Week 7A: Medical Yeasts, Virulence, Antifungals, Brain metabolism Flashcards
HC 37, 38, 39
Tinea capitis
Trinchophyton schoenleinii (fungus) grows in hair follicles > bald head
> Scalp ringworm
> hair contains protein: carbons hydrogen and sulfur as nutrients
Tinea Circinate
Ringworm causes ring inflammed round scaly lesion lower on body (chest)
Tinea pedis
Zwemmerseczeem, between toes
Onychomycosis
Infection of nails by fungi
Risk treating fungi
If one cel survives, it can grow back
Candidas are easier treatable when they are … and not …
superficial and not invading the system
Mycoses meaning
Diseases caused by fungal infection
Pathogenic fungi vs opportunistic fungi
Pathogenic
> cause disease in both immunocompetent and immunocompromised hosts
Opportunistic
> cause disease in immunocompromised hosts
> immune resistance of host compromised
> can cause life threatening disease
Immunocompromised host
Immunity is altered, because
-Presence debilitating disease, like AIDS
-Immunosuppressive drugs
Name the three opportunistic fungi we need to know for the course and their fungal groups
Candida albicans > Hemiascomycetes
Aspergillus fumigatus > euascomycetes
Cryptococcus neoformans > basidiomycetes
Difference ascomycetes and basidiomycetes in secual cycle
During sporulation, ascomycetes form an ascus (sac), and basidomycetes form a basidium (mushroom like)
Ascospore formation
> Haploid + and - cells fuse to diploid
Diploid cell
meiosis
Four nuclei in one PM
ascospore formation
Four spores in one membrane sac (ascus)
autolysis
Ascospores (seperate)
Aspergillus fumigatus asexual spores
Condidia
> Vescicle with a lot of tubes which bud of conidia (ev conidium)
> release A LOT of spores
> conidia are the tiny spores that go into the air from the vesicle.
Hemiascomycetes type
budding yeast like fungi and candida albicans
Aspergillus fumigatus characteristic of infection
Fall on food and grow
> euascomycete
Reproduction aspergillus fumigatus
Sexually (ascospores) and asecual (conidia)
Organ transplantation is a danger for..
fungus infection if spores there
Invasive aspergillosis has a … mortality rate
high
> especially in liver, bone marrow of brain
Candida albicans form …. on blood plates
white colonies
> blood is rich in nutrients for fungal growth
> cryptococcus not white: attracts water
Morphological switch in Candida albicans
Can switch reversibly between yeast form and hyphal form
> hyphal form has directional growth (like tubes of cells)
> fully reversible and essential for virulence
> triggered by conditions
Yeast-to-hypha transition in candida albicans
Yeast cells form germ tubes which grow out
What kind of fungus is C albicans and how where does it reside?
Opportunistic yeast
> commensal life on skin and mucose of GI tract > occupy spaces so that nasty microorganisms cannot grow there
Invasive systemic infection of C. albicans in patients with …, and which organs affected make it life threatening
compromised immune system
> life threatening: infections of liver, kidneys and brain.
Candidiasis
Infection caused by Candida species
Superficial candidiasis
Local infection
Hyphal C.albicans can penetrate …
the epithelium > invasion > switch back to yeast form which can travel through blood
> immune cells do not attack when immunocompromised
Why do males and females with T2DM have risk for Candida infection at penis head or vagina?
