O ensaio de infectividade foi realizado utilizando-se macrófagos alveolares de camundongos BALB/c e leveduras de H.
capsulatum.
Resultados pouco satisfatórios foram obtidos neste tipo de ensaio com leveduras de P. brasiliensis. Após a análise das lâminas coradas duas observações foram possíveis: i) um menor
número de macrófagos permaneciam aderidos às lamínulas; e ii)
dos macrófagos aderidos muitos apresentavam vacúolo que, aparentemente continham parte das leveduras de P.
brasiliensis. Uma possível explicação para esses fatos decorre
provavelmente do tamanho das leveduras de P. brasiliensis. Após a incubação dos macrófagos alveolares com leveduras de P.
brasiliensis, as lamínulas foram lavadas. O procedimento de
lavagem das lamínulas, para retirada das leveduras não aderidas/fagocitadas, favorecia o descolamento dos macrófagos infectados das lamínulas. Consequentemente, a contagem dos macrófagos infectados ficava prejudicada pelo baixo número de macrófagos presentes nas lamínulas.
No entanto, resultados obtidos, utilizando-se leveduras de
H. capsulatum, sugerem que a integridade dos microdomínios de
membrana, dependentes de ergosterol, é fundamental para a infectividade dos macrófagos alveolares, tendo em vista que uma redução significativa (53%) na infectividade pode ser
observada após o tratamento das leveduras de H. capsulatum com mβCD.
Cossart et al. (2004) mostrou que a infecção de células Vero pela bactéria Listeria monocytogenes é reduzida em torno de 50% após a depleção de colesterol das células Vero e que, a infectividade pode ser restabelecida após adição de colesterol. Uma vez que não foi possível a aquisição comercial de ergosterol solúvel em água, adicionou-se colesterol solúvel ao meio de cultura contendo leveduras, em que as membranas apresentam baixa concentração de ergosterol após incubação com
mβCD. No entanto, o colesterol, inserido nas membranas das
leveduras, não foi capaz de restabelecer a infectividade dos macrófagos alveolares, sugerindo a necessidade do ergosterol nos microdomínios para funcionalidade destas regiões com relação a processos de “binding” e invasão da célula hospedeira.
CONCLUSÕES
Os resultados obtidos nessa tese nos permitem concluir que:
1. As análises dos microdomínios de membrana, obtidos através do fracionamento em gradiente de sacarose, demonstram a presença de DRMs em leveduras de P.
brasiliensis e H. capsulatum que, apresentam-se
enriquecidos em glicoesfingolipídeos, ergosterol, fosfolipídeos e proteínas específicas.
2. Os microdomínios de membrana de P. brasiliensis e H.
capsulatum são insolúveis mesmo quando isolados a
temperaturas elevadas como 37°C.
3. As análises protéicas demonstraram o deslocamento de algumas proteínas, como por exemplo, Pma1p e proteína de 30 kDa, das frações de microdomínios de membrana para frações solúveis do gradiente após remoção do ergosterol. Por outro lado, uma proteína de 50 kDa não e deslocado de microdomínios para as frações solúveis de membrana após
tratamento com a mβCD, sugerindo a existência de
microdomínios independentes de ergosterol para sua integridade. Esses dados sugerem fortemente a existência de duas populações de microdomínios de membrana em
leveduras P. brasiliensis e H. capsulatum: i. dependente
de ergosterol; ii. independente de ergosterol.
4. Uma possível função, para os microdomínios de membrana, dependentes de ergosterol, foi demonstrada após o tratamento de leveduras de H. capsulatum com mβCD. Nessas condições foi observada uma redução de 53% na infectividade de macrófagos alveolares por esse fungo.
REFERÊNCIAS
Alvarez FJ, Konopka JB. Identification of an N- acetylglucosamine transporter that mediates hyphal induction in Candida albicans. Mol Biol Cell. 2007;18(3):965-975.
Andreotti PF, Monteiro da Silva JL, Bailão AM, Soares CM, Benard G, Soares CP, Mendes-Giannini MJ. Isolation and partial characterization of a 30 kDa adhesin from Paracoccidioides
brasiliensis. Microbes Infect. 2005;7(5-6):875-881.
Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K. Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A. 2000;97(7):3254-3259.
Bagnat M, Chang A, Simons K. Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast. Mol Biol Cell. 2001;12(12):4129-4138.
Barbosa MS, Báo SN, Andreotti PF, de Faria FP, Felipe MS, dos Santos Feitosa L, Mendes-Giannini MJ, Soares CM. Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides
brasiliensis is a cell surface protein involved in fungal
adhesion to extracellular matrix proteins and interaction with cells. Infect Immun. 2006;74(1):382-389.
