MICR3001 – Fungus

Question Answer
What role do fungal metabolite play in medicine? Play a significant role in pharmaceuticals, ranging from antibiotics to cholesterol lowering statins
Why are fungi good model organisms part 1 Easy to grow, short life cyclesSmall genome relative to other eukaryotesMost fungal genes are homologous to those in other eukaryotes
Why are fungi good model organisms part 2 Most have haploid genomes amenable to mutation They can undergo asexual replication
What are the different types of pathogenic fungi? The pathogenic fungi can be moulds (filamentous) and yeast (unicellular) and dimorphic fungi that can transition between both
What are the three fungal disease types? Superficial and cutaneous mycoses -> skin, hair, nail, mucous membranesSubcutaneous mycoses -> skin, subcutaneous tissue, lymphaticsDeep seated mycoses -> single deep organ, disseminated
What are four examples of systematic mycoses? HistoplasmosisCoccidiomycoses ParacoccidiomcycosisBlastomyoses
Why are yeast cells difficult to treat Homologous to humans
What is the causative species of histoplasmosis? What type of fungus is it. Where does it survive. Are there more than one species, where is it found? What does it have an affinity for, does the infection confer immunity? Histoplasma capsulatumThermally dimorphic haploid fungus -> adopts yeast form in tissuesSurvives within phagolysosomesSeveral species around the worldaffinity for high nitrogen levelsi confers immunity but disease can be reactivated
What are the symptoms of histoplasmosis and what is its route of transmission? Inhalation of conidia. Can cause mild flu like diseaseSerious pulmonary infectionDisseminated (often fatal) infection, can include skin
Why do people who visit caves frequently get histoplasmosis Bat guano contains high levels of NaN03, which promotes growth of microconidia
What is the causative agent of Coccydiomycosis (valley fever). What type of fungus is it, how many species are there, where is it found? Coccidioides immitis and posadasiiA thermally dimorphic haploid fungus that adopts a yeast form in tissues to generate large spherulesRecently recognised as two speciesSouth East Asia, Central America, South America
Describe Coccydiomycosis. What diseases does it cause, how virulent is it? What is its route of infection Most virulen fungusCan cause mild flu like symptoms, serious pulmonary infections or it can disseminate into CNS and skin.Inhalation during sandstorms, relapse is common
What type of people are more susceptible to Coccydiomycosis? Pregnant women, dark skinnned individuals and the immunocompromised
What is the causative species of Paracoccidiodomycoses? What type of fungus is it, where is it found? Thermally dimorphic haploid fungus that adopts a yeast form with multiple buds in tissues.Found exclusively in south america
What diseases can Paracoccidiodomycoses cause? Who is more susceptible to the disease? Mile flu like infectionSerious pulmonary infectionDisseminated infection with skin involvementMales are infected 13-87 times more often than femalesInhalation of spores, reactivation
What is the causative agent of Blastomycosis? What type of yeast is it, where is it found? A thermally dimorphic haploid fungus that adopts a yeast form in tissues, may be a subspecies of H. capsulatumFound in central and South East USA, parts of Africa and India
What types of diseases does Blastomycosis cause, what is its route of transmission Mild flu like infectionSerious pulmonary infectionDisseminated infection with skin involvementInhalation o
What are four typical feature of dimophic fungal pathogens? Primary filamentous in nature, but adopt a yeast form in tissuesUsually geographically restrictedClinical manifestation mild flu respiratory infection through to disseminationm often involving skinroute of infection is usually inhalation
C.elegans mutants who have defects in dorso-ventral axis have mutations in what genes? What exactly is this mutation? Unc-6 and Unc-40. THe AVM mechanosensory neuron does not reach ventral cord, instead it runs directly anterior to the head
What does a penetrance of 30% mean? The phenotype is present in 30% of the organisms
How can you test whether two genes act in the same pathway (axon guidance – C.elegans) Double mutants should have the same phenotype and an increased penetrance
Do unc6 and unc-40 act in the same pathway to control dorso-ventral axon guidance in C.elegans? Yes they do. The defect of the double mutant unc-6 and unc-40 is not worse than the individual mutants. The penetrance of the double mutants was higher as well. Therefore they operate in the same genetic pathway