Glucose in the urine > nutrients
> candida balanitis
Risk factors Candida albicans infection
-T2DM
-(broad spectrum) antibiotics (less competition for fungus, normally all the niches are occupied by bacteria)
-Immune suppressants
-Catheters
-Prosthetic devices
-Abdominal surgery
-Needle: push it into blood
Phagocytosis Candida in vitro by macrophages
After a while the germ tube is formed (morphological switch) aand hyphal penetrates the cell and kills it
> a lot of building blocks in the lysosomes of the macrophages to make fungi grow
Defensive reaction macrophages upon phagocytosis candida
Oxidative stress > respiratory burst
> produce ROS to attack Candida
Triggers of morphological switch in Candida
-Absence nitrogen and carbon: poor conditions
-pH, serums and ingestion by macrophages
> sensing proteins and TFs involved: programs for hyphal form activated
Why is the morphological switch of C. albicans a virulence factor
Otherwise it cannot kill the macrophages by switching to hyphal form
Candida auris
An emerging fungus
> invasive and picked up in the hospital
> treatable with echinocandins
> some strains are multidrug resistant
> hospital-acquired infection
Cryptococcus neoformans defensive structure
Capsule
-Hair like molecules around them (around the PM
> harder to take up by phagocytes
The cell compartments of the fungus makes it clear why it is hard to make fungal drugs,: explain
It resembles the human cell
> neokaryotes: make new nucleus
> eukaryote
> contain peroxisomes (vacuoles in fungi and yeast), lysosomes, ER, mitochondria
> drug should not kill human cells
Melanin in cryptococcus neoformans
Protect cells against oxidative burst inside immune cells
> the ROS of macrophages and neutrophils cannot penetrate it
> in multiple fungi present: virulence factor
Virulence factors
Molecules expressed by pathogenic and opportunistic organisms that are important to cause disease in the host
> can be lipids, carbohydrates, proteins
> ideal target for antifungal drugs
Factors that are involved in basic cellular metabolism are essential for survival of a microorganism in the host, but they are not called virulence factors. Why?
They do not influence the host directly
Why does Cryptococcus neoformans appear glazy on blood plate
Carbohydrate (polysaccharide) capsule
> attracts water
How does Cryptococcus neoformans synthesize melanin: which precursor
From L-DOPA (dopamine precursor)
Melanin production enzyme and reaction of committed step
L-DOPA > Dopa quinone (enzyme: laccase, phenoloxidase involved in melanin formation)
Laccase
Phenoloxidase involved in formation of melanin
Melanin in cryptococcus neoformans protects against:
-Environmental stresses such as ionizing radiation
-Oxidative damage by phagocytes
Melanogenesis in Cryptococcus
-L-DOPA > Dopa quinone (laccase/ phenol oxidase)
-Spontaneous autooxidation steps from Dopa quinone to melanin
Two virulence factors Cryptococcus neoformans
-Melanin
-Capsule
Capsule cryptococcus neoformans
High molecular weight polysaccharide: Glycuronoxylomannan (GXM)
> major repeat unit + serotype
Cryptococcus survival inside macrophages
- Phagocytic uptake
- Phagolysosome formation
- late stage trafficking markers
- rupture phagosomal membrane
> love acidic environment and secrete lipases to break down lysosome > slow digestion macrophage contents for nutrients due to not optimal pH) - growth Cryptococcus in cytosol
- rupture hos cell > releasing cell after 8-18h after ingestion
Mode of action of defense of macrophages and neutrophils
Oxygen-independent mechanism
> Hydrolytic enzymes: proteases and lipases
> Defensins: microbicidal peptides
> nutrient deprivation
Oxygen-dependent mechanism
> ROS and nitrogen species: O2-‘, H2O2, NO’
> HOCl (hypochlorite, only in neutrophils because they have the enzyme)
Identification and testing virulence factors
-Identification
-Gene deletion: KO
-Virulence assays with deletion strain and control (in vitro and in vivo)
-Virulence factor > target for antifungal drug design
Capsule assay cryptococcus as virulence factor: which samples included
-Wild type
-Cap10 mutant (acapsular mutant)
-cap10 mutant transformed with empty vector
-cap10 mutant transformed with cap10 gene
> survival when cap10 mutant or cap10 mutant with empty vector, and quickest death when transformed with cap10 vector: many copies of the gene > more of the limiting virulence factor
How are Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans acquired>
-Aspergillus fumigatus: in air
-Candida albican: commensal in GI tract
-Cryptococcus neoformans: in GI tract of birds > bird poop air breathed in when walked over.
