The Hidden Fortress: A Comprehensive Review of Fungal Biofilms with Emphasis on Cryptococcus neoformans

Abstract

Biofilms are structurally organized communities of microorganisms that adhere to a variety of surfaces. These communities produce protective matrices consisting of polymeric polysaccharides, proteins, nucleic acids, and/or lipids that promote shared resistance to various environmental threats, including chemical, antibiotic, and immune insults. While algal and bacterial biofilms are more apparent in the scientific zeitgeist, many fungal pathogens also form biofilms. These surprisingly common biofilms are morphologically distinct from the multicellular molds and mushrooms normally associated with fungi and are instead an assemblage of single-celled organisms. As a collection of yeast and filamentous cells cloaked in an extracellular matrix, fungal biofilms are an extreme threat to public health, especially in conjunction with surgical implants. The encapsulated yeast, Cryptococcus neoformans, is an opportunistic pathogen that causes both pulmonary and disseminated infections, particularly in immunocompromised individuals. However, there is an emerging trend of cryptococcosis among otherwise healthy individuals. C. neoformans forms biofilms in diverse environments, including within human hosts. Notably, biofilm association correlates with increased expression of multiple virulence factors and increased resistance to both host defenses and antifungal treatments. Thus, it is crucial to develop novel strategies to combat fungal biofilms. In this review, we discuss the development and treatment of fungal biofilms, with a particular focus on C. neoformans.

Keywords: Aspergillus; Candida; Coccidioides; Cryptococcus neoformans; Fusarium; Trichosporon; biofilm; central nervous system; cryptococcosis; extracellular matrix; fungus; meningoencephalitis; polysaccharide capsule; titan cell; virulence factors.

Figure 1

Structure and components of C. neoformans. (A) The proximal cell membrane consists of a phospholipid bilayer composed of various proteins and lipids. The central cell wall consists of a dense conglomeration of β-glucans, mannoproteins, chitin, and melanin. The distal layer consists of a 5–10 µm capsule primarily made of glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). (B) Transmission electron micrograph of a mother cell with a clear dark melanin-containing cell wall and a budding daughter cell. The arrow indicates the disrupted edges of the mother cell. The dark pigmentation is the melanin granules and the fibrillar structures on the cell surface are polysaccharide components of the capsule. Adapted from Mandal et al. [96] under license https://creativecommons.org/licenses/by-nc/4.0/ (accessed on 22 January 2025).

Figure 2

Stages of cryptococcal biofilm maturation and components. (A) (1) Adhesion: C. neoformans yeast cells adhere to a surface with the help of cell wall components. (2) Early formation: cells proliferate to form microcolonies; polysaccharide capsule production increases, providing a protective barrier; and extracellular matrix (ECM) deposition begins, comprising polysaccharides, proteins, DNA, and lipids. (3) Maturation: the biofilm structure becomes three-dimensional, with densely packed cells embedded in an extensive ECM. This ECM comprises key components, including GXM, GalXM, and melanin. (4) Dispersal: some cells revert to a planktonic state to colonize new areas. (B) A 63x immunofluorescent micrograph of C. neoformans 52D adhered to a glass coverslip and stained with Alexa Fluor 647-conjugated 18B7 antibody. Biofilm formation was performed according to Martinez and Casadevall with limited modifications [9].

Figure 3

Pathways of C. neoformans invasion into the brain. Spores from soil or bird excreta are inhaled into the lungs, the primary site of infection. If the infection is not cleared, C. neoformans yeasts may transverse the lungs and enter the bloodstream. This occurs either through direct fungal migration into the blood or via mobile phagocytes that engulf the fungus and transport it across barriers. Once in circulation, the fungus disseminates throughout the body, with a predilection for the central nervous system. C. neoformans crosses the blood–brain barrier via multiple mechanisms, including transcytosis, paracellular crossing, and lateral transfer. Additionally, the fungus employs “Trojan horse” strategies in which phagocytes carry fungal cells crossing the blood–brain barrier via transcellular and paracellular migration.