C5- Virulence Determinants In Candida Flashcards
Give some examples where candida can outgrow
Antibiotics, pid/genetic defect, oestrogen/glycogen increase during pregnancy
What are the 4 stages of infection
Stage 1- colonisation through adhesion
Stage 2 - superficial infection: epithelial penetration and nutrient acquisition through host protein degradation
Stage3 - deep infection : penetrate tissues and vascular system and immune
evasion in blood
4- disseminated disease eg in organs like kidney via endothelial adhesion
- Adhesins
What are the main components of adhesins on outer cell wall
ligand binding domain (effector domain)
Then low complexity domain with ser/thr tandem repeats (often)
Can be anchored by gpi anchor to cell wall
What are the 3 families of adhesins in Candida albicans and what they bind (not in benign yeast)
Als family (9 genes) - epi and endothelium
Hwp1 family (5members) - saliva coated oral cavity and normal epi cells
Iff family (12 members)- epithelium
Looking at genome sequences (from previous lecture). What is evident about these family of genes
There is gene expansion/ duplication events - potentially with different affinity capacities like bspA in trichomonas
What agglutinin like gp are there in als family
Als 1 and 5
Explain their c terminus and the role it plays
C terminus is the low complexity ser/thr glycosylations allowing extension into environment
Threonine rich tandem repeat region forms amyloid like structure which has aggregation capacity (will aggregate with other fungi or bacteria esp in biofilms)
What is their n terminus/ effector domains like
Ig-like domains which swivel and act as a cavity for host peptides/ligands
Are they specific or broad binding
B-road s
Is it held by gpi anchor
Yes
Which adhesins important for biofilm formation on biotic / mucosal surfaces or abiotic like catheters
Als3 and hwp1
What are biofilms for
Evade immune system and increased resistance
Can they interact with other microbes
Yes bacterial-fungi interactions
What protein for drug resistance is upregulated in biofilms
Drug efflux pumps
Give a biofilm example in other candida species suggested important in Ibd (hoarau 2016)
C tropicalis with s marcescens and E. coli
What are invasins
Adhesins als3 and ssa1 which are specifically important for mediating fungal endocytosis for invasion eg for nutrient acquisition
What do they bind
E cadherin on epi or n cadherin on endothelial
What other way to invade is there
Penetration through hyphae
2 - morphogenesis
What sorts of things does morphogenesis occur to (pseudo with septation division or hyphae )
Ph (7 in blood = hyphae) and dfg16 alkaline example thewes 2007
, nutrients, co2 in blood, temperature (37c) , serum in blood
What is the diff between pseudo and hyphae
Pseudo have a construction at site of septation(between mother and daughter)
Cannot see distinct cells in hyphae and they are much thinner
What is filamentation crucial for
Access to blood for systemic
Escape from phagocytosis
Infection of internal organs - thewes found dfg16 for kidneys of mouse and pig
What happens when engulfed in phagosome
Hyphae switch occurs to kill them and escape
What is the ras positive pathway of filamentation
Ras gtp active
Will activate adenylyl cyclase cdc35 to produce camp from atp
Camp activated pka will then phos tf efg1 which induces expression of ume6 hyphae specific gene
What is the mapk pathway/cph1 activation instead for ume6 expression
Mapk pathway activation by ras-gtp
Ste11 mapkkk
Hst7 mapkk
Cek1 mapk
will phosphorylate tf cph1 which also induced ume6 gene
What happens to double mutants of efg1 or cph1 (c4 suggests both mouse and zebra fish survival studies)
Form Budding avirulent forms
Which 2 proteins recruit tup1 txn repressor to block hyphae genes like ume6
Rfg1 and nrg1
What is the evidence dimorphism is required not just hyphae
Mutant tup1, rfg1 or nrg1 also reduces virulence of the fungi
Ume6 and virulence evidence study
Using ume6 regulated under doxycycline (if dox present represses ume6) in mouse models injected with candida strains (engineered)
What we’re the histologixal and survival results
After 30 days - 70% survival in dox+ / 0 Survivial when ume6 wasn’t repressed and was expressed highly in lack of dox presence
Kidney histology showed lack of dox meaning ume6 exp allowed filamentation - showing it promotes virulence
What sorts of genes are hyphae specific/ upreg in hyphae form
Als3 (invasins and biofilms)
saps and hwp1 (biofilm)
So how do we know it isn’t because of these genes not the actual shape causing virulence