> gobbled up by alveolar macrophages
HC38: Manganese, iron and zinc are …
Transition metal
Transition metals are involved in which reactions in biochemistry?
One-electron reduction or oxidation reactions
> can deal with individual electrons
Coordinate bonds in insulin hexamer
Two Zinc ions coordinated each by three His residues
> problem when mutated
Binding manganese cofactor in managese superoxide dismutase
Bound by three Histidine side chains and one aspartate side chain
> water molecule and the inhibitor azide can be coordinated by manganese
Stucture of the antifungal polyene called amphotericin B
ring with two large carbon chains. One has many hydroxyl groups and is hydrophilic. The other has many di-een groups (double bonds) and is hydrophobic)
Function amphotericin B and other polyenes
Bind to sterols
> ergosterol in fungi > in PM
> adheres with hydrophobic side: make hydrogen bonds with carboxylic acid group between hydrophobic and hydrophilic chains (this groups faces towards the hydrophilic head of the phospholipids which lay next to the ergosterol which binds with its hydrophilic OH group to the polyene
> two rings of 8 molecules form hydrophilic channel when hydrophilic chains face eachother
> Pore: content flows out (leakage Mg2+ and K+ and others), cell dies
Polyenes are fungi….
Fungicidal: the fungus is killed
How does ergosterol differentiate from cholesterol
Extra double bonds
Structure azoles
Ring of carbons with a nitrogen in it
> heterocycles of carbon and nitrogen atoms
A diazole is characterized by?
Two nitrogens in the heterocycle
Miconazole is a … and fluconazole is a …
Diazole, triazole
Azoles are fungi….
Fungistatic
> Inhibits growth of fungi
> If the patients stop taking azoles, the fungi will grow again
> take for rest of life
Azoles can bind …
The heme group with the transition metal iron
CYP51 in fungi is involved in…
Ergosterol production from lanosterol
> Lanosterol 14a-methyl demethylation
> and then for two other methyl groups
Azoles are inhibitors of …
Fungal CYP51
Mechanism of action Voriconazole and Posaconazole
Voriconazle binds in binding pocket where lanosterol binds with the O2 (at the iron metal)
Posaconazole binds in a different binding groove and then also to the heme group. > stronger interaction
How many oxygens needed to remove one methyl group?
Three molecular oxygens (O2)
Function terbinafine
Block the conversion of Squalene to Squalene-2,3-epoxide in the synthesis of ergosterol
> inhibits squalene epoxidase: committed step
Azoles inhibit ergosterol biosynthesis. What is the consequence?
Altered structure of function of the plasma membrane in fungi
> ergosterol acts as bioregulator of PM fluidity and asymmetry > membrane integrity
Flucanazole resistance in AIDS patient can be dangerous, why?
Resistance builds up > dose increases > more dangerous side efffects
Drawbacks current antifungals
-Limited number of antifungals available
-Amphotericin B is reversibly nephrotoxic
-Resistance against azoles in increasing
Meningitis characteristics
-Infection with Exserohilum rostratum
-Spinal injections of steroids as pain relief, but steroids were infected with fungi
Treatments meningitis
-Standard: Voriconazole
-Severe: Amphotericin B as well
Why is the cell wall a good target for development antifugnal drugs?
Not in humans, and fungi depend on it for survival
Ideal antifungal target characteristics
-Enzyme that synthesizes reaction product essential for viability funga; cell
-Enzyme is absent in humans or very different than human type
Which cell wall components (polysaccharide components) of fungi can be targeted?
Synthesis of
-alpha-glucan
-beta-glucan
-chitin
Cell wall composition
Long polysaccharides for rigidity and strength on outside of PM, proteins associated as well
Enzymes for polysaccharides in fungal cell wall?
