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Previous Article | Next Article 
Clinical Microbiology Reviews, January 2002, p. 21-57, Vol. 15, No. 1
0893-8512/02/$04.00+0 DOI: 10.1128/CMR.15.1.21-57.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Mycology Reference Centre, Division of Microbiology, University of Leeds and Leeds General Infirmary, Leeds, United Kingdom
SUMMARY INTRODUCTION History Taxonomy Structure, Physiology, and Biochemistry SEROLOGICAL AND ANTIGENIC STUDIES OF MALASSEZIA Serological Studies of Malassezia Species Analysis of the Antigens Present in Malassezia NONSPECIFIC IMMUNITY AND IMMUNOMODULATION BY MALASSEZIA Activation of the Complement Cascade Phagocytosis of Malassezia Immunomodulation by Malassezia COMMENSALISM Distribution of Malassezia Species on Normal Skin Skin Immune System Nonspecific immune responses. Specific immune responses. Humoral Immune Responses to Malassezia in Normal Individuals Cellular Immune Responses to Malassezia in Normal Individuals DISEASES ASSOCIATED WITH MALASSEZIA Pityriasis Versicolor Seborrheic Dermatitis and Dandruff Malassezia Folliculitis Atopic Dermatitis Other Superficial Diseases Systemic Diseases Peritonitis. Catheter-related fungemia. IMMUNOLOGICAL RESPONSES TO MALASSEZIA IN DISEASE Pityriasis Versicolor Humoral immune responses. Cellular immune responses. Lesional infiltrates. Seborrheic Dermatitis and Dandruff Humoral immune responses. Cellular immune responses. Lesional infiltrates. Malassezia Folliculitis Humoral and cellular immune responses. Lesional infiltrates. Atopic Dermatitis Humoral immune responses. Hypersensitivity to Malassezia. Cellular immune responses. Lesional infiltrates. Other Superficial Diseases Acne vulgaris. Psoriasis. Systemic Diseases CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Despite being described in 1846, the first successful isolation of the organism is generally accepted to be by Panja in 1927 (324), although several previous authors claimed to have grown the organism in vitro (83, 114, 233, 282). The difficulty in culturing the organism was explained by Benham in 1939, when she observed the need for a "fatty substance" in the growth medium (45). Once this lipid requirement had been established, it paved the way for the formulation of various culture media that could reliably recover and maintain the organism (135, 245, 288, 325, 444, 445), enabling work on the taxonomy, physiology, and biochemistry of the genus to be undertaken.
Eichstedt (118) was the first to describe the fungus associated with lesions of pityriasis versicolor (PV) in 1846, but no name was given to it until 1853, when Robin designated it "Microsporon furfur" (367). Since then it has also been placed in the genera Cryptococcus (363), Saccharomyces (56), Pityrosporum (381), Dermatophyton (114), and Monilia (452). Sabouraud was the first person to suggest that the yeast and mycelial forms might be related (381), but it was not until 1927 that Panja classified them within the same genus (324). The first official taxonomic classification placed them in the genus Pityrosporum and defined two species—P. ovale and P. pachydermatis, associated with animals (260). Gordon then added another species, P. orbiculare, differentiating this species on the basis of its round cell shape (159). By 1970, three species were recognized, P. ovale, P. orbiculare, and P. pachydermatis, and although it was accepted that there was a relationship between the yeast form and the mycelial form, conversion between them had never been demonstrated and so the two distinct genera were maintained (407). This situation was finally resolved in 1977, when three independent groups succeeding in inducing the yeast to produce hyphae in vitro (115, 303, 383). Using a variety of culture conditions, they produced hyphae that were indistinguishable from those on strains seen on patients suffering from PV. It was also observed that both the round and oval yeast forms could produce hyphae, and this led to the suggestion that the round and oval yeast forms and hyphae were simply stages in the life cycle of a single organism (383). The ability to induce the yeast to form mycelial elements paved the way for the unification of the two genera in 1986, with the acceptance of the species names Malassezia furfur (Robin) Baillon (including P. orbiculare, P. ovale, and M. furfur) and Malassezia pachydermatis (including P. pachydermatis) (79). Despite this, many workers maintained the use of the names P. ovale and P. orbiculare and continued to differentiate strains on the basis of cellular and colonial morphologies (128, 288, 402). In 1990, Simmons and Gueho defined another species, M. sympodialis, on the basis of its lower G+C content (54% compared with 66% for M. furfur) and the presence of sympodial budding (402). Cunningham et al. differentiated three serovars of M. furfur, A, B, and C, which had culture and morphological differences that corresponded to serological differences determined by cell surface antigens (101).
