key study in 2010

Answer 38

674

Question 39

What 3 things can they be screened for

Answer 39

Morphogenesis , infectivty/growth defects eg iv in mouse kidney, proliferation screening (growth defects)

Question 40

How did they measure infectivity in kidney of mice

Answer 40

Inoculated mice with 48 mutants at a time Then removed kidneys later and extracted dna for qpc Determine mutant relative abundance using qpcr after dna extraction from mouse kidney If lower abundance than wt = defective durinf infection and gene would be important

Question 41

How many genes found for impaired infectivity vs morphogenesis vs proliferation and what were the conclusions from this

Answer 41

115 impaired infectivity vs 133 for morphogenesis 68 for proliferation Many of the genes were non overlapping First study which concluded that impaired growth or morphogenesis does not always impair infectivity

Question 42

Give example of the imapired infectivity- specific genes (not overlapping with morphogenesis or prolifedation6

Answer 42

Sphingolipid biosynthesis impairement

Question 43

How was the identification of the sphingolipid parhway mutant important for infectivity been proven relevant for new antimycotics (like hsp90, and gpi deletions/defects in drug lecture)

Answer 43

Screened through HtS many molecules to find 2 potent inhibitors which work in animal models against Cn and ca = new Antimycotics currently in clinical trials

Question 44

Experimental models to study virulence

Question 45

How are cn and af administered to disseminated mice models vs ca

Answer 45

Inteanasally imahlation Vs iv injection into tail vein

Question 46

How can you study virulence form the mouse

Answer 46

Study survival rates mutant vs wt eg dox ume6 and tissue burden (cfu/g) eg kidney effects eg Hgc1 analysis

Question 47

What is galleria mellonella (a mini host alternative to mouse)

Answer 47

Wax moth larvae

Question 48

How can you inject and study survival

Answer 48

Larva taken from galleria and injected through a proleg to monitor survival

Question 49

How can c elegans be given fungi to monitor survival

Answer 49

Can be gfp-linked fungi and fed to nematodes

Question 50

Which 2 genes using celegans found important for killing of c elegans (mutant screening)

Answer 50

Ada2 and cas5 for hyphae formation

Question 51

Which other model can be used which is first vertebrate with both innate and adaptive unlike the other minibost

Answer 51

Zebra fish

Question 52

How are they infected

Answer 52

Injected into larval hindbrain ventricle

Question 53

Which tf for filamentation found important in mice was replicated in zebra fish

Answer 53

Cph1 and efg1

Question 54

What is the best incvitro model to study mucosal surface interactions

Answer 54

Reconstituted human epithelium Cell lines cultured on polycarbonate filters

Question 55

How can you quantify adhesion through RHE

Answer 55

Count cfu when cultured to see how much fungi adhesion

Question 56

How can you quantify invasive ness/ penetrance using rhe

Answer 56

Can assay number of fungi cells that penetrate the rhe matrix (filamentation process)

Question 57

Can you test mutant libraries using rhe for adhesion/penetration

Answer 57

Yes

Question 58

Which pm protein found in the 5 unique genes for sc5314 strain was found to be important (thewes 2007) also in the study for penetration

Answer 58

Dfg16

Question 59

How else was Dfg16 studied for virulence after RHE studies for more evidence of virulence

Answer 59

Disseminated mouse model where mutant showed increased survival time of mouse

Question 60

Through study into ca genetic mutants of mannans (no O linked or n lined) and also prr ko mice what was found

Answer 60

Found tlr4 for o linked, MR for n linked and then decrin 1 are major for CA response

Question 61

What alteration causes overgrowth of candida in ugt

Answer 61

Oestrogen production during pregnancy Hiv immunodeficiency Lactobacillus loss

Question 62

What was the first model which you could study candida/macrophage interactions because they were transparent so could use tags

Answer 62

Zebra fish