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PM3 - Infection and immunity > Fungal infections > Flashcards

Fungal infections Flashcards

1
Q

types of mycelium

A

siphonale mycelium -> a multinucleic giant cell -> usual in archemycota

standard mycelium -> cells ae mono, bi or poly-karyotic -> basido- or ascomycota

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2
Q

morphology filamentous fungi

A

very diverse
can form everything from hyphae to conidia which makes effective immune response and drug development hard

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3
Q

life cycle of filamentous fungi

A

asexual via mitosis, formation of conidiospores (mitospores)
sexual via meiosis, formation of ascospores (meiospores)
parasexual via cell-fusion

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4
Q

KDACs in filamentous fungi

A

lysine-deacetylation of histones
responsible for regulation of many secondary metabolites

Class I:
- HosA: reduces e.g. orsellenic acid TK
- RpdA: essential for viability of A. nidulans

Class II:
- HdA: deletion increases seconary metabolite production
- HosB

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5
Q

Class I KDACs in filamentous fungi

A

Class I:
- HosA: reduces e.g. orsellenic acid TK
- RpdA: essential for viability of A. nidulans

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6
Q

HosA

A

Class I KDAC
regulation of secondary metabolites
reduces e.g. TK of orsellenic acid

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7
Q

RpdA

A

Class I KDAC
regulation of secondary metabolite production
essential for A. nidulans

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8
Q

Class II KDACs in filamentous fungi

A

regulation of secondary metaboolite production

Class II:
- HdA: deletion increases seconary metabolite production
- HosB

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9
Q

HdA

A

Class II KDAC
deletion especially for biotechnology interesting -> increases 2nd metabolite production (AB)

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10
Q

HosB

A

Class II KDAC

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11
Q

spectrum of fungal disease

A

allergic
superficial
mucosal
chronic
acute invasive (life-threatening)

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12
Q

WHO lif-threathening fungi
definition and fungi

A

based on resistances, treatment and diagnosis options, incidence, complications and squelae

Cryptococcus neoformans
Candida albicans
Candida auris
Aspergillus fumigatus

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13
Q

obligatory pathogenic

A

also disease causing in immunocompetent
mild manifestation in immunocompetent
serious manifestation in immunocompromised

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14
Q

opportunistic pathogenic

A

no manifestation in immunocompetent
serious manifestation in immunocompromised

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15
Q

causes for invasion of fungi

A

comination of risk factors

innate immune status (polymorphisms)
underlying conditions (especially NEUTROPENIA)
environment (house, work, etc)
others (DM, high iron, etc.)

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16
Q

Candidiasis
strain, transmission, manifestation

A

C. albicans, tropicans or auris (since 2005)
endogenous or exogenous
transmission via contaminated material or personal contact -> nosocomial!

variable morphology

Manifestation: superficial (cutaneous), subcutaneous (locally invasive) or disseminated (invasive)
- CUTANEOUS: sores with white coating, topical antimycotics
- INVASIVE: can manifest in every organ, 2.-4. cause of death in ICU

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17
Q

Virulence traits Candida

A

morphological switch
adherence: mannans, affinity for plastic
bifilm formation (adherence mediated)
proteases damaging epithelial barriers -> invasion
phospholipases destroy complement and IgG -> immmune evasion

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18
Q

Virulence traits Candida auris

A

develeoped thermoteolerance and haplotolerance in recent years
intrinsic resistance to most antifungals and desinfectants -> rapid spread
causes prolonged hospital stays

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19
Q

Aspergillosis
strains, manifestation

A

A. fumigatus, flabvus, terreus and nidulans

MANIFESTATION: allergic, colonisation of body- or lung cavitis, invasive
- INVASIVE: most common in COPD in western world (previously ICU), administration of prophylaxis but break through infections (resistant)
- CHRONIC PULMONARY ASPERGILLOSIS: > 3 months, diagnosis pf post mortem due to mild symptoms -> aspergilloma formation leads mostly to obstruction, formation in lung after tuberculosis (e.g.) or sinus, can become invasive -> rising mortality

most problematic is ACQUIRED AZOLE RESISTANCE

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20
Q

Virulence traits Aspergillus

A

adaptations to host niche!
morphological switch
thermotolerance
proteases
toxins
immune evasion via RODLET LAYER and MELANIN