Barr K, Laine R A, Lester R L. Carbohydrate structures of three novel phosphoinositol-containing sphingolipids from the yeast Histoplasma capsulatum. Biochemistry 1984;23:5589-5596. Barr K, Lester RL. Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum. Biochemistry 1984;23:5581-5588.
Borges-Walmsley MI, Chen D, Shu X, Walmsley AR. The pathobiology of Paracoccidioides brasiliensis. Trends in Microbiology 2002;10(2):80-87.
Bretscher MS. The molecules of the cell membrane. Sci Am. 1985; 253(4):100-8.
Brown DA, Rose JK. Sorting of GPI-anchored proteins to
glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992;68(3):533-544.
Brown DA, London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol. 1998; 14:111-36.
Brown DA, London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem. 2000;275(23):17221-17224.
Brown DA. Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda). 2006;21:430-439.
Brummer E, Castaneda E, Restrepo A. Paracoccidioidomycosis: an update. Clin Microbiol Rev 1993;6:89-117.
Campbell CC. Problems associated with antigenic analysis of
Histoplasma capsulatum and other mycotic agents. Am Rev Respir
Dis. 1965;92(6):113-118.
Cossart P, Sansonetti PJ. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science. 2004;304(5668):242-248.
de Mattos Grosso D, de Almeida SR, Mariano M, Lopes JD. Characterization of gp70 and anti-gp70 monoclonal antibodies in Paracoccidioides brasiliensis pathogenesis. Infect Immun. 2003;71(11):6534-6542.
Dickson RC, Lester RL. Yeast sphingolipids. Biochim Biophys Acta 1999;1426:347-357
Dickson RC. Sphingolipid functions in Saccharomyces
cerevisiae: Comparison to mammals. Ann Rev Biochem
1998;67:27-48.
Dietrich C, Bagatolli LA, Volovyk ZN, Thompson NL, Levi M, Jacobson K, Gratton E. Lipid rafts reconstituted in model membranes. Biophys J. 2001;80(3):1417-1428.
Djordjevic JT, Del Poeta M, Sorrell TC, Turner KM, Wright LC. Secretion of cryptococcal phospholipase B1 (PLB1) is regulated by a glycosylphosphatidylinositol (GPI) anchor. Biochem J. 2005;389(Pt 3):803-812.
Dubois M, Gillis KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350-356.
Eissenberg LG, Goldman WE. Histoplasma capsulatum fails to trigger release of superoxide from macrophages. Infect Immun. 1987;55(1):29-34.
Eissenberg LG, Goldman WE. Histoplasma variation and adaptive strategies for parasitism: new perspectives on histoplasmosis. Clin Microbiol Rev. 1991;4(4):411-421.
Eissenberg LG, Goldman WE, Schlesinger PH. Histoplasma
capsulatum modulates the acidification of phagolysosomes. J
Exp Med. 1993;177(6):1605-1611.
Fewster ME, Burns BJ, Mead JF. Quantitative densitometric thin-layer chromatography of lipids using copper acetate reagent. J Chromatogr. 1969;43(1):120-126.
Gaigg B, Timischl B, Corbino L, Schneiter R. Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast. J Biol Chem. 2005;280(23):22515-22522
Gildea LA, Ciraolo GM, Morris RE, Newman SL. Human dendritic cell activity against Histoplasma capsulatum is mediated via phagolysosomal fusion. Infect Immun. 2005;73(10):6803-6811.
González A, Gómez BL, Diez S, Hernández O, Restrepo A, Hamilton AJ, Cano LE. Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins. Infect Immun. 2005;73(4):2486-2495.
González A, Gomez BL, Restrepo A, Hamilton AJ, Cano LE. Recognition of extracellular matrix proteins by
Paracoccidioides brasiliensis yeast cells. Med Mycol.
2005;43(7):637-645.
Gustafson KS, Vercellotti GM, Bendel CM, Hostetter MK. Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium. J Clin Invest. 1991;87(6):1896-1902.
Hakomori S. Glycosynapses: microdomains controlling carbohydrate-dependent cell adhesion and signaling. An Acad Bras Scienc. 2004;76(3):553-572.
Hanada K. Sphingolipids in infectious diseases. Jpn J Infect Dis. 2005;58(3):131-148.
Hogan LH, Klein BS, Levitz SM. Virulence factors of medically important fungi. Clin Microbiol Rev. 1996;9(4):469-88.
