Lecture C2 + C3 - The WHO Designated Fungal Critical Pathogens

Question 1

What are the 3 groups of the fungal priority pathogens list?

Answer 1

Critical group
High group
Medium group

Question 2

What are the 4 critical priority fungal pathogens?

Answer 2

Opportunistic pathogens.
Candida albicans
Candida auris
Cryptococcus neoformans
Aspergillus fumigates

Question 3

In what host can critical priority fungal pathogens infect?

Answer 3

Immunocompromised hosts - opportunistic.
Dramatic rise seen during the HIV epidemic.

Question 4

What is the survival rate of the critical fungal pathogens and why?

Answer 4

Low, 40-50% survival rate following systemic infection.
Present with an infection such as sepsis and get given antibiotics that don’t treat the fungal disease and gets rid of the bacterial competitors so fungus can thrive more.

Question 5

Describe Candida albicans.

Answer 5

A commensal yeast.
Fungus in the gut can bypass the dampened mucosal defences and get into the blood stream and compromise almost every anatomical area of the host causing organ failure and then death.

Question 6

What are 4 key virulence determinants?

Answer 6

Adhesins
Morphogenesis
Secreted enzymes
Stress responses

Question 7

Why isn’t most fungi pathogenic to humans?

Answer 7

Most fungi can’t survive at 37 degrees so can’t survive at human body temperature.

Question 8

Describe adhesins.

Answer 8

Cell wall proteins that promote binding to host cells.
Low complexity C-terminal highly glycosylated Ser/Thr rich region extends the adhesion into the environment allowing the N-terminal ligand binding domains to swivel and interact.
N-terminal contains Ig like domains which form a cavity to bind peptides with broad specificity.

Question 9

What are 3 examples of adhesins?

Answer 9

Als family (Als 1-9) - endothelial and epithelial cells, abiotic surfaces.
Hwp1 family (5 members) - saliva coated surfaces, epithelial cells.
Iff family (12 members) - epithelial cells, plastics.

Question 10

What are biofilms?

Answer 10

Microbial communities that develop in association with a surface.
The adhesins Hwp1 and Als3 are essential for biofilm formation.

Question 11

Describe morphogenesis.

Answer 11

Most widely studied virulence trait.
C. albicans capable of reversible morphogenetic switching between budding and filamentous forms.
Filamentous form plays key roles during different stages of the infection process - access to the bloodstream, macrophage escape and infection of internal organs.

Question 12

Describe macrophage escape.

Answer 12

The yeast cells are taken up by the macrophages but once it is in the macrophage, they change shape and form filanments that pierce the macrophage membrane, killing it and allowing the fungus to escape, infecting other tissues.

Question 13

How does morphogenetic switching occur?

Answer 13

Requires the induction of hyphae-specific genes.
MAPK pathway responds to serum or nitrogen starvation.
PKA pathway responds to amino acids, high carbon dioxide, acidic pH and muramyl dipeptide.

Question 14

What are positive regulator signalling pathways for morphogenesis?

Answer 14

Efg1 pathway - Efg1 transcription factor regulated by cAMP dependent protein kinase A (PKA) pathway.
Cph1 pathway - Cph1 activity controlled by a MAP kinase cascade.

Question 15

What are negative regulator signalling pathways for morphogenesis?

Answer 15

Rfg1 and Nrg1 - DNA binding proteins recruit the Tup1 transcriptional repressor to hyphen specific genes.
If the negative regulators are deleted, then the cell becomes constitutively hyphal.

Question 16

How is filamentation linked to virulence?

Answer 16

UME6 is an important regulator of hyphal specific genes.
When placed under control of a promoter that was inhibited by the presence of DOX in the media.
Using this strain, UME6 was expressed at high levels in the absence of DOX but repressed in the absence of DOX.
UME6 expression can trigger filamentation under non-inducing conditions.

Question 17

Describe the experiment performed to link filamentation to virulence.

Answer 17

The tetO-UME6 strain was injected into mice.
Drinking water containing DOX or not containing DOX given to the mice.
In the mice given drinking water with DOX, infection was suppressed as UME6 expression prevented, by day 11 80% survival.
In the mice given drinking water without DOX the infection is not suppressed and UME6 expression was increased, by day 11 survival rate was 20%.
This means stimulating filamentation during infection can promote the virulence of the infection.

Question 17

How can morphogenesis and tissue damage be linked?

Answer 17

How can hyphae but not yeasts damage human epithelial cells.
Hypha-specific genes include adhesins (Hwp1/Als3) which allow epithelial cell binding.
ECE1 is the most highly expressed gene in hyphae and the resulting protein is processed onto 8 peptides.
One peptide - candidalysin - is a pore forming toxin that damages the epithelium cells and induces inflammatory responses.

Question 18

Describe secreted enzymes.

Answer 18

Fungi have to release enzymes to survive.
Proteinases degrade host proteins.
Phospholipases degrade host lipases releasing lipid and phosphate groups allowing for active penetration into the cells, tissue invasions and damage, extracellular nutrient acquisition and virulence.

Question 19

Describe stress responses.

Answer 19

Stress responses and nutrient acquisition strategies are needed for C. albicans to thrive in numerous different microenvironment within the host and counteract host immune defences.
Hog1 stress activated protein kinase is a central stress protector for the years cell,
If Hog1 is deleted from the Canidia then survival rate is less than wt.

Question 20

What are all required for virulence?

