MYCO | 1.3 Ultra- & Internal structures, 1.4 Fungal Growth

T/F: Mycelium to yeast conversions are just as common as yeast dimorphisms

FALSE

Mycelium-to-yeast conversions are not as common as yeast dimorphisms

_ is the most well-studied model for mycelium-to-yeast conversion triggered by heat shock/shift

Paracoccidioides brasiliensis

Mycelium to yeast conversion is usually a response to strenuous conditions, including _

• Heat stress (26-30 C to 37-40 C)

• Oxidative stress (e.g., putting dilute solution of hydrogen peroxide)

Apart from Paracoccidioides brasiliensis, what other species can exhibit mycelium to yeast conversion? Also, describe figure shown

• Time-lapsed transformation of Paracoccidioides brasiliensis hyphae to yeast form

  • Mycelium cultured in liquid medium was induced to undergo transformation from hyphal to yeast form by increasing temp from 26 C → 37 C

• Other species bhsp

  • Blastomyces dermatitidis

  • Histoplasma capsulatum

  • Sporothrix schenckii

  • Penicillium marneffei

T/F: The hyphal wall is composed of chitins, glucans, and different sugars and proteins

TRUE

Fungal walls are composed of _

1. Matrix components (sugars, i.e., mannan; B-1,3- and B-1,6 glucan that are embedded and linked to scaffolds of

2. Fibrous load-bearing polysaccharides (protein-bound chitin)

Explain figure shown

Variations in hyphal & yeast cell wall compositions

• General

  • Matrix components (mannan, B-1,3-, B-1,6)

  • Fibrous load-bearing polysaccharides (protein-bound chitin)

• C. albicans yeast = chitin, B-1,3- & B-1,6-glucans, mannan proteins

• Pneumocystis sp. = no chitin, B-1,3- & B-1,6, mannan (no outer chains)

• Aspergillus conidium = Melanin (UV protection), rodlets

• Aspergillus fumigatus hyphae = GAG, Galactomannan (with antigenic properties)

• Cryptococcus = Capsule (immune defense evasion), GalXM/GXM polysaccharides

• Histoplasma capsulatum, Blastomyces dermatitidis = a-1,3-glucan

_ catalyze chitin formation and transformation across the membrane

Chitin synthases

• _ is the first identified member of the fungal kingdom that does not have chitin in its cell wall

• Describe the figure shown

• Pneumocystis

• Smear of Pneumocystis carinii stained with toluidine blue

Why does Pneumocystis sp. have no chitin?

pin

• It’s because their parasitic lifestyle has adapted them to the nutrient-rich environment of their host tissues, where chitin’s rigidity would be unnecessary and could hinder flexibility.

• Additionally, the absence of chitin reduces immune recognition by host defenses and allows for a more efficient nutrient uptake, which is key to their survival within host’s lungs

Explain the figure shown

• Special wall of Pneumocystis sp.

• (A) Enzymes and accessory proteins (i.e., chitin synthase, chitinase) involved in chitin metabolism in fungi not found in Pneumocystis sp.

• (B) Gas chromatograms of partially methylated alditol acetates of P. carinii and S. cerevisae

• Terminal and 4-linked N-acetylglucosamine signals (T-GlcNAc, 4-GlcNAc) were detected in S. cerevisiae but not in P. carinii

Explain chitin formation to create septum

• Blue = plasma membrane

• Chitosomes deliver chitin synthases within secretory vesicles, fusing to plasma membrane

• Chitin synthases, once proteolytically activated, will start synthesizing chitin

• Chitin then lines the septum, allowing centripetal septal growth (from edge to center)

T/F: Chitin microfibrils provide flexibility to the fungal cell wall.

FALSE

Chitin microfibrils provide rigidity and structural strength to the cell wall, not flexibility. Flexibility is contributed by other components, such as glucans.

T/F: The centripetal growth of the septum in fungi refers to its expansion outward from the center to the edges.

FALSE

Centripetal growth refers to growth starting from the edges and moving inward toward the center E→C

T/F: Chitosomes are responsible for transporting glucans to the plasma membrane during septum formation.

FALSE

Chitosomes are responsible for transporting chitin synthases to the plasma membrane during septum formation.

T/F: The plasma membrane plays no role in septum formation.

FALSE

The plasma membrane is critical for septum formation as it houses enzymes like chitin synthase and coordinates the assembly of wall components.