Thus, in the early 1990s the taxonomy of the genus Malassezia was still chaotic, with different groups tending to favor their own classification scheme, resulting in an inability to compare work carried out by different groups. This chaos was finally resolved with a seminal publication in 1995 by Guillot and Gueho (170). They assembled 104 isolates of Malassezia species encompassing all the different classifications favoured by different groups and carried out sequencing of the large-subunit rRNA and nuclear DNA complimentarity studies. On the basis of their results, they defined, and later named, seven species of Malassezia: M. furfur, M. sympodialis, M. obtusa, M. globosa, M. restricta, M. slooffiae, and M. pachydermatis (167). These currently accepted species and their corresponding names in other classifications are shown in Table 1. Several subsequent molecular studies have confirmed this classification and taxonomic grouping (173, 207, 267). The characteristics of the different species are summarized in Table 2.
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Malassezia species undergo asexual reproduction by monopolar, enteroblastic budding from a characteristic broad base. The mother and daughter cell are divided by a septum, and the daughter cell separates by fission, leaving a bud scar or collarette through which successive daughter cells will emerge (7).
The cell wall of the genus Malassezia is poorly characterized. It is very thick in comparison with other yeasts (about 0.12 µm) and constitutes 26 to 37% of the cell volume (214). The major components of the cell wall are sugars (
70%), protein (10%), and lipids (15 to 20%), with small amounts of nitrogen and sulfur (181, 439). Several workers suggested that the cell wall consisted of two layers (39, 67, 213) with indentations on the inner layer, while other workers have found multiple layers within the wall (401, 426). The most recent work on the cell wall has confirmed the presence of multiple layers, although two main layers were noted, which may explain the previous reports (293). This study also demonstrated the presence of an outer lamellar layer around the cell wall, which had previously been mentioned by other workers (234, 472) but never investigated. The lamellar layer was "membrane-like" with an electron-transparent middle enclosed by two electron-dense lines. The structure of the layer varied with different lipid sources in the medium and stained with Nile blue sulfate, suggesting that it contained lipid. The lamellar layer may play a role in adhesion of the organism to both human skin and indwelling catheters (293). The cytoplasmic membrane adheres closely to the inner surface of the cell wall and follows the indentations present (39, 293, 426, 468).
The number and shape of the mitochondria in each cell may vary (39), differing between the round and oval cell shapes (214). The nucleus has a well-defined limiting membrane (39) surrounded by a granular homogenous nucleoplasm. Vacuoles present in the cell contained lipid and varied in size according to the age of the cell (39).
The physiology of Malassezia species is poorly understood because problems with reliably culturing and maintaining the organism have hindered progress in this area. As early as 1939, Benham noted that Malassezia was unable to ferment sugars (45). The organism can use lipid as the sole source of carbon (301), does not require vitamins, trace elements, or electrolytes (279), and preferentially uses methionine as the sole sulfur source, but it can also use cystine or cysteine (71). It is able to use many amino acids, as well as ammonium salts, as nitrogen sources (279). Although the organism is normally grown in vitro under aerobic conditions, it is also able to grow under microaerophilic and anaerobic conditions (133).
The growth requirement of Malassezia for lipid was first noted in 1939 but was not studied in detail until Shifrine and Marr demonstrated the inability of the organism to form long-chain fatty acids due to a block in de novo synthesis of myristic acid, requiring the addition of preformed fatty acids (395). Subsequent work showed that the addition of most fatty acids with a carbon chain length greater than 10 supported growth and that it did not matter whether odd- or even-numbered carbon chain lengths were used (469). The lipid source used during growth affects the fatty acid composition of the organism, suggesting that the fatty acids are not used as energy sources but, rather, are incorporated directly into cellular lipids without being further metabolized (80). Wilde and Stewart further found that the lipids present on normal human scalps were able to fulfil the lipid requirement of the organism (469).
Malassezia species elaborate a range of enzymes and metabolites. They have lipolytic activity both in vitro (301, 459) and in vivo (85, 272), indicating the production of a lipase. The lipase is located in the cell wall and/or membrane sites in the cytoplasm (85, 349). Ran et al. (349) found that the pH optimum was 5.0 and that lipase production was greatest during the logarithmic phase of growth, perhaps demonstrating its importance in the hydrolysis of lipids for cell growth. In contrast, Plotkin et al. (339) found the pH optimum to be 7.5 but also found that lipase activity was greatest during active cell growth and correlated with substrate concentration. They concluded that there were at least three separate lipases in Malassezia which were essential for cell growth. Mayser et al. (277) studied the lipolytic activity of Malassezia on fatty acid esters and found that it had only minor substrate specificity, with the degree of hydrolysis being determined by the alcohol moiety. In vitro, Malassezia species also produce a phospholipase (360). This phospholipase activity is able to cause the release of arachidonic acid from HEp-2 cell lines (338). Since arachidonic acid metabolites are involved in inflammation in the skin (164), this has been suggested as a mechanism by which Malassezia species may trigger inflammation. Malassezia species produce an enzyme with lipoxygenase activity, as demonstrated by its ability to oxidize free and esterified unsaturated fatty acids, squalene, and cholesterol (304). The resultant production of lipoperoxides may damage cell membranes and consequently interfere with cellular activity—a mechanism that has been proposed to cause the alterations in skin pigmentation associated with PV (108). Cultures of Malassezia produce a characteristic "fruity" smell, first described by Van Abbe (445). Gas chromatographic-mass spectrometric analysis of the gas from the culture headspace of Malassezia grown in lipid containing medium showed it to consist of volatile gamma lactones. This characteristic was unique to Malassezia and was suggested as a possible way to differentiate this genus from others (240). Another metabolite produced is azelaic acid, a C9 dicarboxylic acid. It is produced when Malassezia is grown in the presence of oleic acid and is a competitive inhibitor of tyrosinase, an enzyme involved in the production of melanin (302). In addition to having antibacterial (190, 242) and antifungal (65) activity, azelaic acid inhibits the proliferation of several tumor cell lines (331) and decreases the production of reactive oxygen species in neutrophils by inhibiting cell metabolism (9).