What type of molecule is Unc-6 (C.elegans) A secreted molecule that is attractive. Controls ventral guidance of AVM. Homolog of netrin
How can you tell whether Unc-6 is an instructive or a permissive cue? Ectopically express UNC-6. Choose promotor expressed dorsally to express UNC-6. If rescue = permissive, no rescue= instructive, phenotype should be worse as axon should be attracted dorsally
Is unc-6 and instructive or permissive cue? (C.elegans) It is an instructive cue
What type of molecule is unc-40 (C.elegans) UNC 40 is a transmembrane receptor for UNC-6. THese molcules are attractive cues, that mediate the ventral guidance of AVM . Homolog of DCC
How could you tell whether unc-40 is autonomously required to mediate the ventral guidance of AVM (C.elegans)? Take a mutant for UNC40 and express WT copy UNC-40 selectively in AVM. Rescue: cell autonomous, No rescue – non cell autonomous
Is Unc-40 required cell autonomously? yes it is
In unc-6 and unc-40 mutant animals only a proportion (about 30%)of the animals have a defective AVM axon that cannot reach theventral cord.What is guiding the AVM axon in the 70% of the animalswith a normal phenotype? How did they find these molec? They performed another genetic screen and found the molecules slt-1 and its receptor sax-3(ROBO) induced the same defect as unc-6 and unc-40 mutations
Do slt-1 and sax-3 act in the same genetic pathway? What type of guidance molecules are slit-1 and sax-3? Yes double mutant phenotype same as individual, penetrance increased. SLT-1 is expressed dorsally and Sax-3 is on the AVM axon, thereforethey mediate ventral guidance of AVM via repulsion
Do UNC-6 and SLT-1 act in parallel pathways in regulating AVMdorso-ventral axon guidance? Double mutants (KO one molecule from each pathway) had stronger defects than the individual mutants. Therefore. UNC-6/UNC-40 and SLT-1/SAX-3 act in parallel to regulate the dorso-ventral guidance of the AVM axon
What controls the posterior-anterior axis of the AVM mechanosensory neuron? Wnt molecules and their receptors Frizzleds.
How many Frizzled receptors/Wnt molecules do you need to KO to see an anterior guidance defect in AVM? Why is this? Two. Double mutant analysis revealed that Wnt signals andFrizzled receptors regulate AVM anterior-posterior axonal guidance. Single mutants – no defect. Double – 25-30% penetrance
Through what receptors do unc-6 and unc-4through which
What is the most common fungal pathogen for humans? Candida albicans
What is the causative agent of Candidiasis? What type of yeast is it, describe its sexual cycle. Where does it infect? Most common is Candida albicans. Diploid yeast, no sexual cycle. Can grow in yeast, pseudohyphal or true hyphal form in response to environmental stimuli. Infects mucosal surfaces
What is the difference between yeast cells, pseudohyphae and true hyphae. Yeast – round to oval, Readily seperatePseudophyphae -> remain attached to each other. Elongated, ellipsoid cellsTrue hypahe -> characteristic of moulds, highly polarised cells. No obvious constrictions between cells
What types of disease can Candidiasis cause? Oral thrush, vaginitis, skin infections, nail infections, UTIs, oesophageal infections, disseminated infection in seriously immunocompromised patients
Describe quorum sensing and growth forms The swithc between yeast, pseudophyphae and true hyphae is controlled by enviornmental signals including pH, serum, Co2, temp.Quorum sensing: Small mol farnesol is secreted when culture grows to saturation,. Represses growth and formation of hyphae
Is candida albicans usually pathogenic? No it is a commensal fungus. 80% of pop have this yeast in normal flora
How is Candidiasis often acquired? Often nosocomially acquired, readily forms biofilms on cathethers. Infection common after anti-biotiocs as bacterial competition is absent
Describe the four stages of Candidiasis infections? Adhesion and colonisation of mucosal surfaceEpithelial penetrationVascular disseminationEndothelial colonisation and penetration
What are the four virulence factors of Candida Albicans? Phenotypic switching – enable adaption to microenviornmentsPleomorphism – hyphae aids in invasion of host barriers, biofilmsS adhesins – ALS and integrin family -Hydrolytic enzymes – aspartyl proteinases phospholipases degrade tissue and defences
The ability of C. albicans to switch growth form enables it tonot only form biofilms but but also to escape from macrophages(not neutrophils).