T/F: The matrix component of yeast cell walls are generally similar across different taxonomic groups

FALSE

The matrix component of yeast cell walls varies per taxonomic group

Matrix components of yeast cell walls are critical for various life processes, its main roles include _

isrp

• Interaction with other organisms

• Survival and resistance to immune defense

• Prevention of parasitism (by other fungi)

Some infectious yeasts couple _ to resist phagocytosis; explain figure

• Cell size change with capsule formation

• Spore inhaled into a host shifts to budding,

  • Producing yeast cells of varying sizes and

  • Forming capsule to evade immune defense

2 types of extrahyphal matrix (capsule in cell wall) of yeasts

• Discrete polysaccharide capsule

• Diffuse polysaccharide / glycoprotein

_’s extrahyphal matrix (contains sugary capsule) masks the antigenic components of its cell wall (e.g., glucans) and can proliferate in tissues, inhibiting leukocyte migration and suppressing cytokine production

Cryptococcus

T/F: Cryptococcus is a vertebrate pathogen, while Mucor is a plant pathogen

TRUE

Usually spore → filamentous once inside host, but to evade immune recognition, Cryptococcus & Mucor couples cell size change with capsule formation to resist phagocytosis

T/F: Generally, fungal capsule does not trigger immune defense but fungal cell wall does

TRUE

_ creates a capsule only in liquid culture to defend itself against parasitism from other fungi; other _-causing species use this capsule to protect themselves against environmental stress

• Mucor

• Mucoromycosis-

How is mycelium-to-yeast conversion triggered in Paracoccidioides brasiliensis?

Heat shock/shift, specifically by increasing temperature from 26 C to 37 C

Fungi possess vacuoles of _ for multiple functions, existing in both yeast and hyphal forms

varied shapes and arrangements

4 main function of fungal vacuoles

spia

• Solute storage

• Protein turnover

• Ion homeostasis

• Apoptosis

T/F: Tubular vacuolar systems are unique to fungi

TRUE

Fungal vacuoles are similar to _

mammalian lysosomes & plant vacuoles

Fungal vacuoles have a wide variety of _ in different species, reflecting their ecological specialization

architecture & roles

Explain the figure shown

Pattern of vacuole distribution in yeast and filamentous fungi

• (A) Network of spherical and tubular vacuoles in many filamentous fungi, e.g., Aspergillus oryzae

• (B) Migratory cytoplasm in hyphae of C. albicans

• (C) Empty-looking vacuolated sub-apical compartments of Ustilago sp.

• (D) Oval/spherical vacuoles of S. cerevisiae

• (E) C. albicans vacuole segregation structure extending from mother to daughter cell during vacuole inheritance

• (D) Numerous small vacuoles of fission yeast Schizosaccharomyces pombe

_ is a family of enzymes that synthesize chitin

Chitin synthases (CHS)

Fungi have dynamic _ used for exocytosis

tubular vacuolar system

Explain the figure shown

Dynamic tubular vacuolar system of living fungal hyphae

• a) Hypha showing the presence of septum

• b) Same hypha showing narrow tubule passing through the septum and then branching

• c) Same hypha showing dilated vacuoles after tubules pass through the septum

• def) Hypha photographed at 8-sec intervals showing successive peristalsis-like movements causing vacuolar dilations to travel along hypha from left to right

Without _, growth in the hyphal tip is not possible

tubular vacuolar system that allows the peristaltic motion for the delivery of vesicles

Explain figure shown

• Vacuoles can either fuse together or elongate to create long vacuolar structures extending to hyphal tip

• Tubular vacuolar system, through the peristaltic movement it creates, essentially squeezes nutrients, allowing these to flow towards the apical tip and constantly supply materials SPK needs to extend

• Same is true for branching, semi-circular tubular vacuoles can participate in branching and septation

_ is an important protein-rich region for endocytosis

Sub-apical collar (SAC) of the hypha

T/F: It was previously thought that fungi were incapable of endocytosis

TRUE

But then they discovered SAC of the hypha

_ are enriched with proteins for endocytosis

Sub-apical collar (SAC) of the hypha

T/F: Polarity is entire controlled by SPK

FALSE

SPK and SAC directs polarity of the tip

_ points to evidence for endocytosis using steryl dyes

Tubular vacuolar system

T/F: Fungi have dynamic tubular vacuolar system used for endocytosis

FALSE

Fungi have dynamic tubular vacuolar system used for exocytosis

Explain figure shown

• Hyphal tips of Neurospora crassa treated with 2 steryl dyes to detect putative endocytosis

• Both steryl dyes (A) FM4-64 and (B) FM1-43 became internalized, and both hyphae treated with steryl dyes continued to grow during confocal laser imaging, as shown in photos taken at 30-sec intervals

How are (1) internalization of steryl dyes and (2) subsequent continued growth of hyphal tips evidences for endocytosis actually occurring?

• This is because steryl dyes specifically bind to the plasma membrane and are taken into the cell via endocytic vesicles, thus observing dye internalization indicates active endocytosis occurring at the site.