| SEROLOGICAL AND ANTIGENIC STUDIES OF MALASSEZIA |
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Quantitative immunoelectrophoretic techniques in one study revealed up to 63 antigenic components in Malassezia but failed to reveal any significant antigenic differences between P. ovale and P. orbiculare (72). Thus, by 1984, several studies had confirmed the antigenic identity of P. ovale, P. orbiculare, and M. furfur, lending support to the idea that they were all stages in the life cycle of the same organism. However, Midgley (288, 289) used immunoelectrophoresis and an enzyme-linked immunosorbent assay (ELISA) to study various morphological variants and found some antigens specific for the different species and forms that she defined. Takahashi et al. (428) also found three group-specific soluble antigens, using double diffusion, in three variants of Malassezia that were differentiated on the basis of cell shape and metabolic differences.
In 1986, the weight of evidence led to the unification of the different morphological forms within the species M. furfur (79). However, the ability to grow very distinct colony variants from the same site on human skin led workers to define three serovars of M. furfur (101). The serovars, designated A, B, and C, were distinguished on the basis of growth characteristics, colony morphology, and specific surface antigens. Production of antisera to the three colonial forms and absorbtion with homologous and heterologous strains indicated the presence of both serovar-specific and common surface antigens. Recent molecular work describing the six lipophilic species of Malassezia confirms that stable variants do exist within the genus and that they are sufficiently different to be classified as separate species. The finding that the three serovars of Malassezia and the variants defined by Midgley represent distinct species confirmed the validity of their differentiation.
The capacity of Malassezia species to stimulate the immune system is well documented, but its antigenicity in comparison to other organisms has not been well studied. Sohnle and Collins-Lech (410) examined four antigenic extracts from Malassezia and Candida albicans and compared their ability to stimulate the immune system using the lymphocyte transformation (LT) assay or skin tests. They found that 20 to 100 times more extracted protein from Malassezia than C. albicans was required to stimulate the cellular immune response in the assays. The protein content of the preparations was similar for the two organisms, and they suggested that Malassezia was less antigenic than C. albicans. This limited antigenicity was proposed as a reason for the lack of inflammation seen in PV.
Recently, work has been undertaken to analyze the antigens present on the mycelial phase of Malassezia (380). The mycelial phases of two strains were induced, and antisera were raised to yeast-mycelium mixtures. Absorbtions with homologous and heterologous yeast and mycelial cells were carried out to obtain antiserum specific to the mycelial phase. Antigens common to both the yeast and mycelium were demonstrated, but all the antigens on the mycelium were present on the yeast. Thus, mycelium-specific antigens were not found. The serovar-specific antigens present on the yeast cells were not present on the mycelia, and so the mycelia did not have any phase-specific antigens, at least not on the cell surface.
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The next two antigens to be characterized, Mal f 2 and Mal f 3, were found to have masses of 21 and 20 kDa, respectively, under reducing conditions (480). Under nonreducing conditions, the masses were 42 and 40 kDa, respectively, suggesting that the antigens were dimers of a single protein, linked by disulfide bonds. There was 51% sequence homology between Mal f 2 and Mal f 3, and they had homology to peroxisomal membrane proteins of Candida boidinii and an allergen of Aspergillus fumigatus. There was no sequence homology to Mal f 1.
Mal f 4, identified by two-dimensional polyacrylamide gel electrophoresis (PAGE) and immunoblotting, was cloned and sequenced (320). The 315-amino-acid protein had a molecular mass of 35 kDa and showed 57% sequence homology to mitochondrial malate dehydrogenase from Saccharomyces cerevisiae. Sera from atopic dermatitis (AD) patients reacted with Mal f 4 in 83% of cases, indicating that it is a major antigen.
