Guest lecture Nov 18

Question 1

what is an invasive fungal infectino

Answer 1

when fungi invade the body in varioues ways - bloodstream or inhalation

*annual world wide deaths greater than 1.5 million

Question 2

what is cryptococcus neoformans H99

Answer 2

• yeast like basidiomycetous fungi

ubiquitous in nature: soil, avian habitats, diff tree species

• infects immunocompromised individuals
• causative agent of cryptococcosis: cryptococcal meningitis/meningoencephalitis

*greater than 223,000 cases occuring globally - resulted in approx 15% of aids related deaths

Question 3

what are the current anti-crytococcal therapeutics

Answer 3

target cell membrane include: Polyenes (amphotericin-B), Azoles (fluconazole)

Nucleoic acid synthesis: Pyrimidine analogue (5-flucytosine)

*recommended therapy is a combo of amphotericin and fluconazole

8 most countries that need this cant afford the combo, must use fluconazole for longer period of time - increases resistance to it

Question 4

decribe the infection cycle of C. neoformans

Answer 4

1. inhalation of desiccated cells
2. Colonization of alveolar spaces
3. Blood stream dissemination
4. Central nervous system invasion

Question 5

what is the rate of pulomary infection

Answer 5

• resident alveolar macrophages are the frist to encounter lung tissue

if immunocompetent: eradicate fungus/ maintain in a latent state

if immunocompromised: facultative intracellular replication and extracellular replication

^will disseminate in the blood stream to every region in body

Question 6

what is the main cause of death from this fungus

Answer 6

meningitis/meningoencephalitis

• it crosses the blood brain barrier to get to CNS

3 proposed mechanisms of how this occurs

1. paracellular: though tight junctions
2. transcellular: through endothelial cells via endocytosis
3. trojan horse: trafficking fungal cells ‘undercover’ within phagocytes (hitch a ride with naturally migrating immunecells)

Question 7

what are the virulence properties of neoformans H99

Answer 7

Polysaccharide capsule: antiphagocytic, antioxidant, immune modulation

*only pathogenic fungus that has this

Melainin: antiphagocytic, antioxidant, thermoprotection

thermotolerance: can adapt from ambient temp to host temp - this is the barrier that stands between us and many other pathogens

Extracellular enzymes: ex are urease (hydrolyse uria to ammonia), sueproxidase mutase, phospholipase

*enzymes are potential targets for new antifungals

Question 8

what are anti-virulence strategies

Answer 8

• virulence factors expressed by the pathogen overcome the host immune defense system and promite fungal survival and proliferation
• want to find inhibitor to block interactions so host system and clear and erradicate the fungus itself - this will disarm the fungus and not kill it

*decreases resistance

Question 9

what is the phDs students hypothesis

Answer 9

C. neoformans alters its proteomic profile and secretes new proteins in the presence of host cells as compared to in itro growth conditions influenceing its ability to survive and promote evasion of the host immune system

*found 35 fungal rpoteims demonstrated higher abundance during infections - possibly contributing to ifnection

Question 10

how are mutants constructed

Answer 10

via biolistic transformation - shooting DNA at fungal cells

• when fungus goes to repair its genome it will use homologous recombiantion and put in gene of interest - took out the other thing to makes a deletion

Question 11

macrophage cytotoxicity assay

Answer 11

• infect macrophages with C. neoforman strain let incubate then collect supernatant
• lactase dehydrogenase is in the supernatant and can be quantified - proportional to amount of cell death

Question 12

i didnt do half of it