Fungi

Question 1

Define fungi and the general types of fungi that may be found.

Answer 1

Fungi are eukaryotic organisms that absorb nutrients by breaking down organic material into simple molecules through the secretion of enzymes.

Types of Fungi

• Yeast(non-polymorphic & polymorphic)Unicellular Yeast
  • Saccharomyces cerevisiae is a species of yeast which is instrumental in winemaking, baking, and brewing since ancient times.
  • This yeast is non-polymorphic.
  Polymorphic Yeast; switch between growth forms(reversible)
  • Candida albicans is a commensal organism of the human body which is a fungus & yeast.
  • Candida albicans is a polymorphic organism which undergoes morphologic transition between yeast, pseudohyphal and hyphal forms. The ability of C. albicans to change from yeast to filamentous types is a major virulence determinant of this organism.
    
    • C. albicans in yeast form is benign, however, when it starts forming hyphae it develops the potential to become invasive and cause disease.
  • Candida can also form spores(i.e. chlamydospores) which are very tough and form when the fungus is in a nutrient-depleted environment(difficult survival elicits this survival mode).

Moulds

Enviormental obligate filamentous fungi, produce conidia. (e.g. Aspergillus fumigatus)

The process of these moulds: Starts as a condia spore which germinates into hyphae and continues to grow. 

- This fungus cannot revert its morphology(unlike candida) - once its hyphae it cannot go back to condia — But if it continues to develop it can create a structure called conidiophore which releases condia spores that then propagate into the environment.

Question 2

Explain the function and importance of the fungal cell wall.

Answer 2

• The cell wall is composed of complex polysaccharides that interlink to form a robust structure(the cell walls are unique to each fungus). It is the Interface with the extracellular environment(i.e. it exhibits strength against anything it encounters within the environment or host immune system).
• Functions include:
  
  • Protects – strong.
  • Flexible – dynamic to allow for growth, adaptation.
• Antifungal drug target, immune target, and vaccine target.
  
  • All anti-fungal drugs used clinically, target the fungal cell wall, membrane, or cell wall components(see image).
    
    • Major components are chitin, glucan, and Mennan which are all different types of sugars.
• If you strip the fungus of its cell wall, it will die from osmolarity because it now cannot regulate the extra/intra pressure.

Question 3

Explain, with examples, the types of primary vs opportunistic fungal pathogens.

Answer 3

• Primary Pathogens; (infect healthy individuals, inhalation, geographically restricted)
  
  • Histoplasma capsulatum; is a species of dimorphic fungus. Its sexual form is called Ajellomyces capsulatus. It can cause pulmonary and disseminated histoplasmosis.
  • Blastomyces dermatitidis; Blastomycosis is a fungal infectioncaused by Blastomyces dermatitidis. It presents as a pulmonary infection after the inhalation of spores, and it may be either asymptomatic or have severe life-threatening complications like acute respiratory distress syndrome.
• Opportunistic pathogens; (mainly infect immunocompromised patients, different routes of infection, global)
  
  • Dermatophytes; arefungal organisms that require keratin for growth. These fungi can cause superficial infections of the hair, skin, and nails. Dermatophytes are spread by direct contact from other people, animals, soil, and from fomites.
  • Candida sp.
  • Aspergillus sp.
  • Cryptococcus sp.

Question 4

What diseases are caused by opportunistic pathogens? Name examples

Answer 4

1. Superficial mycoses (non-lethal)
   
   1. Dermatophytes (e.g. Tinea pedis (athletes foot))
   2. Candida spp. (e.g. Thrush)
   3. Malassezia spp. (e.g. Dandruff, Pityriasis versicolor)
2. Subcutaneous mycoses (non-lethal but debilitating)
   
   1. Rare infections:
      
      1. Black moulds (e.g. Chromoblastomycosis)
      2. Madurella mycetomatis (e.g. Madura foot (or Mycetoma))
3. Invasive mycoses (lethal)
   
   1. Predominant in immunocompromised individuals.
   2. Candida spp., Cryptococcus spp., Aspergillus spp., Pneumocystisspp

Question 5

Based on the diseases caused by opp pathogens, discuss/explain each category.

