Sudbery (Fungal genetics) Flashcards
When are fungal infections more likely to be a big problem?
- when IS compromised
Are fungal pathogens big killers?
- yes
- kill more than malaria and influenza together worldwide
What are the characteristics of an Aspergillus infection?
- compact and protected from innate IS
- can break out of lung and infect brain = lethal
- causes Aspergilloma
When can Candida albicans be an indicator of AIDS?
- if in mouth
When can Candida albicans be deadly?
- if IS compromised can cross mucosal barrier, gets into bloodstream and causes sepsis
Where is Candida albicans usually found?
- common (≈70%) and usually benign commensal of GI tract and vaginal mucosa
What do Candida albican infections usually result in?
- opportunistic pathogen
- vaginitis, oral-pharangeal candidiasis, systemic candidemia
Who is most at risk of infections from Candida albicans?
- premature babies
- immuno-compromised, eg. AIDS
- neutropenia (can be result of immunosuppression therapy after organ transplants and blood cancers)
- abdominal surgery
- broad spectrum antibiotics (decreases competition from other bacteria)
- intensive care patients
- ill fitting dentures (food trapped –> infections)
What are the diff morphological forms of Candida albicans, and why are these diff forms important?
- can look exactly like S. cerevisiae
- also forms true hyphae which burrow into epithelial cells
- ability to switch forms critical to pathogenicity
What happens in cdc42 mutants at a restrictive temp?
- mutants grow but fail to bud
- fail to polarise actin cytoskeleton
- fail to form septin ring
Where do Rho type GTPases accum in cdc42 mutants?
- incipient bud sites
- tips of young buds
- cytokinetic ring
What is the role of the diff GTPases involved in the activation of cdc42?
- Ras-type -> oncogene
- Rab –> secretory pathway
- Rho –> polarised growth and actin cytoskeleton
- Rac
How is the GTPase cdc42 activated?
- DIAG*
- GEF is cdc24 (GDP –> GTP)
- GAPs are Rga2 and Bem3 (GTP –> GDP)
What is the role of sec2?
- GEF for the GTPase sec4
* DIAG*
What is the role of the exocyst complex?
- mediates exocytosis
What methods were used to show physical assoc of diff proteins involved in exocyst complex?
- two-hybrid
- immune precipitation
- sucrose density centrifugation
Which proteins physically assoc in exocyst complex?
- sec3
- exo70
- sec5
- sec6
- sec8
- sec10
- sec15
What happens at the restrictive temp to proteins involved in exocyst complex?
- synthetically lethal w/ each other
- can form active config but can’t function
What are the diff forms of the actin cytoskeleton and what are their roles?
- actin cables –> delivery of vesicles to sites of polarised growth
- actin cortical patches –> endocytosis
- contractile actomyosin ring –> septum formation (separates mother and daughter after cell division)
What is the role of cdc42?
- master regulator of polarised growth
* DIAG*
What does cdc42 activate?
- formation of actin cortical patches
- formation of exocyst
- formation of septins for cytokinesis
- formation of actin cables (made of polarisome)
- mating
What activated cdc24?
- cdk1
- mating pheromone
- localisation cues and bud site selection
What occurs during the late secretory pathway in budding yeast?
- secretory vesicles bleb off golgi
- membrane of secretory vesicle supplies new membrane to sites of polarised growth
- in order to leave golgi sec2 activates sec4 (GDP –> GTP)
- travels along actin cables to sites of polarised growth
- myosin 2 is motor, assoc w/ mlc1 (a light chain)
- actin cables made by polarisome
- in budding yeast, secretory vesicle has most components of exocyst complex, but some already present at cell surface
- when reaches site of polarised growth, exocyst complete and vesicle can fuse w/ pm
How can info from studying budding yeast be used to study hyphal formation?
- looking at env cues that cause cell to switch from yeast to hyphal growth –> many diff signal transduction pathways responding to diff external signals, approx 200 genes triggering transcrip but nothing to suggest they trigger hyphal growth
- mutant screen –> candida is obligate anaerobe so harder to spot mutants, so fuse GFP to proteins to track movement in cell, saw 2 patterns of localisation (one group of proteins localised in crescent structure at tip and another as point at tip
What is the Spitzenkorper?
- apical body
Why are proteins assoc w/ secretory vesicles very dynamic?
- continually being delivered and leaving to fuse w/ proteins at cell surface
Are proteins at cell surface dynamic?
- no, more stable than those assoc w/ secretory vesicles
How can it be tested that proteins are continually arriving and leaving Spitzenkorper?
- FRAP
- shining laser at spot bleaches it and removes fluorescence
- fluorescence recovers as new unbleached molecules arrive
- so can look at rate new molecules arrive at particular place in cell
What did FRAP experiments show about the diff proteins?
- sec4 and mlc1 assoc w/ vesicle –> fluorescence recovered quickly (30 secs)
- exo70 part of exocyst so expect to be stable on cell surface –> recovers much slower (as still replenished but not as fast as proteins assoc w/ vesicles)
- spa2 part of polarisome –> recovers v slowly if at all
Can we model this realistically for hyphal growth?
- use lampshade, represents no. hyphal rings
- when expand, rings that start off flat gain a height, can calc w/ Pythagoras
- proposing vesicles fuse w/ hyphal growth according to exocyst density, so amount each ring expands is proportional to density of exocyst
- so need to measure distribution of exocyst density around hyphal tip
What was the initial process carried out for each ring to calc new hyphal shape?
- measure exocyst density from observed normal distribution
- calc amount of synthase deposited in each ring
- calc new amount of synthase in each ring
- calc change in area
- calc new shape of each ring
- re-calc new hyphal shape
What is systems biology?
- recreating what happens in cell w/ a computer
Did the initial model work, and why?
- kind of right idea, but not parallel-sided tube hyphal shape
- need to account for synthase being taken back into cell in sub-apical regions, where actin patches are
What is the structure of a fungal cell wall, from inner to outer?
- chitin (polysaccharide)
- β-glucan (polsaccharide) –> most visible in light microscope, made by enzymes embedded in cell membrane, which are delivered w/ each secretory vesicle
How was the model mod?
- after calc amount of synthase deposited, calc amount of synthase removed by endocytosis from distribution of actin patches
Did the mod model work?
- yes, quantified where actin cortical patches were and saw just behind tip where should be