Answer 20

Stress activated protein kinase Hog1
Catalase
Superoxide dismutases Sod and Sod5
Trehalose biosynthetic enzymes Tps1 and Tps2
pH responsive factors
Phosphate and metal acquisition enzymes

Question 21

Describe candida auris.

Answer 21

Rapid emergence and spread across the world.
Transmitted amongst groups of patients - a communicable disease unlike other fungal disease.
No treatment as resistant to anti fungal treatments.
5 distinct geographical clades that have simultaneously emerged.

Question 22

How did C. auris simultaneously emerge across continents?

Answer 22

Global warming.
Species has adapted and been able to grow at higher temperatures.

Question 23

What do the key virulence factors promote?

Answer 23

Adhesion to host cells (adhesins)
Invasion and damage of tissues (morphogenesis, secreted enzymes)
Adaptation to host environments and evasion of immune defences (morphogenesis, stress responses)

Question 24

What are the key facts for cyptococcus neoformans infections.

Answer 24

Causes disease in immuno-compromised individuals.
Spores inhaled.
Yeast can enter a dormant phase.
When host immunity compromised yeast disseminates to cause systemic infection.
Can infect CNS, skin, eyes and bone.

Question 25

What is the most common CNS infection caused by fungi?

Answer 25

Cryptococcal meningitis

Question 26

What type of yeast is C. neoformans?

Answer 26

Environmental saprophytic.

Question 27

How does C. neoformans have virulence factors and infect humans?

Answer 27

Spores allow C. neoformans to get into the lungs.
Poor immune system in the host means the spores/yeast does not clear.
The physical characteristic of yeast allowing it to survive in the environment also promotes survival in humans.

Question 28

What are the 5 keys virulence factors of C. neoformans?

Answer 28

Capsule
Melanin
Secreted enzymes
Stress responses
Titanization

Question 29

What is the C. neoformans capsule?

Answer 29

Polysaccharide capsule comprised of 2 large glycans GXM and GXMGal.

Question 30

How does the capsule of C. neoformans promote virulence?

Answer 30

Protects it from phagocytosis
Modulates immune response
Acts as a free radical sink.

Question 31

How does melanin promote C. neoformans virulence?

Answer 31

Brown pigment providing UV protection.
Antioxidant that protects from toxic free radicals (phagocyte survival).
Produced by laccase enzyme.

Question 32

How do secreted enzymes promote C. neoformans virulence?

Answer 32

Degradative enzymes like lipases, proteases and nucleases allow for nutrient acquisition, tissue degradation and immune evasion.

Question 33

What is considered a major cryptococcal virulence factor and why?

Answer 33

Urease allows for the C. neoformans to cross the BBB.

Question 34

How do stress responses aid in C. neoformans virulence?

Answer 34

Deletion of oxidative and nitrosative stress detoxifying enzymes attenuates virulence.
SOD required for growth in macrophages, expression is increased at 37 degrees.
Hog1 SAPK promotes reisstance to stressors encountered in the host and the environment.

Question 35

What are titan cells?

Answer 35

Large cells that can be up to 100um in diameter.

Question 36

How do titan cells promote C. neoformans virulence?

Answer 36

Size prevents clearance by macrophages and resistant to a wide range of stresses.

Question 37

Describe aspergillus fumigatus.

Answer 37

A saprophytic fungus growing on dead/decaying matter.
Produces conidia that are easily dispersed.
Conidia are inhaled and cleared by alveolar macrophages in healthy individuals.
In immunocompromised hosts the conidia germinate and hyphae grow and disseminate causing IA.

Question 38

What is invasive pulmonary aspergillosis?

Answer 38

Characterised by the dissemination of fungal hyphae in the lung and surrounding tissue.
Only happens in immunocompromised hosts.
760K-2.2m cases a year.
Symptoms are non-specific.

Question 39

Describe resting (dormant) conidia.

Answer 39

Layer of RodA proteins organised as rodlets which can shield PAMPs from immune recognition.
Layer of melanin to provide oxidative stress protection and shield PAMPs.
FleA and silica acid modified glycan chains mediate adherence onto pulmonary epithelial cells.

Question 40

Describe swollen conidia.

Answer 40

Swelling releases rodlet and melanin outer layers.
Exposes PAMPs to interact with PRRs.
CalA invasion protein interacts with integral a5B1 on host cells inducing endocytosis.
CalA required for maximal host invasion.

Question 41

Describe evasion and germination of A. fumigatus.

Answer 41

Swollen conidia that evade killing terminate into hyphae.
GAG binds to the surface of the hyphae resulting in a polysaccharide sheath forming an extracellular matrix between hyphae.

Question 42

What are the 5 roles of GAG during A. fumigatus infection?

Answer 42

Efficient adherence of hyphae to host.
Immune evasion.
Induces apoptosis of neutrophils.
Promoters resistance against neutrophil extracellular traps (NETs).
Drives T cells to mount non-protective roles.

Question 43

Describe destruction in A. fumigatus.

Answer 43

Hyphal for promotes tissue degradation and destruction via gliotoxin and fumagillin.
Enzymes like cysteine and serine proteases degrade the host epithelial tissue and generate nutrients to support fungal growth.

Question 44

What does gliotoxin do?

Answer 44

Induces apoptosis in macrophages and monocytes

Question 45

What does fumagillin do?

Answer 45

Cytotoxic - clears lymphocytes and destroys pulmonary tissue.