Mal f 5 has a molecular mass of 18.2 kDa and 57 and 58% sequence homology to Mal f 2 and Mal f 3, respectively (254). Mal f 6, with a mass of 17.2 kDa, has 82% sequence homology to cyclophilin from Schizosaccharomyces pombe. Both recombinant proteins were able to bind sera from patients with AD. Three other proteins, with masses of 21.3, 14.4, and 9.7 kDa, were also cloned and expressed. They did not have sequence homology to any known protein and may be incomplete cDNA clones, although the recombinant proteins from them were reactive in immunoblots. Subsequent work, using full-length cDNA clones, resulted in the description of three more antigens, Mal f 7, Mal f 8, and Mal f 9 (351). Mal f 7 encoded a protein of 141 amino acids (16.2 k Da), Mal f 8 encoded a protein of 179 amino acids (19.2 k Da), and Mal f 9 encoded a protein of 126 amino acids (14.0 k Da). None of the proteins had sequence homology to any known proteins, and the recombinant proteins reacted with sera from patients with AD in immunoblots.
An antigen recently characterized from M. globosa with a molecular mass of 46 kDa was found to be a major antigen, reacting with 69% of sera from patients with AD (230). The antigen reacted with concanavalin A in lectin blots, indicating that it was a glycoprotein. A 67-kDa antigen was also noted, that was probably a protein.
From the work reviewed above, several conclusions are apparent. First, different groups have defined a variety of antigens, ranging from low- to high-molecular-mass proteins and also high-molecular-mass carbohydrates. Second, many of the antigens have similar masses and may be identical, differing simply in the accuracy with which the mass could be assigned. While some workers have found bands of certain masses difficult to distinguish (204), others have stated that bands could be distinguished in 1-kDa intervals, especially in antigens of <50 kDa (256). Third, as already mentioned, many workers have used immunoblotting to define antigens. This is generally effective at demonstrating the presence of proteins but may be less so at demonstrating carbohydrates, either because they do not enter the sodium dodecyl sulfate (SDS) gel or because they produce diffusely stained bands. Hence, while many protein antigens have been described, with the consequent assumption that proteins are the most important antigens (200), other workers have disputed this finding. Several investigators found that mannan or other high-molecular-mass polysaccharides are also important antigens (111–113, 256, 385). A seminal paper by Zargari et al. (486) may be key to understanding the relative importance of protein and carbohydrate antigens in Malassezia. These investigators made a variety of preparations of Malassezia, using different culture media, strains, incubation temperatures, durations of incubation, and extraction procedures. These preparations were separated on SDS gels and blotted with four sera from AD patients and two monoclonal antibodies, one specific to a 37-kDa antigen and the other specific to a 67-kDa antigen of Malassezia. The protein and carbohydrate content of the preparations were determined. Several important conclusions were drawn. First, the use of solid or liquid culture media to grow the organisms had little effect on the antigens present. The most important variable was the duration of culture. The number of protein antigens decreased with increased length of culture, from the presence of a wide range of proteins at 48 h to cultures in which most proteins were lost, after 4 days or more. A 37-kDa protein antigen was found to be maintained the longest. In contrast, the carbohydrate antigens were found to remain at relatively constant levels over the 21-day culture period. Therefore, the length of culture will have a significant effect on the antigens present in Malassezia and may alter the apparent importance of the protein and carbohydrate antigens found in various studies. Another important finding is that the stability of antigens varies (258). Solutions of antigens, to be used for skin testing, were stored at different temperatures and under different conditions, and their antigenic composition was then tested by immunoblotting. The major antigens studied were the 9-, 20-, and 96-kDa proteins and mannan. Most of the IgE binding components were labile at room temperature or higher temperatures and were degraded after 1 month of storage. Addition of 50% glycerol, a widely used stabilizer, had little effect on their longevity. However, storage at 4°C preserved most of the antigens, some of them for up to 1 year.
Malassezia therefore appears to be an antigenically complex organism, which alters the antigens expressed throughout its growth cycle. In addition, different strains possess diverse antigens and different methods of extraction release different antigens (200). However, in conclusion, both protein and carbohydrate antigens are likely to be important in Malassezia, although the proportion of each may vary during the growth cycle. The protein antigens are likely to be cell wall or cytoplasmic components that can easily be detected in immunoblotting and are present in the early phase of growth. The carbohydrate antigens, probably mannans or mannoproteins, are less easy to detect by immunoblotting and are maintained throughout the growth cycle. The identification and characterization of nine defined antigens of Malassezia are important steps in our understanding of the antigenic composition of this yeast.
| NONSPECIFIC IMMUNITY AND IMMUNOMODULATION BY MALASSEZIA |
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There is limited information available about phagocytic uptake and killing of Malassezia. In vitro, neutrophils take up Malassezia in a complement-dependent process, which plateaus after 40 min (358). After 2 h of internalization, only 5% of the cells are killed, but this increases to 23% if the yeasts are pretreated with ketoconazole. The ability of neutrophils to kill Malassezia seems limited. In contrast, 30 to 50% of C. albicans yeast cells (86) and up to 80% of the cells of other fungal genera (299) are killed by neutrophils. The mechanisms by which Malassezia may resist or prevent phagocytic killing are discussed below.