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21
Q

Cryptococcosis
risk factor, strains

A

Immunosuppression as major risk factor –> longterm cortisols, Tx, HIV (AIDS defining disease)

C. NEOFORMANS: only immunosuppressed, worldwide in soil, plants and BIRD DROPPINGS
C. GATTII: typically immunosuppressed, new variants also in competent, high adaptation potential, becomes worldwide endemic

HIV: AIDS defining disease in Africa etc. in western world not via HAART and other new therapies

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22
Q

Cycle of infection Cryptococcus

A

spore inhalation into lung alveoli
morphological switch from conidia to yeast form
dissemination into various organs (BRAIN,skin, kidney, bone marrow, etc)

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23
Q

Virulence traits Cryptococcus

A

many -> examples:
capsule
melanin
phenotypic switching

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24
Q

Cerebral infection by fungus
type, mechanism

A

Cryptococcus

BBB-passaging: 3 theories
- Trojan Horse: phagocytosed in neutrophils, macrophages or monocytes -> transport over BBB
- Transcytosis: hyaluronic acid-mediated endocytosis, exocytosis via Mpn-1 (fungi) and annexin A2 interaction
- Paracelular: host pplasminogen activated (plasmin) -> ECM degradation, fungal Urease degrades tight junctions

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25
Q

Mucormycosis
features, infection, forms

A

rising death rates
fulminant progresion, angioinvasion and extensive tissue damage (necrosis)
therapy requires OP and antifungals

INFECTION: primary manifestation
- INHALATION: rhino-orbital or rhino-cerebral, here pulmonary via mechanical ventilation
- PENETRATING TRAUMA: primary cutaneous manifestation
- GUT: contaminated food, etc.

SECONDARY MANIFESTATION: cutaneous as first sign of dissemination, high potential!

INVASIVE: rare but rising with high mortality, caused by ineffective long-term antifungal treatment, UNCONTROLLED DIABETES as major risk factor (CAM -> covid associated mucormycosis endemic in India)

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26
Q

Primary metabolism

A

encoded genes are conserved between species/strains
encode for glycolysis, TCA, respiratory chain etc.

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27
Q

Secondary metabolism

A

encode genes producing secondary metabolites
not-conserved and differ between strains
produce toxins, proteins etc
are a form of microbial warefare

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28
Q

Diagnostic of fungal infections

A

testing for cell wall components or genetic material
specifity and sample acquisition is problematic

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29
Q

morphological characteristics of fungi

A

different morphological stages

CONIDIA: highly stress-resistant infectious propagules, generated for multiplication, no TK response, long-lived and contain DNH-MELANIN and RODLET LAYER

CELL WALL:
- components recognized by Dectin1 and 2
- in conidia masked by rodelt layer and melanin
- used for immune evasion tactics -> shedded galactomannan (diagnostic) and galactosaminogalactan (decoy)

CELL MEMBRANE: contains ergosterol -> target of antifungals, producation activated/controlled by SrbA

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30
Q

Immune evasion by morphology

A
  • rodlet layer
  • melanin
  • binding of complement regulators (FH, FHL-1, CFHR-1, plasminogen)
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31
Q

morphological characteristics as virulence determinants

A
  • morphological switch
  • morphology of conidia (rodlet layer, melanin)
  • cell wall shedding -> galactosaminogalatan
  • binding of complement regulators (FH, FHL-1, CFHR-1, plasminogen)
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32
Q

metabolic characteristics of fungi

A

primary and secondary metabolism

linked and can adapt to host via TK response
-> stress resistance to oxidative burst, pH, hypoxia, tarvation

produce host component dagrading enzymes and other secondary metabolites & toxins

virulence factors can be toxins, nutrients (metals), immune evasion tactics, etc.