Hgc1 (cell cycle protein) protein mutant which is involved in hyphae formation prevents shape change but had no effect on these genes eg hwp1 still high
Still show reduced ability to colonise kidneys of mice / invasiveness and
Reduced ability to kill the mice too (consistent with ume6 study)
How does quorum sensing (of both bacterial and fungal origin) affect hyphae growth aswell
Dense populations will release farnesol quorum molecule which will prevent hyphae formation for better colonisation of mucosal surfaces
How does farnesol block hyphae growth (showing potential importance of dense bacterial populations on inhibiting hyphal formation)
It acts to block adenyl cyclase cdc35 stopping camp activation of pka and efg1 tf
What 3 types of secreted enzymes do they have for nutrient acquisition, and tissue invasions - in candida are expressed on hyphal tip
Phospholipase (a,b,c,d classes) -only B are extracellular
Secreted aspartyl proteinase (saps)
Candidalysins
What 2 Phospholipase B class are shown as virulence factors on the tip of hyphae cells to breakdown cell membranes
Plb1 and 5
How many genes are there for saps (gene duplication example again) and the main differences
10
9+10 are cell surface gpi anchored
Some are expressed in yeast to hyphae growth eg sap6
Some in yeast cells eg sap2 I.e more acidic ph than sap6
Can work at different ph
Different substrates of proteins
How does internalisation of saps 2 and 6 cause apoptosis and inflam response characteristic of vaginal candidiasis
Activation of the nlrp3 inflammasome
Caspases = apoptosis
Which highly expressed hyphae protein is processed into 8 peptides including candidalysin
Ece1
What does candidalysin toxin do
Forms pore within membrane causing permeability
Also induced ap-1 tf c-Fos causing cytokines and chemokine exp for inflammation
Give an example of when this would happen
Inadequate NETs or phagocytosis
4- fitness traits
Give some fitness trait examples needed to survive a lot of environments
Evade host mechanisms eg ros/stress response
Adequate nutrient acquisition/metabolic change
Ph adaptation
Metal acquisition
How is h202 formed at macrophage and neutrophils
NADPH oxidase complex forms and produces superoxide which is converted to h202
What does stress activated c.albicans hog1 kinase do in response to h202 (aswell as cap1 tf)
Upon stress (ox or osmotic) becomes phos and activated by h202
Translocation into nucleus
Induces cell responses downstream crucial for survival (mutants are not able to survive under conditions of ox stress)
What other enzymes to detoxify ros do they have
Sod , tsa (thiol specific antioxidant) and sod5, catalase
Which tf is important for exp of all of these under ox stress and how
Cap1
When it becomes oxidised, the cys rich domains change conformation which mask it’s nuclear export sequence
Starts to accumulate in the nucleus
Similar to hog1 mutants, what happens to cap1 mutants exposed to h202
Reduced survival / killer
What happens in wt hog1 vs mutant systemic mice modes
Also what happens in macrophage modelling when both are knocked out
Hog1 Wt causes reduced survival in mice
Compared to 100% survival in hog1 mutants = avirulent
In macrophages with candida ko for both hog1 and cap1 there is a lack of filamentation and also reduced macrophage killing
Which human kinase similar to hog1 kinase suggests we could potentially use the inhibitors we have for cancer treatment for hog1 inhibition too - currently testing if it works
P38 kinase (inhibitors admin bc involved in tumour progression)
Which adhesin/invasin can steal fe from ferritin from host
Als3
What do they have to acquire zinc
Sincophore pra1
How is it suggested under starvation, cells can induce hyphae growth (for cell invasion/nutrient acquisition) through alkalinisation (dfg16 can induce this)
Production of ammonia from aa uptake by deamination = released and causes alkalinisation
(Dfg16 mediated filamentation thewes 2007)
Although superficial infections like thrush and oral candidiasis are very common; what happens in systemic disease in Immunocompromised
Can overgrow/ colonise different organs like eye, brain, kidney and cause organ failure and death
How was pepstatin used to confirm that saps are virulence factors needed for tissue invasion
Injected candida with pepstatin sap inhibitor into mice
Found reduced tissue invasion and reduced systemic candidiasis
Which protein present in the ecm of fungal biofilms which block neutrophil ros mediated killing
B glucans