• Meanwhile, continued growth at hyphal tips also is evidence for endocytosis because polarized growth at the hyphal tip requires a balance between exocytosis (adding of membrane and materials for expansion) and endocytosis (recycling of excess membrane and retrieving of materials), thus observing continued growth at hyphal tips while dyes are internalized indicates that endocytosis is actively supporting this process of polarized growth along with exocytosis occurring at the tip

The fungal cell membrane has _ that are antifungal targets

sterols (ergosterol) and transporters (cytosine permease)

Fungal cell membrane is similar to a typical Eukaryotic bilayer: _

lipids + proteins

_ is the distinguishing component of fungal cell membrane that functions to module permeability and fluidity and is thus the target of several drugs

Ergosterol (steroid bc it has 4 rings)

T/F: Humans have ergosterol and cytosine

FALSE

Human PM don’t have ergosterol and cytosine deaminase

Enumerate 4 common antifungal substances

faap

1. Flucytosine (work from in → out bc u need all fungi inside u to have their protein synthesis interrupted)

   1. Flucytosine (cytosine analog) enters fungal cell via cytosine permease

   2. Once inside, flucytosine is converted into 5-fluorouracil by cytosine deaminase

   3. Once 5-FU is incorporated into fungal RNA, this results in inhibition of RNA synthesis and, subsequently, protein synthesis, impairing fungal cell functions

   4. Selective drug: Mammalian cells don’t have cytosine deaminase, making flucytosine specific to fungi

2. Allylamine drugs

   1. Inhibit squalene epoxidase that converts squalene → lanosterol, thus *ultimately inhibiting ergosterol synthesis, leading to decreased membrane permeability and thus leakage of ions & essential nutrients, eventually leading to cell death*

3. Azole derivatives (e.g., ketoconazole) topical

   1. Inhibit enzyme converting lanosterol → ergosterol, thus *

   2. (works from out → in, affecting membrane’s resistance to turgor pressure)

4. Polyene antibiotics (e.g., Amphotericin B)

   1. Directly binds to and break down ergosterol, increasing membrane permeability

T/F: Azoles work slower than polyene antibiotics

TRUE

Bc it will take more time to completely inhibit synthesis of all ergosterols in PM than directly breaking it down

T/F: Sub-apical collar (SAC) of hypha is used for exocytosis, while tubular vacuolar system (TVS) is used for endocytosis

FALSE

Sub-apical collar (SAC) of hypha is used for endocytosis, while tubular vacuolar system (TVS) is used for exocytosis

_ is a fungal specific cytosine transporter that serves as mechanism for protein inhibition (via action of competitive inhibitor flucytosine)

Cytosine permease

Fungi utilize _ for their unique nutrient acquisition strategy

cell membrane enzymes, i.e., H+ ATPases and H+ symporters and antiporters

The most common proteins in the fungal plasma membrane function for:

ssca

• Solute transport (bc of constant growth)

• Signal transduction (bc anastomoses)

• Cell wall synthesis (hyphal tip extension)

• Anchorage for cytoskeleton (support for hyphal growth)

_ is the fungal cell membrane enzyme that allows fungi to secrete H+ ions, increasing external H+ concentrations, decreasing pH (more acidic, pH 4 - 5.5)

H+ ATPases

T/F: H+ ATPases do primary active transport

TRUE

H⁺ ATPases perform primary active transport because they directly use the energy from ATP hydrolysis to pump protons (H⁺ ions) across a membrane

_ is the fungal cell membrane enzyme that co-transports important materials, such as amino acids, ions, etc.

H+ symporters (but antiporters are also present)

T/F: H+ symporter is a primary active transport

FALSE

H+ symporter is a form of secondary active transport because it just relies on energy stored in the proton gradient generated by the action of H+ ATPases

Ergosterol is the distinguishing component of fungal cell membrane that is the target of several drugs and functions to _

modulate permeability and fluidity

Fungal cytoskeleton has 3 major polymers, namely _

ams

1. Actin ogsec

   1. Organelle movement, growth polarity = actin cables (polarized transport)

   2. Septation

   3. Endo- & exocytosis = actin patches

   4. Cytokinesis = actin rings

2. Microtubules tism cc

   1. Tip growth, intracellular trafficking, shape & morphology, cell division, chromosome segregation

   2. Very close to nucleus bc during cell division, microtubules make up spindle fibers

3. Septin y pcb f hmbp

   1. Yeast = protein recruitment/scaffolding, cytokinesis, budding polarity

   2. Filamentous/hyphal = hyphal morphology, branching, & polarity

3 steps involved in fungal endocytosis

bis

1. Bending

   1. PM begins to bend inward, forming small depression

   2. Initiated by recruitment of actin and actin-binding proteins to PM, providing structural support to membrane curvature