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33
Q

Gliotoxin

A

by Aspergillus fumigatus
biosynthesis by gene cluster (12 genes) regulated by gliZ
Target = NEUTROPHILS

IMMUNOSUPPRESSIVE:
- reduced phagocytosis, NFkB
- reduced inflammation and cytokines
- reduced mast cell and neutrophile function
- apoptotic death in immune cells (macrophages, monocytes, others)
- elevated PMN-mediated inflammation

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34
Q

metabolic immune evasion tactics

A
  • secretion of proteases (degrade e.g. complement)
  • secretion of gliotoxin
  • shedding galacosaminogalatan
  • metal acquisition tactics
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35
Q

Copper metabolism in host and fungi

A

Cu is essential nutrient but also used as toxin in phagolysosomes by host
fungi need Cu UPTAKE and DETOXIFICATION mechanism

  • Ctr1&4: high affinity Cu transporters of C. neoformans
  • CnMt1&2: Cu binding molecule -> detoxifying
  • Cuf1: Cu sensing TF -> induces Ctr1&4 Tk during starvation and CnMt1&2 during Cu-excess

in A. fumigatus 2 separate TF -> MacA for Cu-uptake, AceA for detoxifying plus CrpA for cellular Cu-export

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36
Q

Virulence determinants involving copper

A

Ctr4 for brain-infection (is Cu starvation niche)
CnMt1/2 in macrophage phagolysosome and lung (Cu excess)
Cuf1 (TF)

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37
Q

Iron metabolism in host
role of iron

A

iron is essential for haeme, iron sulfurclusters and other proteins
toxic via catalyzation of ROS formation

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38
Q

Iron metabolism in host
mammalian iron homeostasis

A
  • transferrin binds Fe in blood
  • transferrin-receptor for uptake into cells
  • storage in/as ferritin
  • regulation via IRP regulating TL by binding IRE on mRNAs
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39
Q

Iron metabolism in host
Antimicrobial strategies

A
  • Aptoferritin lowers free Fe
  • Apolactoferritin in neutrophil granule & body fluids
  • chronic inflammation leads to Fe uptake in macrophages
  • Siderocalins: bind siderophores -> LIPOCALIN1 (bacterial and fungal siderophores) and LIPOCALIN2 (entero- and carbomyxobactins)
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40
Q

Iron metabolism in Aspergillus
regulation

A
  • SreA-Fe downregulates iron-acquisition pathways
  • HapX downregulates iron-consuming pathways
    -> HapX and SreA-Fe repress each other
  • HapX-Fe upregulates iron consuming pathways
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41
Q

Iron metabolism in Aspergillus
components

A

FetD: low affinity Fe uptake

REDUCTIVE Fe UPTAKE: FreB, Fet C (osidase) and FtrA (Fe permease)

SIDEROPHORE Fe UPTAKE: TATFC-Fe uptake via MirB, TAFC dgradation required for uptake -> EstB, TAFC secretion via Abc transporter

Siderophore Fe STORAGE: FC (siderophore storing Fe), can be released if necessary for metabolic activity

VACUOLAR Fe STORAGE: Fe transported into Fe-vacuole via CccA

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42
Q

Virulence determinants involving iron

A

siderophore biosynthesis (sidA)
HapX

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43
Q

Zinc metabolism in fungi

A

high affinity Zn uptake important for virulence

ZrfA&B: high affinity Zn transporter, pH optimum is acidic
ZrfC: high affinity Zn transporter, pH optimum is neutral/alkaline
ZafA: Zn sensing TF, inhibited by Zn, activates transporter TK
PacC: pH sensing TF, activated by OH- (alkaline) -> induces ZrfC and AspF2, represses ZrfA&B
AspF2: secreted Zn bindng protein -> Zincophore, interacts with ZrfC

VIRULENCE DETERMINANTS: ZrfC, AspF2, PacC

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44
Q

Zinc metabolism in host

A

CALPROTECTIN released in response to inflammation or on Zn shortage
-> Zn/Mn specific chelator

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45
Q

Virulence determinants Zn

A

ZrfC, AspF2, PacC

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46
Q

ZrfA

A

acidic Zn transporter
induced by ZafA in low Zn
repressed by PacC in alkaline/neutral conditions