Answer 5

Superficial Mycoses: Dermatophytes

-Caustaive agent; A type of filamentous fungi from soil (geophilic), animals (zoophilic), or people (anthropophilic). It produces keratinase which digests keratin as a growth substrate and, therefore infects keratin-rich tissues.
-infections can occur in/around the mouth, genitals, skin, etc.
-predisposing factors include -> This can occur in immunocompetent individuals, especially those who:
-Prolonged use of antibiotics() -Pregnancy(Hormonal fluctuations can leave women susceptible)
-Diabetes M

Subcutaneous Mycoses
-Affects subcutaneous tissue - Introduced by puncture wounds/ traumatic injury of the skin.
-These infections typically occur in underdeveloped countries/places with poor healthcare facilities. Infection will continue to develop until chronic—by then it would be far too difficult to treat.

Invasive Mycoses;

• High mortality rate.
• Immunocompromised patients.
• Severity depends on the status of the immune system.
• As many people die from the top 10 invasive fungal diseases as from tuberculosis or malaria.
• 90% of all reported fungal-related deaths are from: Cryptococcus, Candida, Aspergillus.

Question 6

Name the three primary opportunistic pathogens.

Answer 6

Candida
Aspergillus
Cryptococcosis

Question 7

In depth notes on Aspergillosis.

Answer 7

• Causative organisms
  
  • Most Aspergillus species (200+) do not cause disease.
  • Causative organisms:
    
    • A. fumigatus, A. flavusniger & A. terreus.
• Route of infection
  
  • Inhalation of spores (normally inhale 100-200/day)
  • “Sick building syndrome”; is used to describea situation in which the occupants of a building experience acute health- or comfort-related effects that seem to be linked directly to the time spent in the building. No specific illness or cause can be identified.
• Types of disease
  
  • Allergy (asthma sufferers)
  • Invasive bronchopulmonary Aspergillosis
  • Disseminated Aspergillosis
• 60-90% Mortality

(REVIEW NOTION)

Question 8

In depth notes on Candida.

Answer 8

• Causative organisms
  
  • Candida albicans (immuno-compromised and immuno-competent individuals)
    
    • Polymorphic (yeast, hyphae, pseudohyphae, opaque and white cells).
    • Commensal organism of the mucosal and skin microbiota.
• Route of infection
  
  • From within due to environmental changes, reduction in immune competence, lesions.
• Types of disease:
  
  • Superficial candidiasis (oropharyngeal tract, penis, vagina)
  • Invasive candidiasis (GI tract, internal organs, bloodstream)

Question 9

In depth notes on Cryptococcosis.

Answer 9

• Causative organisms
  
  • Cryptococcus neoformans (immunocompromised individuals)
  • Cryptococcus gattii (“Healthy” individuals)
    
    • Dimorphic*****
      
      • Usually does not form filaments
      • Form cells of different sizes (titan, titanides, drop cells)
    • Encapsulated yeast during infection
    • Found in soil and avian habitats
• Route of infection
  
  • Inhalation (desiccated yeast or spores)
  • Crosses the blood brain barrier
• Disease
  
  • Pulmonary cryptococcosis
    
    • Often asymptomatic
    • Chronic
      
      • Cryptococcus can become an intracellular pathogen therefore persist
  • Cryptococcal meningitis
    
    • Can occur in “normal individuals”
    • Common in AIDS patients and organ transplant recipients
      
      • High mortality (40-70%)

Question 10

Discuss ALL available treatments options/classes of drugs. Give an example of a specific drug per class, notes on mechanism, and what sort of pathogen it would be used on.

