Citation: Mukaremera L (2023) The Cryptococcus wall: A different wall for a unique lifestyle. PLoS Pathog 19(2): e1011141. https://doi.org/10.1371/journal.ppat.1011141
Editor: Mary Ann Jabra-Rizk, University of Maryland, Baltimore, UNITED STATES
Published: February 23, 2023
Copyright: © 2023 Liliane Mukaremera. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Academy of Medical Sciences/the Wellcome Trust/ the Government Department of Business, Energy and Industrial Strategy/the British Heart Foundation/Diabetes UK/Global Challenges Research Fund Springboard Award [SBF006\1142] to LM, and the Medical Research Council Centre for Medical Mycology at The University of Exeter (MR/N006364/2 and MR/V033417/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The life-threatening fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are differentiated from all other human fungal pathogens by the presence of a gelatinous capsule as well as an unusual underlying cell wall. These organisms have both been listed on the WHO list of fungal priority pathogens with higher disease burdens and unmet research and development needs, with C. neoformans at the top of the critical fungal priority group . The aim of this review is to assess new insights into the unique attributes about Cryptococcus cell wall in relation to the pathogenic lifestyle of these important pathogens.
Cryptococcus isolates have been classified into two species: C. neoformans (serotypes A, D, and AD) and C. gattii (serotypes B and C). Most studies on the Cryptococcus cell wall were performed using C. neoformans strains and, in many cases, without specifying the serotype used. For simplicity, here, I will only use the term “Cryptococcus” to indicate C. neoformans and C. gattii.
Both the capsule and the cell wall contribute to the virulence properties of Cryptococcus and its ability to evade immune detection and killing (Table 1). The capsule and the cell wall are composed of different polysaccharides. While the capsule is mainly composed of glucuronoxylomannan and galactoxylomannan, the cell wall components include alpha- and beta-glucans, chitin, chitosan, and mannoproteins (Table 1). The capsule is an essential virulence factor of Cryptococcus, and, as a consequence, its synthesis and function have been extensively studied (reviewed in [2,3]). In contrast, little attention has been paid to the Cryptococcus cell wall and its role in pathogenesis. Yet, the cell wall plays a crucial role in capsule synthesis and organisation [4,5], and defects in the Cryptococcus cell wall result into dramatic defects in cell division and morphology, increased sensitivity to stresses, and reduced virulence [6–9]. These observations strongly indicate that the cell wall also plays an important role in the biology of Cryptococcus and is a driver of Cryptococcus infection and disease.
Cryptococcus cell wall composition differs from that of other major human fungal pathogens
The Cryptococcus cell wall is mainly composed of glucans (α-1,3-glucan, β-1,3-glucan, and β-1,6-glucan), glycoproteins, chitin, and its deacelylated form chitosan (Table 1 and reviewed in ), but also contains melanin and lipids [10,11]. Although most fungal cell walls consist of similar polysaccharides, differences between Cryptococcus cell wall and the walls of other common fungal pathogens have been observed.
β-1,3-glucans and β-1,6-glucans
Unlike Candida albicans or Saccharomyces cerevisiae, the Cryptococcus cell wall contains more β-1,6-glucan than β-1,3-glucan [3,12]. Cryptococcus β-1,6-glucan is involved in cell wall organisation through its interaction with chitin, β-1,3-glucan, and glycoproteins . In addition, mutants with defects in β-1,6-glucans form diffuse and enlarged capsules with rough edges, contrary to the smooth edges of the wild-type strains . Although Cryptococcus contains reduced amounts of β-1,3-glucan, they are nonetheless important. The one gene FKS1 encoding for β-1,3-glucan in Cryptococcus is essential. Therefore, β-glucans are vital for Cryptococcus viability and capsule organisation.
Compared to other major human fungal pathogens, Cryptococcus cells wall contains relatively high amounts of chitosan. In Cryptococcus, the wall chitosan content is 3 to 5 times higher than chitin during vegetative growth . This is similar to the less clinically common zygomycete pathogens where 65% to 95% of the chitin is deacetylated . Chitosan is also present in the ascospores of S. cerevisiae and the chlamydospores of Candida dubliniensis in small amounts, but absent in the vegetative cell wall of the yeast cells [15,16]. The cell wall of major pathogens Aspergillus fumigatus, C. albicans, and Pneumocystis jirovecii contain little or immeasurable chitosan. In Cryptococcus, chitosan is present in both in vitro-grown cells and cells isolated from infected mice [6,17], and chitosan deficiency has been associated with a reduced virulence . Therefore, the Cryptococcus cell wall is particular in containing chitosan in both vegetative growth and in vivo, and chitosan is required for Cryptococcus pathogenesis.