-Alpha-glucan synthase
-Beta-glucan synthase
-Chitin synthase
Structure beta-glucan synthase and chitin synthase
Both are transmembrane proteins with catalytic domains located intracellularly
Energetically enriched substrates of chitin synthase and beta-glucan synthase
Chitin synthase: UDP-N-acetylglucosamine
Beta-glucan synthase: UDP-glucose
Chain growth chitin and beta-glucan
growth of the chain intracellularly
> useful UDP stays inside cell
> made chain (not the ends) are translocated to extracellular between the two transmembrane domains of either chitin/b-glucan synthase
Competitive inhibitor of chitin synthase
Nikkomycin
Nikkomycin function
Peptidyl nucleoside produced by a bacterium
> competitive inhibitor chitin synthase by mimicking UDP-acetylglucosamine
> contains reactive peptide bond
> potent antimycotic activity
Caspofungin function
Prevent translocation of growing beta-glucan chain to extracellular side PM by the beta-glucan synthase
Caspofungin is a … and these inhibitors work ….
Echinocandins > non-competitive inhibitors of beta-glucan synthase
> other used echinocandins: Anidulafungin and Micafungin
Echinocandins kind of molecules ?
Lipopeptides
> many reactive peptide bonds
> integrate into channel and bind
> attaches to the synthase
Alpha-glucan synthase at 37 degrees?
Is inactive, fungal cell dies
> 19 degrees: normal shape alpha-glucan synthase
Structure alpha-glucan synthase
-Domain for linking alpha-glucan (extracellular)
-Transmembrane domain for alpha-glucan transport across membrane
-Domain for synthesis alpha-glucan (intracellular)
The putative catalytic domains of polysaccharide synthases for the fungal cells wall are located …
intracellularly
What if there is no alpha-glucan in the cell wall. Which fungus has a disadvantage if this happens?
The (protective) capsule cannot bind
> In Cryptococcus neoformans: required for normal growth and capsule binding: becomes invirulent
> In S. pombe: required for maintaining structural integrity
HC39: Of what cells is the blood-brain barrier composed?
Endothelial cells of brain capillaries form the blood-brain barrier
Blood-brain barrier forms barrier between ..
Blood and cerebral spinal fluid
Where is the blood brain barrier located?
Throughout the whole brain: needs a lot of nutrients
> 20% oxygen consumption
> expensive organ
Between the endothelial cells of the Blood-brain barrier, there are ….
Tight junctions
GLUT transporters of ECs in blood brain barrier
GLUT1 (both sides)
Transport FAs across blood brain barrier
Polyunsaturated FAs transported, not saturated
Which molecules can pass the blood brain barrier by diffusion
Small molecules and gasses like CO2, H2O and O2
Ion transport across blood brain barrier
Use of channels
Transport from blood to brain
Blood > ECs > interstitial space > endfoots astrocytes
How can drugs be transported across the blood brain barrier?
Piggyback transport with ABC transporters
Transporters in blood brain barrier
-Glucose: GLUT1
-Lactate: Monocarboxylated co-transporter 1 (MCT1)
-Amino acids
MCT1 transport
Lactate and proton cotransport (neutralize charge)
How do transferrin, insulin and other proteins cross the blood brain barrier?
Receptor-mediated transcytosis
> endocytosis and exocytosis on other side EC
> requires receptors
Membrane potentials neurons
-Lots of ion fluxes
-Resting membrane potential: -60 to -80 mV
-Cell interior has baseline negative voltage
-Graded potentials (dendrites and soma) and axon potentials (axons) transiently increase or decrease membrane potential
Effect increased expression receptor proteins and ion channels on energy consumption of brain
Increases it
Neuron-astrocyte metabolic coupling using lactate
-Glucose uptake by ECs in blood brain barrier using GLUT1
-Uptake by either Astrocyte or neuron
Astrocyte (transastrocyte path)
> uptake by GLUT1
> Storage as glycogen
> or glycolysis to pyruvate to complete oxidation or to lactate
> Lactate cotransport with proton via MCT1 to interstitial space and again with proton into neuron via MCT2
Neuron
> Uptake glucose via GLUT3 or lactate via MCT2
> Glycolysis glucose or Lactate to pyruvate
> complete oxidation for energy
Why the neuron-astrocyte metabolic coupling of glucose metabolism?