The receptors involved in phagocyte-yeast cell binding have been characterized in a human monocytic cell line as the mannose receptor, ß-glucan receptor, and complement receptor type 3 via the alternative complement pathway (423). Uptake of heat-killed Malassezia yeast cells was more efficient than that of live Malassezia yeast cells, although the reason for this is unknown. Recently, it has been reported that when a monocytic cell line, THP1, was stimulated with either live or heat-killed Malassezia, the production of interleukin-8 (IL-8) was increased, while stimulation of a granulocytic cells line, HL-60, resulted in increased levels of both IL-8 and IL-1
(424). Opsonized and live Malassezia yeast cells were more stimulatory than were nonopsonised or heat-killed Malassezia yeast cells. The effects of IL-1 include the activation of lymphocytes, chemotaxis and activation of neutrophils and induction of inflammation (33). IL-8 also induces chemotaxis and activation of neutrophils and T cells. Therefore, the interaction of Malassezia with phagocytic cells may serve to amplify the inflammatory response and encourage further recruitment of phagocytic cells.
Within the skin, Langerhans’ cells are able to take up antigen and then present it to T cells, providing a link between nonspecific immunity and the specific immune response. Using monocyte-derived dendritic cells, Buentke et al. (73) recently demonstrated that whole cells of Malassezia, mannan, and an allergen, r Mal f 5, were taken up more effectively by immature dendritic cells than by mature cells. Significantly more uptake occurred at 37°C than at 4°C, indicating an active process, and IgE-mediated uptake was excluded. The uptake of the whole cells and mannan was inhibited by methyl--D-mannopyranoside, indicating involvement of the mannose receptor, while uptake of the nonglycosylated r Mal f5 was by pinocytosis.
In contrast to these findings, a study by Walters et al. (458) demonstrated that Malassezia was also able to downregulate the immune system. Various preparations (formalized whole cells, culture supernatant, and a cellular fraction) of Malassezia serovar B (synonym, M. globosa) were coincubated with keratinocytes or peripheral blood mononuclear cells (PBMC), and release of IL-1 was determined at various time points. The levels of IL-1ß released by PBMC coincubated with formalized whole cells were significantly lower than those of the negative control. The authors suggested that this depression of IL-1ß release might contribute to the lack of inflammation seen in diseases such as PV and help Malassezia to evade detection by the immune system.
A subsequent study (217) examined the effect of the three serovars of Malassezia on the production of IL-1ß, IL-6 and tumor necrosis factor alpha (TNF-) by PBMC. PBMC were isolated from four healthy volunteers and cocultured with formalized whole cells of the Malassezia serovars at different ratios (yeast cell-to-PBMC ratio = 1:1, 10:1, and 20:1). The levels of IL-1ß, IL-6 and TNF- were measured in the culture supernatants at 0, 24, and 48 h. PBMC were also cocultured with viable Malassezia cells at a ratio of 20 yeast cells to 1 PBMC, and cytokine levels were determined at 0 and 24 h. In general, although exponential-phase formalized Malassezia cells at some yeast-to-PBMC ratios stimulated increased cytokine production over background levels, stationary-phase cells caused no change or significantly depressed cytokine production at every ratio tested. Viable yeast cells of all three serovars of Malassezia also significantly depressed IL-1ß, IL-6 and TNF- production.
A recent study examined the mechanisms by which this depression of cytokine production by PBMC might be mediated (216). Stationary-phase Malassezia cells were treated with solvents to remove some of the lipid present in the cell wall and the capsule-like layer around the cells. Untreated and solvent-treated cells were then cocultured with PBMC at a ratio of 20 yeasts to 1 PBMC, and cytokine levels in the culture supernatants were determined after 24 h. PBMC cocultured with solvent-treated Malassezia produced amounts of IL-1ß, IL-6 and TNF- that were similar to or significantly greater than constitutive levels. Thus, the removal of lipid from Malassezia ablated its ability to suppress cytokine production by PBMC, and so the lipid in the cell wall and capsular-like layer may be responsible for the lack of inflammation associated with Malassezia in its commensal state.