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47
Q

ZrfB

A

acidic Zn transporter
induced by ZafA in low Zn
repressed by PacC in alkaline/neutral conditions

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48
Q

ZrfC

A

alkaline/neutral Zn transporter
induced by ZafA in low Zn
induced by PacC in alkaline/neutral conditions
virulence determinant

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49
Q

ZafA

A

Zn sening TF -> induces transporter ZrfA,B & C in low Zn

50
Q

PacC

A

pH sensing TF
activated by OH- (alkaline/neutral)
induces ZrfC and AspF2
represses ZrfA and B
virulence determinant

51
Q

AspF2

A

sevcreted Zn-binding protein -> Zincophore
interacts with ZrfC
induced by PacC in alkaline/neutral conditions
virulence determinant

52
Q

FetD

A

low affinity iron uptake

53
Q

FreB

A

involved in reductive iron uptake with FetC and FtrA

54
Q

FetC

A

iron oxidase
involved in reductive iron uptake with FreB and FtrA

55
Q

FtrA

A

iron permease
involved in reductive iron uptake

56
Q

TAFC

A

iron siderophore
secreted via ABC transporter
TAFC-Fe uptake via MirB
degradation via EstB

57
Q

EstB

A

TAFC-Fe degradation

58
Q

MirB

A

transporter for TAFC-Fe uptake

59
Q

FC

A

siderophore involved in iron storage

60
Q

CccA

A

trasnporter for Fe in Fe vaculoar storage

61
Q

sidA

A

inducing siderophore biosynthesis
virulence determinant

62
Q

HapX

A

regulator of Aspergillus iron homeostasis
represses iron cosuming pathways in low Fe
induces iron consuming pathways when HapX-Fe

virulence determinant

63
Q

SreA

A

regulator of Aspergillus iron homeostasis
represses iron acquisition pathways when SreA-Fe

64
Q

Ctr1

A

high affinity Cu trasporter of C. neoformans
induced by Cuf1 during Cu starvation

65
Q

Ctr4

A

high affinity Cu trasporter of C. neoformans
induced by Cuf1 during Cu starvation

virulence determinant for brain infections (brain = Cu starvation niche)

66
Q

CnMt1

A

C. neoformans
Cu binding, detoxifying
induced by Cuf1 during Cu-excess

virulence determinant in macrophageal phagolysosome and lung

67
Q

CnMt2

A

C. neoformans
Cu binding, detoxifying
induced by Cuf1 during Cu-excess

virulence determinant in macrophageal phagolysosome and lung

68
Q

Cuf1

A

Cu sensing TF in C. neoformans
induces Ctr1/4 in Cu starvation
induces CnMt1/2 in Cu excess

virulence deteminant

69
Q

MacA

A

TF in A. fumigatus for Cu regulation
induces for Cu uptake

70
Q

AceA

A

TF in A. fumigatus for Cu regulation
induces proteins for Cu detoxification

71
Q

CrpA

A

ABC transporter in A. fumigatus for Cu regulation
cellular export of Cu

72
Q

innate immune evasion of fungi

A

conidia: rodlet layer and melanin masks cell wall, protects against acidification

73
Q

Innate immune defence against fungi
PRR

A
  • DECTIN 1: rec. beta-glucan -> A.f. spores, not mucormycotes
  • DECTIN 2: rec. alpha-mannan -> cell wall
  • DC-SIGN: rec. mannose-type carbohydrates
  • TLR2&4: rec. mannans -> outher layer of A.f., conidia
  • Ptx3: pentraxin, soluble PRR and opsonin for conidia, not pulmonar expressed
74
Q

Dectin 1

A

CLR type II
recognizes beta-glucan
A.f. spores, not mucormycotes

75
Q

Dectin 2

A

CLR type II
recognizes alpha mannan -> cell wall

76
Q

DC-SIGN

A

recognizes mannose-type carbohydrates
PRR against fungi

77
Q

Ptx3

A

pentraxin
soluble PRR and opsonin
rec. conidia
no pulmonary expression

78
Q

innate cellular defence against fungi
mechanism and cells

A

phagocytosis or direct killing

epithelial cells
alveolar macrophages
NEUTROPHILS: key effector cell -> direct kill conidia and hyphae (neutropenia major risk factor)
DC uptake and presentation activates adaptive IS