Storage glucose as glycogen in astrocytes (not so much in neurons)
Why can T2DM induce mood disorders?
Insulin resistance
> Serotonin (5-HT) neurons express the insulin (Ins) receptor
> good mood
Effect insulin on serotonin neurons
Promotes the synthesis of serotonin
> in T2DM: less synthesis because of insulin resistance
T1DM and serotonin production
No insulin: no serotonin production in untreated T1DM
Lipid metabolism in brain
Only uptake polyunsaturated FAs which are preferentially oxidized to form lipid peroxides
> Lipoxygenase (Fenton chemistry) makes hydroperoxide (5-HPETE) from arachidonic acid (shift extra double bond and oxidize -CH to -OOH)
> Intermediates: redicals.
> Steps: Hydrogen abstraction, rearrangement, O2 uptake with H+)
Why are polyunsaturated FAs prone to oxidation
They contain methylene bridges > sensitive
> in the brain; only transporters polyunsaturated FAs, and a lot of oxygen available
Why is it important that the lipoxygenase is in neurons?
So that the oxidation forming radicals does not occur
Coupling lipid metabolism neurons-astrocytes
-Highly active neurons exocytose toxic lipid peroxides in apolipoprotein E (apoE) particles
> Astrocytes endocytose ApoE particles, incorporate the lipid peroxides into lipid droplets, detoxify them and use them as fuel in beta-oxidation (mitochondrial metabolism upregulated)
-In the highly active neurons, high levels of ROS induce ??? of FAs
peroxidation
Astrocytes produce ??? and detoxify and break down toxic fatty acids
anti-oxidants
Important risk factor Alzheimers Disease, and protective factor?
Apolipoprotein E gene for late-onset AD
> Two cysteines (Cys) at 112 and 158 of ApoE: protection
> Both mutated to arginine (Arg): higher risk AD
APOE2 and APOE4
-APOE2: ApoE isoform: 112 Cys 158 Cys > protective effect
-APOE4: ApoE isoform 112 Arg 158 Arg > most likely to develop AD
> most are APOE3 with one of each
Glutamine-glutamate cycle in brain
Glutamine neurons take up glutamine and convert it to neurotransmitter glutamate using enzyme glutaminase
> Astrocytes can take up excess glutamate and convert it to glutamine using enzyme glutamine synthase (transamination).
> Glutamine cycled back to glutamine neuron using transporters
> Glutamate in synaptic cleft can also be taken up by glutamine neurons themselves (presynaptic)
Why is glutamate important?
Crucial in cells
Brain glucose level
Independent of blood glucose levels
> 2.0-2.5 mM: stable
Formation glutamate and oxaloacetate in brain. What happens in hypoglycemic brain damage
From a-ketoglutarate and aspartate
> Hypoglycemic
> balance other way: oxaloacetate accumulates because glycolysis slows down and acetyl-CoA decreases
> Oxaloacetate + Glutamate > aspartate + a-ketoglutarate
(enzyme aspartate transaminase)
> promotion aspartate excretion by glutaminergic neuron
Lafora disease
Carbohydrate binding phosphatase called Laforin is deficient
> accumulation highly phosphorylated less branched and water-insoluble glycogen called Lafora bodies in neurons and other glycogen synthesizing tissues.
Problem phosphorylation glycogen
It is not normal
> due to mutation in carbohydrate binding domain of the phosphatase laforin
> abnormal glycogen
> accumulation Lafora bodies –> neurodegenerative disease Lofora disease
Glycogen storages in brain
Astrocytes, and a little in neurons