This ability of Malassezia species to downregulate the production of proinflammatory cytokines is in marked contrast to the effects of most other organisms. Gram-negative bacteria are known to cause overproduction of TNF-, IL-1, and IL-6, both in vitro and in vivo. This is primarily the cause of septic shock in gram-negative infections and is mediated by lipopolysaccharide (442). Cell wall preparations of gram-positive bacteria, consisting mainly of teichoic acid and peptidoglycan, have also been shown to induce the synthesis of TNF- and IL-6 by monocytes (187). C. albicans induces TNF- and IL-6 production by human monocytes (201) and arachidonic acid release from alveolar macrophages (84). Viable C. albicans cells preferentially induce TNF-, while heat-killed cells preferentially induce IL-1 (201). Aspergillus fumigatus hyphae and conidia also induce the production of IL-1 and TNF- from mouse macrophages, but in contrast to C. albicans, viable and nonviable organisms do not differ in their effects (433). From this, it can be seen that the effects of Malassezia on cytokine production by mononulear cells appear to be atypical in comparison to most organisms. Malassezia, however, does have similarities to another pathogenic fungus, Cryptococcus neoformans. The effects of C. neoformans on cytokine production by phagocytes are variable, depending on the presence of the polysaccharide capsule. Acapsular mutants induce significant levels of TNF- and IL-1 (449), while the presence of the polysaccharide capsule downregulates this effect (96). In many ways, this parallels the effects documented with Malassezia, with the lamellar layer downregulating the production of proinflammatory cytokines in much the same way as the capsule of C. neoformans does.
As discussed above, phagocytic killing of Malassezia is a very inefficient process, with only 5% of internalized cells killed after 2 h (358). One possible reason for this limited killing ability of phagocytes may be the production of azelaic acid by Malassezia. Akamatsu et al. (9) examined the effects of azelaic acid on chemotaxis, phagocytosis, and production of reactive oxygen species by neutrophils. They found that chemotaxis and phagocytosis were not affected by azelaic acid, but that the production of O2- and OH· was decreased in a dose-dependent manner and H2O2 production was also reduced. The effects were due to inhibition of cellular metabolism. Azelaic acid has also been shown to scavenge oxygen radicals (146). At present it is not known if azelaic acid is produced in vivo, but it is interesting to speculate that if it is, it may well be involved in protecting the organism from the oxidative killing mechanisms utilized by phagocytes.
A recent preliminary report has also suggested that the lipids associated with the cell wall of Malassezia may be antiphagocytic and involved in protection against killing by neutrophils (H. R. Ashbee, Z. L. Alvarado-Alvarez, Z. Whitehead, and E. G. V. Evans, Proc. Fourth Congr. Eur. Confed. Med. Mycol., abstr. 5, 1998). Yeast cells that had been treated with solvents had significantly greater uptake by neutrophils, leading to an increase in nitroblue tetrazolium reduction, a measure of oxygen radical release. Therefore, the lipid-rich layer around Malassezia may have parallels with the capsule of C. neoformans, which is known to protect the organism against phagocytosis (231). Although Malassezia cells on normal skin are unlikely to come into contact with professional phagocytic cells, neutrophils are present in the inflammatory infiltrates of SD (334) and macrophages are present within the infiltrate of PV (66). Therefore, Malassezia may be exposed to phagocytic cells at certain times.
The apparently contradictory ability of Malassezia to either upregulate or suppress the immune response directed against it has only recently been studied in detail. Understanding how this immunomodulation occurs may well be key to understanding how species of Malassezia occur both as commensals and as pathogens.
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Several recent studies have examined the distribution of the newly defined species of Malassezia on healthy adult human skin. The findings, summarized in Table 4, vary significantly among studies, and there are two possible explanations for this. First, there are genuine differences in the distribution of species on the skin of individuals in different countries, and this has been previously suggested (290). However, even the two studies carried out in Spain show very different results. A second explanation is that the use of swabbing, a nonquantitative and relatively insensitive method, is simply not able to produce the quantitative data needed to determine which species predominate at the different sites studied. Quantitative data on the distribution of the new species on human skin is therefore still awaited.
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The increasing recognition of Malassezia as a cause of catheter-related fungaemia in premature neonates has provided the impetus to study colonization rates in premature and full-term neonates. The colonization rates recorded range from 37% (344) to 100% (247) in hospitalized neonates. Factors such as young gestational age (8, 29, 44, 344), low birth weight (8, 344), and extended periods of hospitalization (8, 29, 44, 344) may predispose to colonisation in this group. To date, however, no systematic survey of colonization rates has been undertaken in healthy newborns, for whom the picture remains unclear.
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In addition to the barrier function of the skin and its commensal flora, phagocytic cells are important in the nonspecific cutaneous immune response. In skin diseases where significant inflammation occurs, neutrophils occur within the lesions and may lead to an accumulation of mononuclear cells in the dermis (455). Phagocytic cells may then attack organisms by using either oxidative or nonoxidative mechanisms, leading to their removal (454). Several organisms, including dermatophytes, Candida species, and propionibacteria, activate the alternative pathway of the complement cascade, causing the production of molecules with chemotactic activity for neutrophils (106, 353, 462). In this way, neutrophils may be recruited into the skin by the presence of microorganisms.
