Fungal Virulence Factors

Fungal virulence is multifactorial. Few fungi produce a single dominant toxin. Three categories of trait contribute:

• Fitness attributes that allow survival in the host, such as growth at 37 degrees Celsius, stress resistance, nutrient acquisition and morphogenesis.
• True virulence factors that directly damage the host or subvert defence, such as capsule, melanin, secreted enzymes and toxins.
• Host damaging properties that depend on the interaction, such as cell wall components that provoke excessive inflammation.

Cryptococcus is the only common human pathogen with a prominent polysaccharide capsule. The capsule consists chiefly of glucuronoxylomannan (about 90 percent) with glucuronoxylomannogalactan and mannoproteins.

Functions:

• Impairs phagocytosis by masking wall components and blocking complement deposition.
• Quenches reactive oxygen species and protects against desiccation and oxidative stress.
• Shed capsular polysaccharide suppresses cytokine production, inhibits leukocyte migration, depletes complement and promotes apoptosis of T cells.
• Raises intracranial pressure by obstructing cerebrospinal fluid drainage when polysaccharide burden is high.

Capsule size is regulated: it enlarges in the host in response to carbon dioxide, low iron and serum. Acapsular mutants are avirulent. Shed glucuronoxylomannan is the antigen detected by cryptococcal antigen assays, and capsule size explains why antigen titres track fungal burden. Rare capsule deficient strains stain poorly with mucicarmine and are identified by Fontana Masson staining of the cell wall.

Melanin is a dark pigment polymer deposited in the cell wall.

• Cryptococcus: laccase oxidises host catecholamines such as dopamine to melanin. Brain tissue is rich in these substrates, which is one proposed reason for brain tropism. Melanin protects against oxidative killing and against amphotericin B and other drugs.
• Aspergillus fumigatus: conidial dihydroxynaphthalene melanin gives the blue green colour, protects from reactive oxygen species and inhibits the phagocytic pathway (LC3 associated phagocytosis) that normally kills conidia. Melanin deficient conidia are less virulent.
• Dematiaceous fungi (Exophiala, Fonsecaea, Cladophialophora, Hortaea werneckii): melanin gives pigmented cell walls that resist phagocytosis, oxidative damage and some antifungals.

Pigmented walls can be detected in tissue because they stain brown on haematoxylin and eosin, and melanin stains with Fontana Masson.

Classical protein exotoxins are rare in fungi. The recognised exceptions:

• Candidalysin of Candida albicans is a pore forming peptide toxin that damages epithelial membranes and drives neutrophil recruitment. It is required for oral mucosal infection in experimental models.
• Gliotoxin of Aspergillus fumigatus belongs to the epipolythiodioxopiperazines. It inhibits neutrophil NADPH oxidase, induces apoptosis in immune cells and suppresses phagocytosis.
• Mucoricin of Mucorales is a ricin like toxin implicated in tissue necrosis.
• Mycotoxins (aflatoxin, ochratoxin, fumonisin, ergot alkaloids) cause disease through ingestion of contaminated food and not through infection.

Iron is a limiting nutrient in the host. Transferrin, lactoferrin and ferritin bind iron tightly, so free iron is extremely low. Fungal pathogens overcome this with several systems:

Consequences for clinical practice: free iron in diabetic ketoacidosis (acidosis weakens transferrin binding), iron overload and deferoxamine treatment promote Mucorales growth. Zinc is also required. Candida albicans uses the secreted protein Pra1 to capture zinc, and Pra1 also binds complement factor H.

• Heat shock proteins and calcineurin: support growth at 37 degrees Celsius and under febrile stress. Hsp90 also stabilises drug resistance pathways.
• Oxidative stress defences: catalases, superoxide dismutases and glutathione systems neutralise reactive oxygen species released by phagocytes. Catalase peroxidase supports intramacrophage survival of Talaromyces marneffei.
• Hypoxia adaptation: the sterol regulatory element binding protein SrbA allows Aspergillus fumigatus to grow in hypoxic, necrotic lung. Loss of SrbA causes loss of virulence and hypersusceptibility to azoles.
• pH sensing: the Rim101 pathway of Candida albicans controls responses to neutral and alkaline pH in tissue.
• Cell wall integrity pathway: remodels the wall after echinocandin exposure with increased chitin.

Beta 1,3 glucan is the main immunostimulatory component, recognised by Dectin 1. Pathogens limit its exposure:

• Candida albicans hides glucan beneath outer mannan, and environmental cues such as lactate, hypoxia and iron limitation alter masking.
• Aspergillus fumigatus resting conidia are covered by melanin and the rodlet layer, and exposure of glucan accompanies swelling and germination.
• Histoplasma and Blastomyces yeasts increase alpha 1,3 glucan in the outer wall.
• Cryptococcus hides the wall under capsule.
• Galactosaminogalactan of Aspergillus promotes adhesion and suppresses inflammation.

Exposed glucan from cell wall remodelling after echinocandin therapy can sometimes provoke intense inflammation, a reason why host response and drug effect interact in tissue.

Biofilm is a virulence trait of Candida, Cryptococcus, Aspergillus and others on devices. The matrix of polysaccharide, protein and extracellular DNA binds drugs and shields cells from phagocytes, and persister cells survive treatment. Biofilm on central venous catheters, urinary catheters and shunts sheds yeast cells into the bloodstream. Echinocandins and liposomal amphotericin B retain more activity against biofilm than fluconazole and conventional amphotericin B.