Medical Mycology & Biotechnology – Detailed Study Notes

Overview

• Speaker: Dr. Megan Lenardon (Senior Lecturer, School of BABS), 30 June 2025, 11:05 PM
• Purpose: illustrate the 3-step medical biotechnology framework using medical mycology (esp. Candida albicans) as a worked example
• Lecture roadmap
  • Define medical biotechnology
  • Quick primer on fungi and fungal disease
  • Deep dive into C. albicans biology, diagnostics, and therapy
  • Show how basic knowledge ➜ new biotech solutions (antibody-based tools)

The 3 Steps of Medical Biotechnology

1. Understand the disease
   – Anatomy, physiology, microbiology, pathogenesis
2. Assess the clinical needs
   – What is missing/deficient in current diagnosis, treatment, prevention?
3. Exploit biological knowledge
   – Translate discoveries into diagnostics, drugs, vaccines, preventives

Learning outcome: be able to quote C. albicans–focused examples for each step.

Definition of Medical Biotechnology

• Australian Government (≈ 2017):
  “Use of living cells & cell materials to research and produce pharmaceutical or diagnostic products that help treat & prevent human diseases.”
• Within Applied Biomolecular Sciences, this is biomolecular knowledge → medical setting.

Fungal Biology Primer

• Mycology = study of fungi (Gr. mykes = fungus + -logy = study)
• Classical fungus definition:
  • Chemo-organo-trophic eukaryote (oxidises organic electron donors)
  • Lacks chlorophyll; forms spores
  • Cell wall of polysaccharide (chitin/cellulose)
  • Absorptive nutrition
  • Membrane sterol = ergosterol (vs. human cholesterol)
  • Traditional classification by morphology: mushrooms, moulds, yeasts; now DNA based
• Key terms
  • Chemotroph: derives energy by oxidising chemical compounds
  • Organotroph: uses organic molecules as e⁻ source
  • Polysaccharide: polymeric sugar; examples chitin, β-glucan

Diversity & Beneficial Roles

• Visual gallery: button mushrooms, delicate Pleurotus-like forms, fruit moulds, spore structures, yeasts
• Ecosystem services: nutrient recycling, even plastic degradation → most fungi are harmless

Fungal Pathogenicity Snapshot

• Pop-culture link: “The Last of Us” (fictional Cordyceps zombies) raised public awareness
• Real host range: plants, insects, amphibians, mammals
  • Plant diseases: corn smut (Ustilago maydis), rice blast (Magnaporthe grisea), potato blight (Phytophthora infestans, an oomycete)
  • Insects: zombie ants (Ophiocordyceps unilateralis), bee mycoses
  • Amphibians: chytridiomycosis (chytrid fungus)
  • Mammals: bat white-nose syndrome (fungus grows during hibernation)
• Human infections
  • Superficial (surface, non-lethal): ringworm, athlete’s foot/tinea, onychomycosis; affect ≥ 25\% of global population
  • Invasive/systemic (internal, lethal): sepsis, organ invasion

WHO 2022 Fungal Priority Pathogens List (Top 5)

Global burden (all five together):
• ≥ 6.5\text{ million} invasive cases / yr → ≥ 3.75\text{ million} deaths
• Mortality: untreated \approx 90\%; even with therapy 35–60\%
• Exceeds deaths from TB, malaria, breast or prostate cancer

Candida albicans Biology & Pathogenesis

• Pleomorphic: yeast ↔ pseudohypha ↔ true hypha
• Normal commensal of GI tract; 60–80\% of people carry it from birth
• Kept in check by:
  • Bacterial microbiota (nutrient competition)
  • Innate immunity (neutrophils & macrophages; video shown of phagocytosis)
• Superficial diseases: oral & vaginal thrush
• Invasive candidiasis (at-risk groups):
  • Cancer chemotherapy, organ/stem-cell transplant recipients
  • Immunosuppressive drugs, AIDS, major abdominal surgery, ICU patients
• Disease course: gut escape → bloodstream (candidemia) → dissemination to organs → sepsis → death; mouse-kidney lesions shown
• Rapid diagnosis + therapy markedly improves survival

Current Clinical Needs

Diagnostics

• Gold standard = blood culture
  • Sensitivity only \approx 50\% (misses tissue-only cases)
  • Time to result \approx 72\text{ h} → patient may succumb first
  • CHROMagar Candida: chromogenic agar for species ID once grown
• Molecular tests (PCR/sequencing)
  • Faster but hampered by tough fungal cell wall (manual bead-beating needed)
  • Difficult to distinguish harmless colonisation vs disease-causing invasion

Therapy

• Terminology: antibiotics = antibacterial only; antifungals target fungi
• Need exploitable fungal-specific features → only a few drug classes

Approved systemic classes vs Candida

1. Polyenes (e.g. amphotericin B) – bind ergosterol; potent but nephro-/hepato-toxic
2. Azoles (e.g. fluconazole) – inhibit ergosterol biosynthesis; widespread resistance
3. Echinocandins (e.g. caspofungin) – block β-1,3-glucan synthase (cell-wall); low toxicity but resistance emerging; first approved > 20 years ago, only derivatives since