The cell wall structure varies between Cryptococcus yeast cells of different sizes
The fungal cell wall is a dynamic and flexible structure that change significantly in composition during normal cell growth, environmental adaptation, or during morphological transitions. When grown in standard laboratory growth conditions, Cryptococcus cells appear as a homogenous population of 5 to 7 μm “normal-sized” yeasts . In contrast, yeast cells extracted from infected tissues are of varying sizes and morphological characteristics [26,27]. This dynamic population includes greatly enlarged cells called “titan cells” (10 to 100 μm in diameter), “normal-sized” yeasts, and smaller cells (less than 4 μm of diameter) called titanides, seeder cells, and micro/drop cells [26–29]. Titan cells are so large that they may present challenges to efficient immune cell phagocytosis . In addition to differences in cell sizes, these cell populations present differences in the structure of their cell wall. Titan cells have a significantly thicker cell wall (2 to 3 μm) than normal-sized cells (0.05 to 0.1 μm)  and have increased chitin and mannose contents [17,31]. Titanides, seeder, and micro/drop cells are small, and their cell wall structure also differs significantly from normal yeasts. Drop cells are round and have a thicker cell wall , while titanides are oval and have a thin cell wall . The newly characterised seeder cell population is similar in size to titanides and have more exposed mannan than larger cells . Therefore, the host immune system must be capable of recognising Cryptococcus yeast cells with significant differences in their size as well as their capsule and cell wall composition. However, the precise role of each morphotype in the immune recognition of Cryptococcus is not fully understood.
Both the capsule and the cell wall of Cryptococcus wall influence the host immune response
Because it is enveloped by the capsule, it is not clear how the cell wall actively engages in immune activation. However, it is clear that the wall also contributes to immune recognition and the immune response to this fungus (Table 1; [17,32]). Chitin and chitosan have been associated with nonprotective immune responses [17,18], although they are in the inner layer of the cell wall and covered by other wall components and by the capsule. This is problematic in understanding how the interaction between chitin/chitosan and immune cells occurs, or whether it is triggered by intact cells or by cell wall fragments that are shed by the yeast cell. Cell wall β-1,3-glucans have been detected in the cerebrospinal fluid and serum of HIV+ patients with Cryptococcus meningitis and were associated with pro-inflammatory chemokine responses . In addition, mannoproteins recovered from the Cryptococcus culture supernatant stimulate T-cell immune responses [22,23]. Cryptococcus releases capsule polysaccharides into the extracellular space during infection and in in vitro culture, and the shed polysaccharides modulate the host immune responses . Similarly, cell wall components may be shed and interact with the host cells indirectly. It is not known whether cell wall components, other than β-1,3-glucans and mannoproteins, are also shed during Cryptococcus infection and contribute to immune stimulation.
The cell wall and limitations in the use of antifungal drugs
Echinocandin antifungal drugs (caspofungin, anidulafungin, and micafungin) inhibit β-1,3-glucan synthesis, resulting in the disruption of cell wall integrity and, ultimately, fungal cell death . Although echinocandins are active against most Candida and Aspergillus species, they are largely ineffective against Cryptococcus in vivo [34,35]. This is surprising in so far as the FKS1 gene that encodes for β-1,3-glucan synthase is essential in Cryptococcus , and this enzyme is sensitive to echinocandins in vitro . The mechanisms behind the resistance to echinocandins are not well understood.
In comparison to other yeasts, Cryptococcus cell wall contains more β-1,6-glucans than β-1,3-glucans. Could this difference in β-glucans impact the resistance of Cryptococcus to echinocandins? A previous study showed that treating Cryptococcus with caspofungin resulted in the reduction of both β-1,3-glucans and β-1,6-glucans, and concluded that inhibition of β-1,6-glucans may be an additional mechanism of action of pneumocandin . Therefore, increased β-1,6-glucans in Cryptococcus cell wall does not explain its resistance to echinocandins.
Another possibility is that in vivo cell adaptations such as the capsule and the thick cell wall could prevent access of echinocandins to their target enzyme. Studies using acapsular and melanin-deficient mutants found that the capsule and melanin were not required for the caspofungin resistance . However, lipid flippase defects in the cell membrane were associated with higher caspofungin penetration into the cell and increased caspofungin susceptibility . In response to caspofungin, Cryptococcus increased its chitin and chitosan contents , a compensatory mechanism similar that observed in Candida species and A. fumigatus . Therefore, both the cell wall and plasma membrane integrity may play a role in Cryptococcus resistance to echinocandins.
Chitin synthase inhibitors have been investigated as antifungal drugs and some (e.g. Nikkomycins) have shown in vitro and in vivo activity against fungal pathogens such as Coccidioides and Blastomyces species . These chitin synthase inhibitors do not have a strong activity against Cryptococcus, and currently, there is no chitin synthase inhibitor in clinical use.