Answer 10

• Membrane disrupting agents(e.g. Polyenes (amphotericin B))
• [Mechanism of Action] - Amphotericin B is a good example of a polyene antibiotic that,via the binding of ergosterol, has a dual mode of action by inhibiting membrane proteins and permeabilizing the plasma membrane, causing cell death(fungicidal).
  - Because it targets the membrane its toxic to human liver cells.
  
  • [A wide range of use] - Candida spp., Aspergillus spp. & Cryptococcus spp.
    
    • Amphotericin B - Serious / systemic infections
    • Nystatin – Superficial infections
  [Amphotericin B Problems] -
  • Not available orally – I.V. use only (oral formulations under studies)
  • Nephrotoxicity
    
    • New lipid formulations less toxic, but have variations in term of clinical effectiveness
• Ergosterol synthesis inhibitors (e.g. Azoles, allylamines)

[Mechanism of Action] -
- Inhibits ergosterol synthesis
- Inhibits 14α sterol demethylase (ERG11)
- Alters membrane fluidity
- Build up of 14α sterols
- Mainly fungistatic (inhibit the growth but do not kill fungi)

[Clinical Use] - 

- Spectrum of activity varies with drugs
- Typically broad spectrum
    - Exception fluconazole with no activity against *Aspergillus* spp.

[Problems] - 

- **Drug interactions**
    - Azoles are metabolized by and inhibitors of, human liver cytochrome P450 enzymes
    - Human liver cytochrome P450 involved in drug metabolism
    - Drug-azole interactions can result into decrease or increase in exposure of both interacting drugs
        - **Reduced efficacy** (decreased drug) or **increased toxicity** (increased drug)
- **Resistance**
- ALLYMINES →
- Synthetic compound, 1970’s
- Inhibits ergosterol biosynthesis
    - Inhibits squalene epoxidase (*ERG1*)
- Cells accumulate squalene
- **Fungicidal**
- **Spectrum of activity**
    - Fungicidal against dermatophytes
    - Poor activity against *Candida* sp., *Aspergillus* sp. & *Cryptococcus* sp.
    - Drug of choice for dermatophyte infections

• DNA synthesis inhibitor (e.g. Flucytosine)
  
  • Synthetic analogue of cytosine
    
    • Initially developed as anti-cancer drug
  • Enters fungal cells via cytosine permease
  • Converted to 5-Fluorouracil
  • Inhibits protein synthesis and DNA synthesis
  Spectrum of activity
  • Yeasts only
  • Candida spp. & C. neoformans
  • Fungicidal
  Problems
  • Resistance is very common
    
    • Not administered as a single agent (used in combination Amphotericin B for treating systemic mycoses)
• Glucan synthesis inhibitors (e.g. Echinocandins)
• Semisynthetic
• 3 candins recently approved as antifungals
  
  • Caspofungin (2001)
  • Micafungin (2005)
  • Anidulafungin (2006)
• First new class of antifungals in 20 years
• Inhibit beta1,3-Glucan synthase (FKS1)
  
  • beta1,3-Glucan essential component of fungal cell wall
• Spectrum of activity
  
  • Candida spp.– fungicidal
  • Aspergillus spp. – fungistatic
  • No activity against C. neoformans
  • No problem of cross resistance
  • Low toxicity
  Problems
  • Not available orally – I.V. use only
  • Expensive…
  • The Caspofungin cost for treating 1 patient with Candidemia/invasive candidiasis ~£5400 (14 days treatment) – 2015 study in Spain

Question 11

Discuss/explain the mechanisms of fungal drug resistance.

Answer 11

• Primary / Natural resistance; Intrinsic resistance linked to species or strain (population heterogeneity). Resistance without prior exposure to the drug.
  
  • E.g.
    
    • Allylamines – poorly active against Candida spp.
    • Candida krusei and Candida auris – intrinsically resistant to Fluconazole
    • Cryptococcus neoformans resistance to Echinocandins
• Secondary / Acquired resistance; The susceptible strain becomes resistant. Acquired through mutation and selection.
  