Kawai G, Ikeda Y, Tubaki K. Fruiting of Schizophyllum commune induced by certain ceramides and cerebrosides from Penicillium
funiculosum. Agric Biol Chem 1985;49: 2137-2146.
Kawai G, Ikeda Y. Chemistry and functional moiety of a fruiting-inducing cerebroside in Schizophyllum commune. Biochim Biophys Acta 1983;754:243-248.
Kawai G, Ikeda Y. Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune. Biochim Biophys Acta 1982;719:612-618.
Kimberlin CL, Hariri AR, Hempel HO, Goodman NL. Interactions between Histoplasma capsulatum and macrophages from normal and treated mice: comparison of the mycelial and yeast phases in alveolar and peritoneal macrophages. Infect Immun. 1981;34(1):6-10.
Klotz SA, Pendrak ML, Hein RC. Antibodies to alpha5beta1 and alpha(v)beta3 integrins react with Candida albicans alcohol dehydrogenase. Microbiology. 2001;147(Pt 11):3159-3164.
Kügler S, Schurtz Sebghati T, Groppe Eissenberg L, Goldman WE. Phenotypic variation and intracellular parasitism by
Histoplasma capsulatum. Proc Natl Acad Sci U S A.
2000;97(16):8794-8.
Leimann BC, Pizzini CV, Muniz MM, Albuquerque PC, Monteiro PC, Reis RS, Almeida-Paes R, Lazera MS, Wanke B, Pérez MA, Zancopé-Oliveira RM. Histoplasmosis in a Brazilian center: clinical forms and laboratory tests. Rev Iberoam Micol. 2005;22(3):141-146.
Leipelt M, Warnecke D, Zähringer U, Ott C, Müller F, Hube B, Heinz E. Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J Biol Chem. 2001;276(36):33621-33629.
Levery SB, Momany M, Lindsey R, Toledo MS, Shayman JA, Fuller M, Brooks K, Doong RL, Straus AH, Takahashi HK. Disruption of the glucosylceramide biosynthetic pathway in Aspergillus
nidulans and Aspergillus fumigatus by inhibitors of UDP-
Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth. FEBS Lett. 2002;525(1-3):59-64.
Levery SB, Toledo MS, Straus AH, Takahashi HK. Comparative analysis of glycosylinositol phosphorylceramides from fungi by electrospray tandem mass spectrometry with low-energy collision-induced dissociation of Li(+) adduct ions. Rapid Commun Mass Spectrom. 2001;15(23):2240-2258.
Levery SB, Toledo MS, Straus AH, Takahashi HK. Structure elucidation of sphingolipids from the mycopathogen
Paracoccidioides brasiliensis: An immunodominant -
galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen. Biochemistry 1998;37:8764-8775.
London E, Brown DA. Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts). Biochim Biophys Acta. 2000;1508(1- 2):182-195.
Long KH, Gomez FJ, Morris RE, Newman SL. Identification of heat shock protein 60 as the ligand on Histoplasma capsulatum that mediates binding to CD18 receptors on human macrophages. J Immunol. 2003;170(1):487-494.
Malínská K, Malínský J, Opekarová M, Tanner W. Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol Biol Cell. 2003;14(11):4427-36.
Mañes S, del Real G, Martínez-A C. Pathogens: raft hijackers. Nat Rev Immunol. 2003;3(7):557-568.
Martin SW, Konopka JB. Lipid raft polarization contributes to hyphal growth in Candida albicans. Eukaryot Cell. 2004;3(3):675-684.
Maza PK, Straus AH, Toledo MS, Takahashi HK, Suzuki E. Interaction of epithelial cell membrane rafts with
Paracoccidioides brasiliensis leads to fungal adhesion and
Src-family kinase activation. Microbes Infect. 2008;10(5):540- 547.
McMahon JP, Wheat J, Sobel ME, Pasula R, Downing JF, Martin WJ 2nd. Murine laminin binds to Histoplasma capsulatum. A possible mechanism of dissemination. J Clin Invest. 1995;96(2):1010-1017.
Mendes-Giannini MJ, Andreotti PF, Vincenzi LR, da Silva JL, Lenzi HL, Benard G, Zancopé-Oliveira R, de Matos Guedes HL, Soares CP. Binding of extracellular matrix proteins to
Paracoccidioides brasiliensis. Microbes Infect.
2006;8(6):1550-1559.
Opekarová M, Malínská K, Nováková L, Tanner W. Differential effect of phosphatidylethanolamine depletion on raft proteins: further evidence for diversity of rafts in Saccharomyces
cerevisiae. Biochim Biophys Acta. 2005;1711(1):87-95.