   3. Cargo molecules bind to PM

2. Invagination

   1. Membrane deepens into vesicle-like structure

   2. Facilitated by membrane coat proteins & actin nucleation-promoting factors to stabilize curvature and drive invagination

   3. Cargo molecules are selectively captured during this stage

3. Scission

   1. Scission proteins, e.g. dynamin-related proteins, cut neck of invaginated vesicle, releasing into cytoplasm as fully formed endocytic vesicle

   2. Vesicle transports cargo for processing / recycling

   3. Shed out actin & actin-binding proteins

When making small batches of fungi culture, it’s important to do it less than 2 weeks because _

As nutrients in the medium get depleted and as the fungus is continuously secreting H+ ions, the medium will get hyperacidified and fungi dies due to lack of nutrient supply and hyperacidity (unless u add buffer to medium to deal with excess pH)

Apical hyphal growth is unique in its _

structural plasticity, complex organization

No other living group of organisms exhibits _ through a localized synthesis of cell wall components

continuous tubular growth

No other living group has extreme structural plasticity, displayed through _

• Ballooning of structures to form spores/yeasts,

• Tapering hyphae to penetrate plants

_ can give rise to complex tissues and infectious structures, growing fast by being supplied with protoplasm continuously

Hyphae

Explain the growth patterns in fungal hyphae

• Growth occurs in an isotropic (equal) fashion during spore germination (1)

• Specification of polarity axis ultimately results in formation of hypha that continues to grow at the tip (2-3)

• While tip growth is maintained, specification of additional polarity axes and compartmentalization enables formation of septa and lateral branches

• While septum formation is transient, branching results in the formation of secondary hypha that also continues to grow at the tip

Structural variation in Mycota are made from _

hyphal growth + fusion

• Elongation, compartmentalization, fusion ecf

Describe figure shown

Structural variations resulting from hyphal growth + fusion (micro- & macroscopic fungal forms)

• a) Unicellular yeasts

• b) Pseudohyphae

• c) Filamentous mycelia

• d) Fungal parenchyma (hyphae anastomoses in parallel direction)

• e) Biofilm aggregates (not all fungi can form)

  reproductive structures f-g

• f) Compartmentalized macroconidia

• g) Conidiophores

• h) Spherules

  fruiting bodies i-j

• i) Perithicium

• j) Fruiting bodies

• k) Brackets

• L) Cages

• M) Nests

Apical tip growth and its plasticity involves _

cell wall expansion from secreted vesicles

Explain Tip Growth Model of the Hyphal Apex

tsvc

1. For the hyphal tip to continuously grow, 3 important components are necessary:

   1. Turgor pressure (fluid in protoplasm pushing against CW)

   2. SPK (determines direction) & constant delivery of vesicles (release chitin synthases and other materials) to hyphal tip

   3. Cytoskeleton & TVS to direct all vesicles to proper area

So what happens is:

1. Actin, microtubules, motor proteins deliver diff vesicles from internal protoplasm to the tip where there’s SPK, hence SPK accumulate lots of vesicles at tip

2. Once these vesicles cluster at tip, containing materials for PM + cell wall synthesis, vesicles specifically containing transporters (CW synthase) fuse with PM, movement of materials commence, including chitosomes embedded into CW

3. Hyphal tip usually becomes thinnest point of CW bc its components move to sides, causing strain on hyphal tip, which forces elongation to move forward, coupled with turgor pressure bc protoplasmic movement also exerts pressure against CW

4. Strain is important to determine whether hypha continues to grow because once it becomes very low, in the case that fungus hits a wall, then no more vesicles are brought to the tip.

5. But with the strain constant, vesicles also continue to move forward.

1. Exocytotic vesicles (EV) cluster in the SPK and radiate to promote secretion of cell wall polysaccharides + remodeling enzymes

2. This allows CW to thicken and deform under stresses by turgor pressure from cytoplasm, at a strain rate

3. The pool of EVs is fed by polarized trafficking and other sources of recycling in proportion to the strain rate of the CW at the cell tips (mechanical feedback)

T/F: It’s not only turgor pressure and CW formation driving hyphal motion forward

TRUE

There’s also actin

T/F: Polarized vesicle trafficking ensures that the majority of materials are delivered to the hyphal sides for wall expansion.

FALSE

Materials are delivered to the tip, not directly to the sides, but are redistributed from the tip.

Area where germ tube comes out and hyphae develops

Polarity axis

(3) When 2 polarity axes go against one another, this can trigger _

septum formation

T/F: Septum formation is more transient than secondary hypha / branching formation

TRUE

Bc secondary hypha will also continue to grow at the tip