Other factors involved in the nonspecific immune system of the skin are the lipids found on adult scalps and adult hair, which are fungistatic for certain dermatophytes (54), fungicidal proteins present in the epidermis (210), and the inhibitory effect of unsaturated transferrin on various fungi (23, 222).
Specific immune responses. In addition to these nonspecific immune functions, the skin is involved in specific immune responses. The skin immune system consists of both cellular and humoral components. The cellular skin immune system includes keratinocytes, Langerhans’ cells, mononuclear cells, mast cells, endothelial cells and T lymphocytes, while humoral components include complement proteins, IgG and IgA, and various cytokines.
The way in which antigens from the skin surface, including those from commensal organisms and superficial pathogens, elicit a specific immune response has been the subject of intense research, and a clearer picture of the mechanisms involved has now emerged (60–63, 396, 419, 420). Langerhans’ cells are bone marrow-derived dendritic cells that form a network within the epidermis and are capable of presenting antigen. Immature Langerhans’ cells express low levels of major histocompatibility complex class II (MHC II) and are capable only of presenting antigen to primed T lymphocytes. Under the influence of TNF- and granulocyte-macrophage colony-stimulating factor released by keratinocytes, Langerhans’ cells that have processed antigen undergo maturation and migrate to the draining lymph nodes. Here they become potent immunostimulatory cells and prime antigen-specific T lymphocytes. Vascular endothelial cells, lining blood vessels present in that area of the skin, begin to express intercellular adhesion molecule 1, due to the release of IL-1 and TNF- by keratinocytes. The primed T lymphocytes are then able to adhere to the endothelial cells and migrate out of the veins, via diapedesis, into the surrounding dermal tissue. Once in the dermis, the T lymphocytes secrete gamma interferon, which causes further expression of intercellular adhesion molecule 1 on the endothelial cells and increased MHC II expression on keratinocytes and Langerhans’ cells. Macrophages are drawn into the skin and function as antigen-presenting cells to primed T lymphocytes, so amplifying the response.
One group of cells that are noticeably absent from the skin are B lymphocytes. Despite this, it is known that immunoglobulins specific to the commensal flora are produced in normal individuals (30, 100, 194) and that IgG, IgM, IgE, and secretory IgA are present in human sweat (150, 196, 322), providing a readily available route to gain access to the organisms on the skin. Metze et al. (283) used immunohistochemical techniques to demonstrate that commensal organisms (Malassezia species, Corynebacterium, and cocci) present on the skin surface of normal individuals were coated with immunoglobulins. Thus, antibodies are produced against the commensal flora and are able to reach and attach to these organisms on the skin.
In 1983, the serum antibody titers to Malassezia, C. albicans, and Trichophyton rubrum were determined in 21 young subjects (aged 23 to 44 years) and 20 elderly subjects (aged 70 to 88 years), none of whom had a "significant" history of superficial fungal infections (413). A tube ELISA was used to determine the titers of IgG, IgA, and IgM and found that all three classes of immunoglobulins to all three organisms were present in both the young and elderly groups. Comparison of the data for young and elderly subjects revealed that the responses were very similar, except for Malassezia, where the levels of IgM were significantly lower in the elderly subjects (P < 0.01).
Faergemann examined titers of antibodies in normal subjects, measured using an indirect-immunofluorescence (IIF) technique (123). Sera were included from 21 adults and 36 children aged 6 months to 15 years. The titer in adults was significantly higher than that found in the children (P < 0.01), possibly due to the sparsity of Malassezia on the skin of the children.
Levels of Malassezia-specific IgG were determined by IIF in the sera of normal subjects ranging from 29 to 81 years of age (48). The titers decreased with increasing age (P = 0.002), with the highest titer being found in 29- to 31-year-old subjects and lower titers being found in older subjects. This paralleled the decreasing population densities of Malassezia on the skin, and so the authors suggested that antibody titers reflected the level of skin colonization.
The sensitivities of IIF and ELISA were compared for their ability to detect IgG specific to Malassezia (203). Sera from 10 healthy adults and five healthy 6-month-old children were assayed using both methods. Titers in the ELISA were much higher than the corresponding titers in the IIF technique, demonstrating that ELISA was the more sensitive method. Additionally, titers of IgG specific to Malassezia were substantially lower in the 6-month-old children than in the adults, although no statistical analysis was carried out on the results.