Paniago AM, de Oliveira PA, Aguiar ES, Aguiar JI, da Cunha RV, Leme LM, Salgado PR, Domingos JA, Ferraz RL, Chang MR, Bóia MN, Wanke B. Neuroparacoccidioidomycosis: analysis of 13 cases observed in an endemic area in Brazil. Trans R Soc Trop Med Hyg. 2007;101(4):414-420.
Pasrija R, Prasad T, Prasad R. Membrane raft lipid constituents affect drug susceptibilities of Candida albicans. Biochem Soc Trans. 2005;33(Pt 5):1219-1223.
Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res. 2003;44(4):655-67.
Pike LJ. Lipid rafts: heterogeneity on the high seas. Biochem J. 2004;378(Pt 2):281-292.
Puccia R, Schenkman S, Gorin PA, Travassos LR. Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen. Infect Immun 1986;53(1):199-206.
Rajendran L, Simons K. Lipid rafts and membrane dynamics. J Cell Sci. 2005;118(Pt 6):1099-1102.
Rappleye CA, Eissenberg LG, Goldman WE. Histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by thebeta-glucan receptor. Proc Natl Acad Sci U S A. 2007;104(4):1366-1370.
Restrepo A, Salazar ME, Cano LE, Stover EP, Feldman D, Stevens DA. Estrogens inhibit mycelium-to-yeast transformation in the
fungus Paracoccidioides brasiliensis: implications for
resistance of females to paracoccidioidomycosis. Infect Immun. 1984;46(2):346-353.
Riethmüller J, Riehle A, Grassmé H, Gulbins E. Membrane rafts in host-pathogen interactions. Biochim Biophys Acta. 2006;1758(12):2139-2147.
Salgado PR, Domingos JA, Ferraz RL, Chang MR, Bóia MN, Wanke B. Neuroparacoccidioidomycosis: analysis of 13 cases observed in an endemic area in Brazil. Trans R Soc Trop Med Hyg. 2007;101(4):414-420
Schuck S, Honsho M, Ekroos K, Shevchenko A, Simons K. Resistance of cell membranes to different detergents. Proc Natl Acad Sci U S A. 2003;100(10):5795-5800.
Silveira TG, Yoneyama KA, Takahashi HK, Straus AH. Isolation of Leishmania (Viannia) braziliensis glycolipid antigens and their reactivity with mAb SST-1, specific for parasites of Viannia subgenus. Parasitology. 2005;131(6):737-745.
Simons K, Ikonen E. Functional rafts in cell membranes.
Nature. 1997; 387(6633):569-572.
Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science. 1972; 175(23):720-731.
Singer SJ. The structure and insertion of integral proteins in membranes. Annu Rev Cell Biol. 1990; 6:247-96.
Straus AH, Travassos LR, Takahashi HK. A monoclonal antibody (ST-1) directed to the native heparin chain. Anal Biochem. 1992;201(1):1-8.
Straus AH, Levery SB, Jasiulionis MG, Salyan ME, Steele SJ, Travassos LR, Hakomori S, Takahashi HK. Stage-specific glycosphingolipids from amastigote forms of Leishmania (L.)amazonensis. Immunogenicity and role in parasite binding and invasion of macrophages. J Biol Chem. 1993;268(18):13723- 13730.
Straus AH, Freymüller E, Travassos LR, Takahashi HK. Immunochemical and subcellular localization of the 43 kDa glycoprotein antigen of Paracoccidioides brasiliensis with monoclonal antibodies. J Med Vet Mycol. 1996;34(3):181-186
Suzuki E, Tanaka AK, Toledo MS, Levery SB, Straus AH, Takahashi HK. Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies. Biochim Biophys Acta.;1780(3):362-369
Tagliari L, Toledo MS, Straus AH, Takahashi HK. Function of Fungal Glycosphingolipid Enriched Microdomains in the Macrophage Invasion. Glycobiology, 18(11), 1002-1002, 2008).
Takahashi HK; Toledo, MS; Suzuki E; Tagliari L; Straus AH. Current relevance of fungal and trypanosomatid glycolipids and sphingolipids: Studies defining structures conspicuously absent in mammals. An Acad Bras Scienc. 2009 (in press).
Taylor PR, Tsoni SV, Willment JA, Dennehy KM, Rosas M, Findon H, Haynes K, Steele C, Botto M, Gordon S, Brown GD. Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol. 2007;8(1):31-38.
Toledo MS, Suzuki E, Straus AH, Takahashi HK. Glycolipids from
Paracoccidioides brasiliensis. Isolation of a galactofuranose-
containing glycolipid reactive with sera of patients with paracoccidioidomycosis. J Med Vet Mycol. 1995;33(4):247-251.