Where else can you find C.albicans. Can it be passed from human to human Nowhere else other than humans. Yes human to human transmission is possible
In general what are fungal virulence factors? Adhesion to host surfacesBiofilm formationinvasion of epithelial surfacespenetration beyond epithelial opposition to host defences
What are the four drug targets in fungi? Affect membrane function, cell wall synthesis, ergosterol biosynthesis, nucleic acid synthesis
How does the anti-fungal polyenes act? cell membrane ergosterol binding, causes lysis. Provide an example.
What is the problem with diagonising Candida albicans Most people assume the fungi is candida albicans, not a safe assumption anymore. Could be due to other species
What is better at penetrating tissue? Yeast or hyphae. What is better for travelling in blood stream hyphae. Yeast
Describe how Amphotericin B work as an anti-fungal. What type of anti-fungal is it, what is its MOA. Describe its potency, spectrum, side effects It is a secondary metabolite of Streptomyces nodsusIt is a polyene -> associates with ergosterol to form transmembrane channel ->lysisMost potent anti-fungal. Fungicidal. Broad spectrum. Renal side effects
Describe the anti-fungal flutocytosine? HOw doe it work? It is a pyrmidine analogue. A prodrug that is converted into fluorouracil by cytosine deaminase (absent in humans). Inteferes with nucleic acid synthesis.
Describe the anti-fungal flutocytosine? What infections is it used for, how effective is it, is there resistance? LImited efficacy against moulds. Synergism with Amphotericin B. Used for C.neoformans and C.gattii infections only. Resistance is rapidly emerging during monotherapy in 80s
Describe the anti-fungal Fluconozale. What is it and what is its mechanism of action. A synthetic azole. Inhibit P450-dependent ergosterol production (by binding to it). Targets ERG 11 gene.. It is fungistatic
Describe the anti-fungal Fluconozole. What is its spectrum, side effects, which infections are intrinsicially resistant Borad spectrum of activity, side effects are infrequent. Candida krusei and candida glabrata are intrinsically resistant
What factors contribute to fungal resistance Long term exposure and low concentrations such as prophylaxis, patient non-compliance
describe how resistant fungal strains from AIDS patience have become resistant to fluconazole Targert of fluconazole is ERG11. Strains isolated from AIDS patients have duplications associated with left arm of chromosome 5, wher eyou find ERG11
What are the three most common pathogenic fungi? Candida albicans, cryptococcus neoformans and aspergillus fumigatas
is resistance to anti-fungals a problem GPs giving anti-fungals out to readilyPatient non-compliance -> selects for resistant strains
Why does candida albicans exhibit higher genomic plasticity than other fungals? It is diploid
Resistance to fungals typically occur drug export and import mechanisms
Is resistance to anti-fungals acheived through horizontal gene transfer like in bacteria? No it occurs in a case by case basis to due evolution and presence of anti-fungals (and intrinsic resistance)
What is the most common pathogenic fungus and mould Fungis -> candida albicansMould – Aspergillus fumigatas
which phylum does aspergillus fumigatas, the endemic mycoses and candida albicans fall under? Ascomycota
Describe aspergillus species in the environment. What is there role, are they common where do they reach, what is the most common pathogenic species. Saprophytic moulds, recycle carbon and nitrogen. Common in the environment, frequently inhaled but easily eliminated. Ubiqutous, found everywhere in the world. Small enough to reach alveoli. Aspergillus fumigatas
Describe the transmission of A. fumigatus The conidia are inhaled, which germinate into hyphae in the lungs (not yeast). it is angioinvasive
What 5 clinical manifestations can A. fumigatus cause? Superficial mycoses, angioinvasion, vascular damage, invasive pulmonary aspergillosis (lesions), cerebral aspergillosis (lesions)
When does A.fumigatus disease occur? Pulmonary macrophages engulf conidiaNeutrophils attack escapes hyphae. When both of these are down, disease occurs
The increase in A.fumigatus in the 1980s and 1990s is caused by… New intensive chemotherapy, increase in organ transplantation, immunosuppressives, AIDS
Are there any A. fumigatus strains with increase virulence? No -> most infections caused by single genotype.
