C4- Fungi-immunity And Genetics Flashcards
What do most prr use as adaptors for nfkb txn
Myd88
What is the importance of prr in fungi (opportunists depend on defects in these responses)
Th1 diff, th17, treg(if tlr2)
Also induce respiratory burst
What does tlr4 recognise and in heterodimer with what
Glucoronoxylomannan gxm
particularly hyphae responsive
Also outer cell mannans from cell wall (o linked vs MR n linked)
In the tlr4-md2 complex
What does this signalling help differentiate important to stop cryptococcal meningitis
Th1 through ifny and il12 (via nfkb)
What recognises n linked mannans
MR
What are the 2 major co receptors of tlr which work together
Decrin 1 ctl for b1,3 glucan and mr
What’s the diff between tlr ,7 and 9
Tlr7 recognises ssrna of candida
Tlr9 recognises all dna e
Eg during phagocytosis
Phagosome maturation and acidification stopped by ca and cf means what
Immune evasion through tlr7 and 9
Why is tlr2 useful
Recognition of phosholipomannans
It helps to dampen responses through treg differentiation
Does this through mapk signalling (rather than nkfb) tgfb / il10 induction
Which th is required for fungal clearance eg during allergic aspergillosis
Th17 through il23 induction by prr
Which damps proteins released during inflammation or tissue damage and what sensed by to promote clearance
S100b protein
Tlr2
What is the evidence that over stimulation of tlr9 or any is bad
Snp in tlr9 sensing dna can increase nfkb binding affinity and is linked to ABPA
Snp in what inflammasome associated with recurrent vaginal candidiasis
Nlrp3
What changes happen during Ibd
Increased candida and sach in gut
colonisation in gut and asca antifungal ab
What sort of diet alterations have impact
and plant based increase candida vs penecilum in meat diet
High carb diets cause overgrowth
What complex is recruited by syk downstream of decrin 1 through signalling
Card9 adaptor protein, bcl10, malt1
What tf is induced by this and what differentiation primarily does this cause
Nfkb
Il23/6 for th17 differentiation
For NETs, phagocytosis and b defensin via il22
What does syk induce directly for clearance
Respiratory burst and phagocystosis
What does resp burst link to
Inflammasome nlrp3 activation and binding to Asc and caspases 1 for Il1b
Dysbiosis linked to antibiotics causes what which can trigger Ibd aswell as genetics
Candida outgrowth due to loss of commensal control
Give 2 genetic Ibd examples of poor fungal control
Decrin 1 snp linked to severe form of UC
Card9 linked to uc and crohns
Which immune response impaired by the card9 response
Th17 also phagocytosis in decrin snp
Fungal virulence determinants
How can comparative genomics be used to determine virulence traits
Identify species specific genes in the genomes of the 3 fungal opportunists
How many genes with no known function or orthologue in sacharomyces found in c albicans which could be for virulence
1000
How many af specific genes with pathogenic function are there not in benign yeast like neosartorya fischeri
Around 700
Do they have shared genes for virulence like the spi in diff bacteria
Non are identified yet
What is it suggested they do instead to cause virulence
Adapt conserved parhways eg carbohydrate metabolism pathways
What 2 other approaches can be done to find virulence traits
In vivo indetificatikn of genes
Or study of mutant libraries from insertational mutagenesis
How did thewes 2007 examine transcriptomic profiles of a highly invasive candida strain vs less invasive
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
What did they find comparing virulent strain with non virulent
How could you identify genes expressed at diff time points of the sc5314 strain
Take rna samples at diff time points and sequence them
How many genes found expressed during time points of infection in ex and in vivo both models and how many were sc5314 specific
63
5 specific
Give example of one gene and how they tested this further. Also give example of a protein downregulated in these mutant types
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
Does transcriptional profiling prove it is involved in virulence
No only looks at expression
Why is insertational mutagenesis easier with glabrata than albicans
Haploid so only one copy of interest gene vs diploid need to ko 2
What sort of marker gene from yeast doen for IM
Ura3
A library of how many homozygous mutants of c albicans used for key study in 2010
674
What 3 things can they be screened for
Morphogenesis , infectivty/growth defects eg iv in mouse kidney, proliferation screening (growth defects)
How did they measure infectivity in kidney of mice
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
How many genes found for impaired infectivity vs morphogenesis vs proliferation and what were the conclusions from this
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
Give example of the imapired infectivity- specific genes (not overlapping with morphogenesis or prolifedation6
Sphingolipid biosynthesis impairement
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)
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
Experimental models to study virulence
How are cn and af administered to disseminated mice models vs ca
Inteanasally imahlation
Vs iv injection into tail vein
How can you study virulence form the mouse
Study survival rates mutant vs wt eg dox ume6 and
tissue burden (cfu/g) eg kidney effects eg Hgc1 analysis
What is galleria mellonella (a mini host alternative to mouse)
Wax moth larvae
How can you inject and study survival
Larva taken from galleria and injected through a proleg to monitor survival
How can c elegans be given fungi to monitor survival
Can be gfp-linked fungi and fed to nematodes
Which 2 genes using celegans found important for killing of c elegans (mutant screening)
Ada2 and cas5 for hyphae formation
Which other model can be used which is first vertebrate with both innate and adaptive unlike the other minibost
Zebra fish
How are they infected
Injected into larval hindbrain ventricle
Which tf for filamentation found important in mice was replicated in zebra fish
Cph1 and efg1
What is the best incvitro model to study mucosal surface interactions
Reconstituted human epithelium
Cell lines cultured on polycarbonate filters
How can you quantify adhesion through RHE
Count cfu when cultured to see how much fungi adhesion
How can you quantify invasive ness/ penetrance using rhe
Can assay number of fungi cells that penetrate the rhe matrix (filamentation process)
Can you test mutant libraries using rhe for adhesion/penetration
Yes
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
Dfg16
How else was Dfg16 studied for virulence after RHE studies for more evidence of virulence
Disseminated mouse model where mutant showed increased survival time of mouse
Through study into ca genetic mutants of mannans (no O linked or n lined) and also prr ko mice what was found
Found tlr4 for o linked, MR for n linked and then decrin 1 are major for CA response
What alteration causes overgrowth of candida in ugt
Oestrogen production during pregnancy
Hiv immunodeficiency
Lactobacillus loss
What was the first model which you could study candida/macrophage interactions because they were transparent so could use tags
Zebra fish