Toledo MS, Levery SB, Straus AH, Suzuki E, Momany M, Glushka J, Moulton JM, Takahashi HK. Characterization of sphingolipids from mycopathogens: Factors correlating with expression of 2-
hydroxy fatty acyl (E)-Δ3-unsaturation in cerebrosides of
Paracoccidioides brasiliensis and Aspergillus fumigatus.
Toledo MS, Levery SB, Straus AH, Takahashi HK. Dimorphic expression of cerebrosides in the mycopathogen Sporothrix
schenckii. J Lipid Res 2000;41:797-806
Toledo MS, Levery SB, Suzuki E, Straus AH, Takahashi HK. Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Δ3-unsaturation correlates with yeast-mycelium phase transition. Glycobiology 2001; 11:113-124.
Toledo MS, Suzuki E, Handa K, Hakomori S. Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13. J Biol Chem. 2004;279(33):34655-64.
Tomazett PK, Cruz AH, Bonfim SM, Soares CM, Pereira M. The cell wall of Paracoccidioides brasiliensis: insights from its transcriptome. Genet Mol Res. 2005;4(2):309-25.
Vicentini AP, Gesztesi JL, Franco MF, de Souza W, de Moraes JZ, Travassos LR, Lopes JD. Binding of Paracoccidioides
brasiliensis to laminin through surface glycoprotein gp43
leads to enhancement of fungal pathogenesis. Infect Immun 1994;62(4):1465-1469.
Warnecke D, Heinz E. Recently discovered functions of glucosylceramides in plants and fungi. Cell Mol Life Sci. 2003;60(5):919-41.
Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin North Am. 2003;17(1):1-19, vii.
Xu X, Bittman R, Duportail G, Heissler D, Vilcheze C, London E. Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide. J Biol Chem. 2001;276(36):33540-33546.
Yoneyama KA, Tanaka AK, Silveira TG, Takahashi HK, Straus AH. Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity. J Lipid Res. 2006;47(10):2171-2178.
Zajchowski LD, Robbins SM. Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains. Eur J Biochem. 2002;269(3):737-752.
ABSTRACT
Biological membranes are constituted by a mixture of several classes of lipids. In this enviroment, sphingolipids and sterols pack tightly to form together with specific proteins a complex membrane organization known as membrane microdomains.
In order to detect the presence of membrane microdomains in yeast forms of pathogenic fungi, such as Paracoccidioides
brasiliensis and Histoplasma capsulatum, yeast forms of both
fungi were lysed by vortexing with glass beads and then incubated with Brij 98 at 4ºC. Fractions containing membrane microdomains were isolated by ultracentrifugation on sucrose gradient, and their components were analyzed by HPTLC, SDS- PAGE and Western blotting. Analysis of membrane lipids showed that about 40% of ergosterol from both P. brasiliensis and H.
capsulatum was present in the membrane microdomains, whereas
the percentage of glycosphingolipids present in P.
brasiliensis and H. capsulatum was 42% and 25%, respectively.
Analysis by SDS-PAGE and Western blotting clearly showed a protein enrichment in the membrane microdomains fraction of both fungi. It is noteworthy the presence of Pma1p, a fungal microdomain marker, and also the presence of a 30 kDa (glyco)protein which binds to laminin.
To investigate the requirement of ergosterol to mantain the integrity of membrane microdomains, it was used methyl- beta-cyclodextrin (mβCD) an agent able to efficiently extract membrane sterols. After treatment of yeasts using mβCD, it was observed the removal of 80% and 70% of ergosterol in P.
brasiliensis and H. capsulatum, respectively. After treatment
with mβCD it was observed a shift of 25% of the
glycosphingolipids from the insoluble to the soluble fraction, conversely the distribution profile of phospholipids remained
unmodified after treatment with mβCD. The protein analysis
showed the displacement of a few proteins to soluble fractions of the sucrose gradient, such as Pma1p and the (glyco)protein
of 30 kDa. On the other hand, using an anti-α5-integrin
antibody it was detected, even after the mβCD treatment, the
presence of a 50 kDa protein in membrane microdomains, suggesting the existence of microdomains that do not depend on ergosterol for their integrity. These data strongly suggest the existence of two population of microdomains: i) dependent of ergosterol for integrity maintenance of microdomains and ii) microdomains non-dependent of ergosterol for the maintenance of their functional role.
Furthermore, the biological importance of membrane microdomains was clearly demonstrated by a 53% reduction of infectivity of alveolar macrophage infectivity when yeast forms of H. capsulatum were treated with mβCD.