What are the diagnostic problems with A. fumigatus? No single test sensitive or specific enough for diagnosisvariable and non-specific presentation -delay in initiation of therapy often fatal -> grow so fast
Why can A. fumigatus spread so well Asexual spores blow in the wind everywhere and germinate
What is the most potent anti-fungal Amphotericin B
How does amphotericin B affect aspergillosis not that well
Which drug do they use to treat A. fumigatus? Are there strains resistant to this drug? Voriconazole (more useful than Fluconazole). Yes but the resistant strains were found to be a cryptic species Aspergillus lentulus
What are the 4 virulence factors of A. fumigatus? Adhesins – can adhere to many host proteinsPigments -> asexually produce condidia are dark green -> protectiveHigh temp growth -up to 45Toxins -> produces gliotoxin, which is potent and has immunosuppressant properties
How do you show that A. fumigatus gliotoxin is a virulence factor? part 1 KOCH's postuoates The phenotype must be associated with pathogenic members of a genus or pathogenic strains of a speciesInactivated of genes associated with virulence should reduce pathogenicity/virulence
How do you show that A. fumigatus gliotoxin is a virulence factor? part 2 Complementation of mutated gene should lead to restoration of pathogenicity (complementation)Gene which causes virulence must be expressed during infectionImmunity must be protective (anti-bodies produce against factor)
A. fumigatus infections are most common in cancer and organ transplant patients
What phylogeny does cryptoccocus neoformans belong to? Basidiomycota.
What are the causative species of cryptococcus? Where are they found, which is more common Haploid basidomycete yeasts that preferentially disseminate to CNSCryptoccoccus neoformans and cryptococcus gattii.Neo-formans found around the world in association with pigeon guano. gattii in the tropics
What diseases can C. neoformans and cryptococcus gattii cause? What is their route of entry? Cutaneous infection, respiratory infection (pneumonia), meningoencephalitis (fatal if untreated)Disease can be reactivated, relapse commoninhalation of sexually produced bassidospores or dessicated yeast
What type of yeast is C neoformans? It is really common in? What distribution does it have? What does it cause? Haploid basidiomycete yeast. worldwide distribution. Most common life threatening fungal infection in AIDS patients. Causes systemic mycoses ->commonly meningoencephalitis
What are the virulence factors of C. neoformans? Capsule -> interferes with phagocytosis by macrophages, protects cell from dessicationMelanin -> protects against toxic radicals, UV radiationUrease ->Promotes sequestration in microcapillaries, converts urease to ammonia
Why do cryptococcus makes good models? Easy to cultureHaploid, amenable to molecular studiesdefined sexual cyclegene deletions easily created5 genomes sequencedrobsust animal models
What is the treatment of cryptococcus neofromans for AID patients? Induction therapy: First diagnosis, lasts 6-10 weeks. Combination therapy of Flucytosine and amphotericin BMaintenance Therapy: after induction. lifelong, Fluconazole
THe dimorphic species like to disseminate where, cryptococcus neoformans likes to disseminate where, candida albicans, A, fumigatus skin, CNS, blood stream and liver, lung
What treatment complications exist for cryptococcus neoformans? Delay in initial diagnosis,Elevated intracranial pressure and associated manifestationsThe presence of enlarging or new cryptococcomas during therapyprolonged treatment timesrelapse (sometimes due to non-compliance)
What are cryptic species Different species, that mimic each other, Important for treatment and epidemiology
Which causative species of cryptoccocus prefers to cause pneumonia and which species prefers to cause meningoencephalitis pneumonia – gattiiMeningoencephalitis – neoformans
Describe relapse and karyotypic change If patients survive initial infection, 80% suffer relapse within six monthsIsolates from different stages of infection often display gross chromosomal rearrangmentsTranslocations, duplications, inversions and deletions may provide selective advantage
pigeon guano is rich in nitrogen
What are the 5 techniques to transform fungi part 1 Electroporation – electrical field creates transient pores, enabling polar molecules (DNA) to enterLithium acetate -> competent cells are induced to take up DNA following addition of PEG, heterologous carrier DNA and heat shock