79
Q

innate defence against airborne fungi/spores

A

mucocilliary clearance
pulmonary macrophages
activation of PMN -> NETosis
DC activating T cells

80
Q

adaptive defence against fungi

A

Ab inefficient!
(neutrophils as key effector cell)
helped by TH1 and TH2

81
Q

primary resistance
definition, example

A

intrinsic resistance
target of drug is low/not expressed in the organism

E.g. A. terreus resistant to ROS stress -> AmpB resitance
C. neoformans have low b-glucan content -> echinocandine resistance

82
Q

secondary resistance
definition, example

A

acquired or developed resistance under the application of an antifungal in sub-lethal concentration via selective pressure

e.g Azole resistance via point mutation in CYP51A or 34mer duplication

83
Q

biofilm in resistance

A

mechanism of drug resistance
- lowers accessibility
- lower ergosterol content
- drug efflux elevated
- elevated stress response genes (via calcineurn)

84
Q

causes for fungal resistance

A

usual adaptations to the environment (less common geneic) -> metabolic/TK changes

GENTICS:
- mutator strains (e.g. DNA repair defect)
- chromosmal aneuploidy
- parasexual or sexual reproduction
- horizontal gene transfer

85
Q

genetic causes for fungal mutations

A
  • mutator strains (e.g. DNA repair defect)
  • chromosmal aneuploidy
  • parasexual or sexual reproduction
  • horizontal gene transfer
86
Q

genomic hotspots for drug resistances

A
  • target proteins
  • drug importers
  • drug exporters
  • regulatory proteins
87
Q

Echinocandins
target, effect, efficac, resistance mechanism

A

target 1,3-b-glucan synthase (Fsk1 enzymes)

EFFECT: b-glucan is component in the CELL WALL, when decreased chitin content increases to maintain high intrracellular pressure, only temporal solution and osmotic sensitivity rises

EFFICACY: fungistatic against A. fumgatus, fungicidal against singel cell fungi

RESISTANCE: mutations in target enzyme -> hotspots are extracellular loop connecting TM domains that are important for drug binding

88
Q

target echinocandins

A

1,3-b-glucan synthase (Fsk1 enzymes)

89
Q

effect echinocandins

A

target 1,3-b-glucan synthase (Fsk1 enzymes)

EFFECT: b-glucan is component in the CELL WALL, when decreased chitin content increases to maintain high intrracellular pressure, only temporal solution and osmotic sensitivity rises

90
Q

efficacy echinocandins

A

EFFICACY: fungistatic against A. fumgatus, fungicidal against singel cell fungi

91
Q

resistance mechanisms against echinocandins

A

RESISTANCE: mutations in target enzyme (1,3-b-glucan synthase -> Fsk1 enzyme)
-> hotspots are extracellular loop connecting TM domains that are important for drug binding

92
Q

Polyenes

A

Amphotericin B

93
Q

amphotericin B
effect, side effects, resistance mechanisms

A

EFFECT: pore formation via 2 parallel domains and positive charge -> efflux of ions and later fluids (cell lysis), massive oxidative stress
- Polyene domain: binds ergosterol with higher affinity than cholesterol
- polyol domain: aligns with other polyoldomains and forms inner wall of pore

SIDE EFFECTS: not exclusive ergosterol-binding -> problems with esp liver and kidney, application in lipid vesicles or as combination reduces side effects

RESISTANCE:
- stress tolerance (e.g. Hsp and catalase, A. terreus)
- cell wall alterations reducing cell membrane access (e.g. biofilms)
- altered membrane sterole composition (intrinic)

94
Q

AmpB
effect

A

EFFECT: pore formation via 2 parallel domains and positive charge -> efflux of ions and later fluids (cell lysis), massive oxidative stress
- Polyene domain: binds ergosterol with higher affinity than cholesterol
- polyol domain: aligns with other polyoldomains and forms inner wall of pore