The fungal wall is an ideal target for the development of new antifungal drugs. Cryptococcus cell wall differs in design and composition from that of other major human fungal pathogens. Although substantial work is still needed to fully understand the role of each wall component in immune recognition/evasion and/or antifungal drug resistance, information presented here emphasizes that the cell wall is a key player in Cryptococcus pathogenicity and could be a potential target of new anti-Cryptococcus drugs.
WHO. Fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization; 2022 Oct 25. p. 48.
- 2. Casadevall A, Coelho C, Cordero RJB, Dragotakes Q, Jung E, Vij R, et al. The capsule of Cryptococcus neoformans. Virulence. 2019;10(1):822–831. pmid:29436899
- 3. Wang ZA, Li LX, Doering TL. Unraveling synthesis of the cryptococcal cell wall and capsule. Glycobiology. 2018;28(10):719–730. pmid:29648596
- 4. Reese AJ, Doering TL. Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Mol Microbiol. 2003;50(4):1401–1409. pmid:14622425
- 5. Fonseca FL, Nimrichter L, Cordero RJB, Frases S, Rodrigues J, Goldman DL, et al. Role for chitin and chitooligomers in the capsular architecture of Cryptococcus neoformans. Eukaryot Cell. 2009;8(10):1543–1553. pmid:19617395
- 6. Baker LG, Specht CA, Lodge JK. Cell wall chitosan is necessary for virulence in the opportunistic pathogen Cryptococcus neoformans. Eukaryot Cell. 2011;10(9):1264–1268. pmid:21784998
- 7. Kumar P, Heiss C, Santiago-Tirado FH, Black I, Azadi P, Doering TL. Pbx proteins in Cryptococcus neoformans cell wall remodeling and capsule assembly. Eukaryot Cell. 2014;13(5):560–571. pmid:24585882
- 8. Baker LG, Specht CA, Donlin MJ, Lodge JK. Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot Cell. 2007;6(5):855–867. pmid:17400891
- 9. Gilbert NM, Donlin MJ, Gerik KJ, Specht CA, Djordjevic JT, Wilson CF, et al. KRE genes are required for beta-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans. Mol Microbiol. 2010;76(2):517–534. pmid:20384682
- 10. Wang Y, Aisen P, Casadevall A. Cryptococcus neoformans melanin and virulence: mechanism of action. Infect Immun. 1995;63(8):3131–3136. pmid:7622240
- 11. Longo LV, Nakayasu ES, Pires JH, Gazos-Lopes F, Vallejo MC, Sobreira TJ, et al. Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67. J Eukaryot Microbiol. 2015;62(5):591–604. pmid:25733123
- 12. James PG, Cherniak R, Jones RG, Stortz CA, Reiss E. Cell-wall glucans of Cryptococcus neoformans Cap 67. Carbohydr Res. 1990;198(1):23–38. pmid:2191777
- 13. Banks IR, Specht CA, Donlin MJ, Gerik KJ, Levitz SM, Lodge JK. A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell. 2005;4(11):1902–1912. pmid:16278457
- 14. Crognale S, Russo C, Petruccioli M, D’Annibale A. Chitosan Production by Fungi: Current State of Knowledge, Future Opportunities and Constraints. Fermentation. 2022;8(2).
- 15. Briza P, Ellinger A, Winkler G, Breitenbach M. Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan. J Biol Chem. 1988;263(23):11569–11574. pmid:3042773
- 16. Bemena LD, Min K, Konopka JB, Neiman AM. A Conserved Machinery Underlies the Synthesis of a Chitosan Layer in the Candida Chlamydospore Cell Wall. mSphere. 2021;6(2):e00080–e00021. pmid:33910989
- 17. Wiesner DL, Specht CA, Lee CK, Smith KD, Mukaremera L, Lee ST, et al. Chitin recognition via chitotriosidase promotes pathologic type-2 helper T cell responses to cryptococcal infection. PLoS Pathog. 2015;11(3):e1004701. pmid:25764512
- 18. Upadhya R, Lam WC, Maybruck B, Specht CA, Levitz SM, Lodge JK. Induction of Protective Immunity to Cryptococcal Infection in Mice by a Heat-Killed Chitosan-Deficient Strain of Cryptococcus neoformans. mBio. 2016;7(3):e00547–e00516.