Describe cryptococcus and cryptic species part 1 Initially thought there was n, Through analysing morphology and later sequencing, found out there were neoformans had 4 distinct molecule types and a second specie Gatti with 2 distinct molecular types. Up for debate whether MT are 6 different species
What proportion of AIDS patients will be infected with cryptococcus neoformans? 40%
Describe cryptococcus and cryptic species part 2 95% of isolates were C neoformans v grubbi (immunocompromised), 5% c neoformans v neoformans (found in europe not in aus). Less than 1% gatti – immunocompromised, endemic to Aus
What are good examples of cryptic species? Candida species which do not respond to fluconazole A. fumigatus that does not respond to Voriconazolecryptic species of endemic mycoses that show very distinct geographical distribution
why do they have to stop induction treatment after 6-10 weeks in c neoformans amphotericin B causes renal failure
Why has the rate of cryptococcus neofromans gone down? Crag lateral flow assay. Fluid sample from patient, stick in dip stick and it detects presence of cryptococcus capsule antigen
What does melanin protect against? Amphotericin Bmacrophage mediated phagocytosisnitrogen and oxygen derived oxidantsmicrobial peptidesultraviolet light
How did melanin fulfill the first of koch's postulates? Non pathogenic cryptococcus lacked melaninThe strains that reached the brain had melain -> melanin ghostTherefore gene is expressed during infectious process
How did they find the gene that encoded melanin production in cryptococcus neoformans knew from other species that laccase can create melaninsearched for laccase via chromatography (nowadays you would perform sequencing)Purified the LAC1 gene then performed southern blot
how does a southern blot work Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
What are the 5 techniques to transform fungi part 3 Protoplast-mediated fusion: following digestion of the cell wall,DNA uptake is driven by the presence of calcium ions and highconcentrations of PEG
What are the 5 techniques to transform fungi part 4 Agrobacterium: a Gram negative species of the Agrobacteriumgenus is used to transfer DNA horizontally into the fungal cell
What are the 5 techniques to transform fungi part 5 Biolistic: Gold or tungsten particles are coated with DNA and shotinto the cell under vacuum using a short burst of high-pressure helium
What is the common method of transforming c elegans biolistic method
How do you delete a gene? You need to create a construct that contains a marker for selection of transformed cells (marker cannot be in organism naturally – eithe rneed to create mutant or different species) flanked by regions of homology to the gene of interest
what are the benefits and cons of using an auxotrophic marker media is cheapgreater number availablecan be counterselectablerestricted to use in defined auxotrophic strainsmay affect virulence
what are the benefits and cons of using an dominant marker can be used in any strainunlikely to affect virulencemedia is expensivefew available
Compare gene disruption vs gene deletion part 1 Disruption -> selectable marker is inserted into gene of interest. Gene still there just interrupted. Constract can be made by transposition. Can create loss of function or partial. Old method of doing it
Compare gene disruption vs gene deletion part 2 Gene deletion – new waygene of interest is deletedconstruct easily made by PCRgenerates a guaranteed loss of function alelle
How do you confirm deletion southern blotting
knocking out of LAC 1 ablated what melain production and virulence but they didn't complement ot
geographical distribution between c immitis and c paradasii i – californiaparadassi – texas south america
What is the relationship between chocolate and candida kruiseii scrape kakou nibs out and place them in fermentation bins. Candida kruiseii associates with fermentation process and it is one of the most anti-fungal resistant candida species
What are the different types of antifungals part Polyenes – ergosterol binding, cell wall lysis Pyrimidine analogues Azoles – inhibit p450 ergosterol productionEchinocandinds – cell wall 1,3 glucan synthase inhibitors

Leave a Reply

Your email address will not be published. Required fields are marked *