Fungal Growth and Reproduction

Fungi grow as budding yeasts, as hyphae, or as both.

• Budding: a daughter cell grows from a site on the mother cell, receives a nucleus and separates after septum formation. Each division leaves a bud scar on the mother cell and a birth scar on the daughter. Polarity is established by septins and the actin cytoskeleton.
• Fission: a cell elongates and divides by a central septum into two equal cells. This is the mode of division of Talaromyces marneffei in tissue.
• Hyphal growth: hyphae extend from the tip only. Secretory vesicles carrying wall synthetic enzymes concentrate at the tip, in filamentous fungi at the Spitzenkörper, and new wall material is inserted at the apex. Branches arise behind the tip, and a mass of hyphae is a mycelium. Cross walls (septa) of ascomycetes and basidiomycetes carry pores that permit cytoplasm and organelle flow. Mucoromycota hyphae form few septa and are essentially coenocytic.
• Pseudohyphal growth: elongated buds fail to detach and form chains with constrictions at the septa.

Tip growth has clinical importance for two reasons.

First, hyphae can exert pressure and secrete enzymes at the tip, which drives tissue penetration and angioinvasion.

Second, beta glucan synthase is concentrated at the growing tip, so echinocandins act on hyphal tips of Aspergillus and produce stunted, branched growth without killing the mycelium. For this reason the endpoint of susceptibility testing for echinocandins against moulds is the minimum effective concentration, reading altered hyphal morphology, rather than the minimum inhibitory concentration.

Asexual reproduction is the commonest route of propagation and produces most clinically relevant spores. Conidiogenesis follows two broad patterns:

• Thallic: an existing hypha fragments or differentiates into conidia (arthroconidia, chlamydoconidia).
• Blastic: a conidium swells from a conidiogenous cell and is then cut off (phialoconidia, annelloconidia, aleuroconidia, blastoconidia).

The fruiting structure is the main morphological basis of mould identification:

Spore size determines how deeply inhaled propagules travel. The conidia of Aspergillus fumigatus (2 to 3 micrometres) reach the alveoli. Mucorales sporangiospores are larger (3 to 11 micrometres). Cryptococcus basidiospores and desiccated yeast cells (1 to 3 micrometres) also reach distal airways.

Dormant conidia are metabolically quiet and are protected by melanin, trehalose and a hydrophobic surface layer. Germination begins with swelling, followed by emergence of a germ tube after several hours at 37 degrees Celsius in permissive conditions. Swollen and germinating conidia expose cell wall beta glucan, whereas resting conidia are shielded, a point that matters for immune recognition.

Sexual reproduction involves plasmogamy (cell fusion), karyogamy (nuclear fusion) and meiosis, and generates genetic diversity. Many clinical isolates are known only as asexual forms.

Pneumocystis jirovecii and Paracoccidioides loboi do not grow on routine laboratory media.

The Pneumocystis life cycle takes place entirely in the alveolar space, where organisms attach to type 1 pneumocytes through major surface glycoprotein. Haploid trophic forms multiply by binary fission. Conjugation of two trophic forms yields a zygote, then a precyst, then a cyst containing eight intracystic bodies that are released when the cyst opens. Trophic forms greatly outnumber cysts.

This is why Giemsa and Diff Quik staining of trophic forms and cyst wall stains such as Grocott methenamine silver are complementary, and why diagnosis rests on microscopy, PCR and biomarkers rather than culture.

Paracoccidioides loboi has never been cultured, so its thermal behaviour has not been studied. It is recognised only in tissue, as chains of lemon shaped cells joined by short tubular bridges.

Biofilms are surface attached communities embedded in an extracellular matrix. Candida biofilm develops in stages: adherence of yeast cells, proliferation with hyphal formation, matrix production and maturation over 24 to 72 hours, and dispersal of yeast cells.

• Mature biofilm is tolerant of azoles and amphotericin B, because the matrix beta 1,3 glucan sequesters drug and efflux pumps are upregulated. Echinocandins and liposomal amphotericin B retain more activity.
• Dispersal seeds the bloodstream from catheters, which is why catheter removal is a central intervention in candidaemia.
• Biofilm forms on central venous catheters, urinary catheters, prosthetic valves, endotracheal tubes, dentures and ventriculoperitoneal shunts (Cryptococcus).
• Aspergillus grows as a hyphal mass in air filled cavities such as an aspergilloma, where it also sporulates; this explains why a fungus ball can be seen on imaging as a mobile intracavitary mass.

Cryptococcus also changes cell size in the host. Titan cells of 50 to 100 micrometres arise in the lung, are polyploid, and resist phagocytosis, whereas small cells of 2 to 4 micrometres disseminate.

Cultures are held for at least 4 weeks when dimorphic fungi or dermatophytes are suspected.

Medium composition affects recovery. Sabouraud dextrose agar at pH about 5.6 with chloramphenicol suppresses bacteria. Cycloheximide in the medium inhibits Cryptococcus, many Candida species, Aspergillus and Mucorales, and is therefore avoided for general diagnostic cultures. Dermatophytes and dimorphic fungi are cycloheximide tolerant, so cycloheximide containing media are used selectively for them.