- 19. Rodrigues ML, Alvarez M, Fonseca FL, Casadevall A. Binding of the wheat germ lectin to Cryptococcus neoformans suggests an association of chitinlike structures with yeast budding and capsular glucuronoxylomannan. Eukaryot Cell. 2008;7(4):602–609. pmid:18039942
- 20. Thompson JR, Douglas CM, Li W, Jue CK, Pramanik B, Yuan X, et al. A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function. J Bacteriol. 1999;181(2):444–453. pmid:9882657
- 21. Reese AJ, Yoneda A, Breger JA, Beauvais A, Liu H, Griffith CL, et al. Loss of cell wall alpha(1–3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. Mol Microbiol. 2007;63(5):1385–1398. pmid:17244196
- 22. Levitz Stuart M. Nong S-h, Mansour Michael K, Huang C, Specht Charles A. Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans. Proc Natl Acad Sci. 2001;98(18):10422–10427. pmid:11504924
- 23. Biondo C, Messina L, Bombaci M, Mancuso G, Midiri A, Beninati C, et al. Characterization of two novel cryptococcal mannoproteins recognized by immune sera. Infect Immun. 2005;73(11):7348–7355. pmid:16239533
- 24. Nosanchuk JD, Casadevall A. Impact of Melanin on Microbial Virulence and Clinical Resistance to Antimicrobial Compounds. Antimicrob Agents Chemother. 2006;50(11):3519. pmid:17065617
- 25. Pukkila-Worley R, Gerrald QD, Kraus PR, Boily M-J, Davis MJ, Giles SS, et al. Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade. Eukaryot Cell. 2005;4(1):190–201. pmid:15643074
- 26. Zaragoza O, Nielsen K. Titan cells in Cryptococcus neoformans: cells with a giant impact. Curr Opin Microbiol. 2013;16(4):409–413. pmid:23588027
- 27. Denham ST, Brammer B, Chung KY, Wambaugh MA, Bednarek JM, Guo L, et al. A dissemination-prone morphotype enhances extrapulmonary organ entry by Cryptococcus neoformans. Cell Host Microbe. 2022;30(10):1382–400.e8. pmid:36099922
- 28. Alanio A, Vernel-Pauillac F, Sturny-Leclere A, Dromer F. Cryptococcus neoformans host adaptation: toward biological evidence of dormancy. mBio. 2015;6(2). pmid:25827423
- 29. Dambuza IM, Drake T, Chapuis A, Zhou X, Correia J, Taylor-Smith L, et al. The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLoS Pathog. 2018;14(5):e1006978. pmid:29775474
- 30. Zaragoza O, Garcia-Rodas R, Nosanchuk JD, Cuenca-Estrella M, Rodriguez-Tudela JL, Casadevall A. Fungal cell gigantism during mammalian infection. PLoS Pathog. 2010;6(6):e1000945. pmid:20585557
- 31. Mukaremera L, Lee KK, Wagener J, Wiesner DL, Gow NAR, Nielsen K. Titan cell production in Cryptococcus neoformans reshapes the cell wall and capsule composition during infection. Cell Surf. 2018;1:15–24. pmid:30123851
- 32. Vecchiarelli A, Pericolini E, Gabrielli E, Kenno S, Perito S, Cenci E, et al. Elucidating the immunological function of the Cryptococcus neoformans capsule. Future Microbiol. 2013;8(9):1107–1116. pmid:24020739
- 33. Rhein J, Bahr NC, Morawski BM, Schutz C, Zhang Y, Finkelman M, et al. Detection of High Cerebrospinal Fluid Levels of (1→3)-β-d-Glucan in Cryptococcal Meningitis. Open Forum. Infect Dis. 2014;1(3):ofu105–ofu.
- 34. Letscher-Bru V, Herbrecht R. Caspofungin: the first representative of a new antifungal class. J Antimicrob Chemother. 2003;51(3):513–521. pmid:12615851
- 35. Abruzzo GK, Flattery AM, Gill CJ, Kong L, Smith JG, Pikounis VB, et al. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrob Agents Chemother. 1997;41(11):2333–2338. pmid:9371329
- 36. Maligie MA, Selitrennikoff CP. Cryptococcus neoformans resistance to echinocandins: (1,3)beta-glucan synthase activity is sensitive to echinocandins. Antimicrob Agents Chemother. 2005;49(7):2851–2856. pmid:15980360
- 37. Feldmesser M, Kress Y, Mednick A, Casadevall A. The Effect of the Echinocandin Analogue Caspofungin on Cell Wall Glucan Synthesis by Cryptococcus neoformans. J Infect Dis. 2000;182(6):1791–1795. pmid:11069257
- 38. Huang W, Liao G, Baker GM, Wang Y, Lau R, Paderu P, et al. Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans. mBio. 2016;7(3):e00478–e00416. pmid:27165800
- 39. Pianalto KM, Billmyre RB, Telzrow CL, Alspaugh JA. Roles for Stress Response and Cell Wall Biosynthesis Pathways in Caspofungin Tolerance in Cryptococcus neoformans. Genetics. 2019;213(1):213–227. pmid:31266771
- 40. Walker LA, Gow NAR, Munro CA. Fungal echinocandin resistance. Fungal Genet Biol. 2010;47(2):117–126. pmid:19770064
- 41. Larwood DJ. Nikkomycin Z-Ready to Meet the Promise? J Fungi (Basel). 2020;6(4). pmid:33143248