Comparative numbers: \text{antifungal classes}=5 vs >200 antibacterial classes

C. albicans Cell-Wall Architecture

• Electron micrograph: plasma membrane (ergosterol-rich) + layered wall
• Structural polysaccharides
  • Chitin microfibrils (brown in model)
  • β-1,3-glucan triple helices (green)
• Outer fibrillar layer = highly mannosylated proteins (mannan chains)
• Wall uniqueness → prime drug/diagnostic target (absent in humans)

From Basic Biology to Biotech Solutions

• Lab programmes
  • Map wall biosynthesis → novel drug targets
  • Collaborate with chem-eng on antifungal polymers
  • Study GI colonisation for microbiome-based prevention
• Today’s focus: antibody-based diagnostics/therapies targeting wall mannans

Antibody Fundamentals (Rapid Recap)

• Natural antibody = Y-shaped protein produced by B cells
  • Variable (V) region: epitope-specific; 3 Complementarity-Determining Regions (CDR1-3)
  • Constant (C) region: species-specific effector domain
• Antigen: protein, carbohydrate, small molecule, nucleotide
• Epitope: precise part of antigen bound by antibody
• Preparations
  • Polyclonal sera: mix of antibodies → multiple epitopes; varied CDR DNA
  • Monoclonal (mAb): identical, single-epitope binder; cloned B-cell or recombinant
• Engineered fragments: Fab, F(ab')₂, scFv / scAb (single-chain variable fragment) – retain CDRs, easier recombinant expression

Why Monoclonal Antibodies for Fungal Disease?

• Can exploit even subtle fungal-human differences → high specificity
• Intrinsically non-toxic to humans; off-target effects minimal
• Circumvent mechanisms of small-molecule resistance (novel mode of action)

Discovery of Anti-Mannan Monoclonals

• Strategy: identify antibodies that bind C. albicans wall mannans
• Phage-display library panning (detailed multistep cycle)
  • Output: DNA sequences of CDRs for candidate monoclonals
• Recombinant production: scAb format
  • Human κ light chain spacer + CDRs + 6\timesHis tag
  • Expression in E. coli → rapid, low-cost screening

Key Clones: 1A2 & 1H6

• Naming arbitrary (plate wells); both bind C. albicans mannan exclusively
• Specificity ELISA: high OD only with C. albicans mannan; zero cross-reactivity to
  • Other fungal mannans (e.g. Saccharomyces, Cryptococcus)
  • Non-mannan polysaccharides / monosaccharides
• Affinity: single-chain antibodies showed stronger binding than some full-length anti-mannan mAbs (sub-nanomolar K_D)

Functional Activity

• In vitro fungicidal assay
  • Both scAbs killed C. albicans, C. dubliniensis, C. tropicalis, C. krusei
  • Viability ↓ vs untreated and irrelevant scAb controls
• In vivo protection (Galleria mellonella larvae model)
  • Inject larvae: one pro-leg Candida, opposite pro-leg treatment
  • Survival curve: 1H6 (orange) prolonged survival vs saline; median survival day 5 → day 7+
  • Live larvae remain white; dead turn black (easy scoring)

Practical Considerations: Full mAb vs Fragment

• Fragments (scAb/scFv)
  • Pros: simple bacterial expression, cost-effective, ideal for discovery & diagnostic kit integration
  • Cons: rapid renal clearance in humans, potential immunogenicity if non-human framework, lack Fc effector functions
• Full-length humanised mAb
  • Required for therapeutic injection (longer half-life, complement/ADCC)
  • Production in mammalian systems, higher cost/time
• Development pipeline: fragment for screening ⟶ humanised IgG for clinic

Mapping to the 3 Biotechnology Steps

Key Takeaways

• Invasive fungal disease = major, under-recognized killer (> 3.75\text{ M} deaths/yr)
• C. albicans normally harmless commensal can become lethal in immunocompromised hosts
• Current gold-standard diagnostics & drugs have serious limitations
• Fungal-specific cell wall components (e.g. mannan, β-glucan) are rich biotech targets
• Monoclonal antibody technology offers:
  • Highly specific detection (diagnostics)
  • Novel therapeutic modality (direct fungicidal activity, immune engagement)
• Phage display + recombinant engineering rapidly deliver candidate antibodies (1A2, 1H6) with proof-of-principle efficacy in vitro & in vivo
• Illustrates full medical biotechnology pipeline: discovery → need identification → translational product design

Possible Exam/Revision Prompts (as provided by lecturer)

• State & exemplify the 3 steps of medical biotechnology
• Define “medical biotechnology” and contrast with basic research
• Describe key differences between fungal & human cells exploitable for therapy
• List classes of systemic antifungals, their targets, and limitations
• Explain why blood culture has only 50\% sensitivity for invasive candidiasis
• Draw/annotate C. albicans cell wall layers, indicating drug/antibody targets
• Compare polyclonal vs monoclonal antibodies; give clinical uses of each
• Outline phage-display workflow for antibody discovery
• Discuss advantages & caveats of antibody fragments vs full IgG in therapy