95
Q

AmpB
side effects

A

SIDE EFFECTS: not exclusive ergosterol-binding -> problems with esp liver and kidney, application in lipid vesicles or as combination reduces side effects

96
Q

AmpB
resistance mechanisms

A

RESISTANCE:
- stress tolerance (e.g. Hsp and catalase, A. terreus)
- cell wall alterations reducing cell membrane access (e.g. biofilms)
- altered membrane sterole composition (intrinic)

97
Q

Azoles
target, effect, efficacy

A

TARGET: ergosterol biosynthesis (cell membrane) -> Lanosterol (C14) demethylase -> CYP51A or ERG11

EFFECT: need to enter cell and be active during critical step
- inhibition of lanosterol demethylase
- accumulation of lanosterol
- incorporation of lanosterol instead of ergosterol -> toxic
- destabilization and disruption of cell membrane

EFFICACY: fungistatic -> resistance develpment possible and common!

98
Q

Azoles
efficacy

A

fungistatic –> resistance development!

99
Q

Azoles
target

A

ergosterol biosynthesis
inhibits lanosterol (C14) demethylase -> CYP51A or ERG11

100
Q

Azoles
effect

A

EFFECT: need to enter cell and be active during critical step
- inhibition of lanosterol (C14) demethylase -> CYP51A, ERG11
- accumulation of lanosterol
- incorporation of lanosterol instead of ergosterol -> toxic
- destabilization and disruption of cell membrane

101
Q

Azoles
resistance mechanisms

A
  • target mutation
  • target overexpression (TR34, HapE)
  • exporter overexpression (YAP1)
  • further processing of lanosterol

usually develop over time, especially agricultural usage leads to accumulation in harvest, animals and humans -> resistances develop

TR34: tandem repeat of 34mer (bp) sequence, is regulator of CYP51A via containing SrbA binding sites -> duplication or multiplication leads to increased target expression

HapE-P88L: Hap complex (CBC) composed of HApB, C and E -> repressor of synthesis by binding 34mer and directly regulating 4 enzymes in pathway -> mutation (lof) leads to target overexpression

YAP1-L588W: AA exchange in NES sequence of TF YAP1 -> stays nucear and increased expression of AtrF (drug exporter)

102
Q

TR34

A

azole resistance mechanism

TR34: tandem repeat of 34mer (bp) sequence, is regulator of CYP51A via containing SrbA binding sites -> duplication or multiplication leads to increased target expression

103
Q

HapE-P88L

A

azole resistance mechanism

HapE-P88L: Hap complex (CBC) composed of HApB, C and E -> repressor of synthesis by binding 34mer and directly regulating 4 enzymes in pathway -> mutation (lof) leads to target overexpression

104
Q

YAP1-L588W

A

azole resistance mechanism

YAP1-L588W: AA exchange in NES sequence of TF YAP1 -> stays nucear and increased expression of AtrF (drug exporter)

105
Q

nucleoside analogs
effect, resistance mechanism

A

EFFECT: incorporation influences DNA, RNA and protein metabolism
- uptake of 5FC via fcyB -> cytosine permease
- fcyA -> cytosine deamnase to 5FU (not present in humans)
- uprt -> to 5FUMP

RESISTANCE:
- intrinic: low expression of fcyB in neutral conditions (high at pH5) via CBC (Hap-complex, repressor at pH7) and PacC (pH sensor, represses fcyB at pH7)
- acquired: deletions/lof for fcyB (prevented via AmpB combination), fcyA or uprt

106
Q

nucleoside analogs
resitance

A

INTRINSIC: low expression of fcyB in neutral conditions (high at pH5)
- CBC (Hap-complex, repressor at pH7)
- PacC (pH sensor, represses fcyB at pH7)

ACQUIRED: deletions/lof for
- fcyB (prevented via AmpB combination)
- fcyA
- uprt

107
Q

Combination therapy of fungal infections

A

timing is essential
depletion theory (azoles reduce target for AmpB)
enhancement theory (increased effiicacy of azoles via AmpB-increased access)

combination of 5FC with AmpB prevents rsistance via fcyB mutation -> pore formation of AmpB guarantees entry for 5FC, allows dose reduction for AmpB (reduced side effects)

108
Q

problems of in vivo models for fungi

A
  • Murine: intrinsic resistance, limited sampling
  • Chicken egg: not really (HTS, no ethical regulation till day 21, ideal temp)
  • Zebrafish: temp not well tolerated by larvae
  • invertebrae: ev. no thermotolerance (D. melanogaster), no cytokines or adaptive IS, route of infection not comparable, no defined protocols, strain lines or mutants, size can be problematic (D. melanogaster)
109
Q

Why are fungi an object of research?