  • Through modification / overexpression of target.
  • NOT through horizontal gene transfer in fungi.

Question 12

Explain/discuss fungal virulence factors and how they act together to escape the immune system.

Answer 12

• Ability to grow in hostThermotolerance, nutrient acquisition, adaptation to different niches.
  • Non-pathogenic Cryptococcus species cannot grow at 37°C
    
    • Genes required for growth at 37C
      
      • Calcineurin A (CNA1): phosphatase activated by calmodulin
    • Gene disruption blocks growth at 37C and reduces virulence
    • Allelic replacement restores virulence
  • Nutrient Acquisition
    
    • Fungal pathogens are exposed to multiple host niches, and metabolic adaptation is essential for virulence
    • Use sources of carbon and nitrogen found within the host. In addition they have specialised mechanisms to acquire micronutrients (e.g. iron, zinc, etc)
    • Secreted hydrolytic enzyme families play a combined role in virulence
      
      • Nutrient acquisition, combat host defence, adhesion
      • Examples: proteinases, lipases, phospholipases
    • Hallmark of hyphae (polymorphism)
    • Fungal pathogens display multiple strategies to acquire micronutrients (e.g. zinc, iron) from the host (resistance to nutritional immunity)
      
      • Nutritional immunity: host mechanisms to deny access to essential nutrients such as iron, zinc or other metals
• Adhesion and invasionBiofilm, polymorphism, translocation.
  • Biofilm Formation in C. albicans:
    
    • Form on a variety of biotic and abiotic surfaces(catheters are a big problem)
    • Structured assemblies encased in extracellular matrix
    • Display inherent resistance to antifungals and host defences
    • Quorum sensing and polymicrobial interactions impact on formation
  • Stages of Biofilm Development:
    
    • Attachment – adherence of yeast cells to substrate
    • Initiation – formation of micro-colony
    • Maturation – hyphal development & Extracellular matrix production
    • Dispersal - release of non-adherent yeast cells
• Stress resistance

Cell surface changes, detoxification, Biofilm.
-Phagocytosis;
Fungi can actually withstand the stres caused by the immune response.
When cells are phagocytoses by macrophages there will be a change in pH, low amino acids, glucose, and iron.

-respiratory burst;
ROS and RNS are produced these are damaging and toxic.

-cytokine burst
The incited immune repose obviously through cell signaling summons pro-inflammatory cells and thus an increased temperature(fever) - some fungal species are good at withstanding heat.

The pathogen response is immune evasion or stress response.

• Immune evasionPolymorphism, escape from immune cells, masking of PAMPs (e.g. capsule, pigment).
  • Intrinsic structure of the outer surface of the fungus (the cell wall) that allows them to hide their PAMPs (pathogen associated molecular patterns) in addition to providing stress resistance
  • Secretion of enzymes and other components modulate the immune response – dampening
  • Cell surface changes depending on available nutrients
• Damage to the host

Physical damage, secreted enzymes, toxins.

• C. albicans glucose to Lactate:
  
  • Intrinsic structure of the outer surface of the fungus (the cell wall) that allows them to hide their PAMPs (pathogen associated molecular patterns) in addition to providing stress resistance
  • Secretion of enzymes and other components modulate the immune response – dampening

Question 13

Why are environmental fungi very good at infecting human hosts?

Answer 13

because they have the same amount f stresses in the environment. they’re good at digesting things in the soil. they compete w bacteria in soil for nutrients and other things. they’re quite robust and have high survivability . high fungal infections are a result of high use of azoles and azoles in environment which creates resistance. the increase in immunocomporosed patients will also add to the problem.

Question 14

Why is there an increase in anti-fungal resistance?

Answer 14

There are more tools to be used to be aware of the increase/presence of resistance.

The amount of immunocompromised patients has increased in the past 20-30 years. The treatment options for cancer and HIV patients has increased therefore survivability of these patients increases, however fungal pathogen can be opportunistic.