A
  • medical research for fungi as pathogens
  • agricultural pathogen
  • bitechnology for fungi as producer of e.g. AB
  • model organism for fundamental research
110
Q

Benefits of model organism A. nidulans

A

50% of genes encode secondary metabolites
sequenced genome
GRAS
sexual and asexual cycle -> easy growth and crosss between strains possible

111
Q

Types of generated mutations in fungi

A

CONVENTIONAL: caused by physical or chemical agents
- no transfection/transformation -> not classified as GMO
- random and not directed -> screening of 1000s

GENETIC MANIPULATION:
- transformation/transfection of organism -> GMO
- in coding or regulatory sequences
- targeted deletions, truncations, point mutations, promotor fusion, tags for purifications, flourescence, …

112
Q

generating a deletion mutant fungi

A

replacement of a target sequence with a maker via HR
HR rare in fungi -> increased by bigger size of homologous sequences, deletion of NHEJ genes (ku70, ku80)

efficacy of uptake is low -> selection markers

113
Q

Transformation (DNA uptake) strategies in fungi

A

transformation requires removal of cell wall and cell embrane
- Electroporation
- protoplastation (enzymatic removal in hyposomotic conditions)
- biolistic (gene gun -> metal particles coated with DNA)

114
Q

Knock down strategies

A
  • insertion of recombinant promotor that is suboptimal for GOI
  • 2 promotor system: one inducible promotor initiates full GOI, others (inducible) initiate only fragments
115
Q

positive vs negative selectable markers

A

positive: growth on selection media is possible if the marker is present
negative: growth on selection media is possible if the marker is absent

116
Q

positive selectable markers

A

auxotrophy markers:
- auxotrophic reciptien strain (PyrG-)
- PyrG + GOI transfer
- selection media = depleted of uracila and uridine

Drug resistance marker:
- prototrophic recipient strain
- hph + GOI transferred
- selection media contains hygromycine

117
Q

negative selectable markers

A

used for marker excision -> recycling of markers
- insert = GOI + FRT-hdh-cd-FRT
- positive selection via hygromycine
- excision of hdh and cd (fcyA) via FRT
- negative selection via 5FC

ENDOGENOUS negative selectable markers:
- genes preventing growth in certain conditions -> e.g. 5FC
- replacement of fcyA with GOI via HR
- selection media containing 5FC kills all with functional fcyA
- limited number of transformations

118
Q

Reporter
usage

A

used for:
- multicolour imaging: organism contains specific promotor coupled with flourescent reporter (e.g.)
- subcellular compartment label: transport sequence coupled to reporter -> MTS for mitochondria

119
Q

Reporter
types

A
  • colorimetric reporter: induces colour change (e.g. lacZ) -> indirect quantification possible
  • bioluminescent reporter: e.g. promotor analysis by coupling o luciferase genes
  • flourescent proteins
120
Q

promotor
types

A

CONSTITUTIVE: from housekeeping genes, could generate toxic concentrations!

INDUCIBLE: better growth and reduced toxicity -> PxylP, PTet-ON, PTet-OFF

121
Q

promotor analysis

A

replacement of original gene with reporter -> reports promotor activity
- mRNA/protein level detection
- flourescent reporters: HIGH background (cheap, real time, longtime)
- colorimetric reporter: outdated (cell lysis and substrate necessary)
- bioluminescent reporter: LOW background, long-time difficult (substrate often expensive)

122
Q

screening for positive transformants

A

selection media
PCR: primer placement is essential
Southern blot: also shows multiple integrations!

PM3 - Infection and immunity (9 decks)

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