Cunningham et al. also used ELISA to determine antibody titres to Malassezia serovars A, B, and C in normal individuals of various ages (100). Sera were obtained from 50 nonatopic females with no history of dermatoses. Five age groups were included: 2 to 3 years, 7 to 10 years, 20 to 24 years, 33 to 40 years, and 60 to 64 years, with 10 individuals in each. Titers of IgG and IgM were determined for all the subjects, and titers of IgA were determined for 36 subjects. IgM was present in the sera of 2- to 3-year-old children at levels comparable to those in adults. The titers of IgM were similar for all age groups, except the 60- to 64-year-old group, where they were significantly lower (P < 0.05). IgG titers did not differ significantly between age groups. IgA was not detectable in 18 sera, and its levels were low in all groups, with no differences between age groups or serovars.
The most recent study to define humoral immunity to Malassezia included 868 serum samples from subjects ranging from 0 to 80 years of age (139). However, the subjects were included "independently of the presence or absence of signs of disease attributable to the fungus," so the results may not be representative of healthy individuals. Antibodies to Malassezia, detected by immunoelectrophoresis, were present in 31% of the samples, with none in children younger than 11 years and the highest prevalence in the 31- to 40-year-old group. No statistical analysis was performed and so it is not known whether the differences were statistically significant. These results contrast with the findings of other groups, who have detected immunoglobulins in children, and may be a reflection of the relative insensitivity of immunoelectrophoresis.
Despite the variety of methods and different antigen preparations used in these studies, some consensus has emerged from the results. The majority of individuals have some antibodies to Malassezia, even from a relatively young age, although a few studies perhaps did not detect them because of the methods used. Antigen is presented to the immune system over a sufficient period to initiate both naive (IgM) and anamnestic (IgG) responses. Levels of IgA are generally low, suggesting that mucosal sensitisation by Malassezia is not an important route.
In the commensal state, Malassezia usually occurs as yeast cells, although mycelium may also be seen (288). Because of this and because of the difficulty in producing the mycelial phase, no investigators have determined the humoral response to mycelium. Recently, this issue was addressed by Saadatzadeh (380), who induced the mycelial phase and used whole mycelial antigens in IIF. Titers of total immunoglobulins, IgM, IgG, IgG subclasses, and IgA were determined in sera from 12 normal healthy adults. All the classes of immunoglobulins were detected, with the highest titers being found for IgG. Appreciable levels of IgM, IgA, and the IgG subclasses were also found. Thus, although the relatively insensitive method of IIF was used, significant levels of humoral immunity to the mycelial phase of Malassezia could be detected in normal individuals. Despite the limited amount of mycelium on normal skin, the immune system recognizes and responds to mycelial antigens. This may be due to the presence of common antigens, shared either with the yeast phase of Malassezia or with other commensal organisms.
In an attempt to gain a clearer picture of cellular immunity to Malassezia in normal individuals, data from control subjects in various studies have been collated in Table 5. From this it can be seen that Malassezia elicits significant cellular immunity in normal individuals. The similarity in responses seen in the different age groups studied (from 8 to 61 years of age) suggests that the levels of cellular immunity remain fairly constant throughout life, although its persistence in individuals older than 61 years has not been studied. It may be that, as occurs with humoral immunity, the level of cellular immunity falls in elderly individuals.
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The presence of Malassezia on the skin as a commensal and the measurable humoral and cellular immune responses to Malassezia in healthy individuals with no history of skin disease present unusual problems when studying patients with Malassezia-associated diseases. To provide convincing evidence that Malassezia may be involved in the disease, the immune response in patients must be significantly different from that in healthy controls. However, the response seen in healthy controls will vary according to their age and possibly the amount of Malassezia that they carry on their skin. Therefore, it is essential in any study of the microbiology or immunology of Malassezia that controls be carefully selected to match both the age and sex of the study population. Comparison of results between groups not matched in this way is meaningless.
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The mycology of PV has been extensively studied. The association of the mycelial form of Malassezia with PV lesions was made as early as 1871 (308), when Neumann described hyphae from lesions. Moore successfully cultured Malassezia from PV lesions in 1938 (296), and Gordon described the round yeast form associated with the lesions of PV and normal skin (159). In 1969, Roberts (365) examined 25 patients with PV at presentation and included a further 62 patients retrospectively. Lesions were scraped in 25 patients, and Malassezia was found in all of them by microscopy; however, the proportion of yeast and mycelium varied. For the 27 patients for whom samples were available, 25 samples were culture positive on malt agar overlaid with olive oil. P. orbiculare was isolated in all 25 cultures; in 6 of them it coexisted with P. ovale. Roberts found filaments in some of the samples from clinically normal skin of patients and suggested that filament production had to occur on a "massive scale" to produce clinical lesions of PV.
The following year, McGinley et al. (280) studied 31 patients with PV to assess the proportion of mycelium and yeasts present on normal and lesional skin. The mean count of mycelial elements on lesions was 295,300 cm-2, compared to 155,900 cm-2 yeast forms, i.e. a 2.1:1 ratio. On normal skin this pattern was reversed, with 18,900 yeasts cm-2 and 5,800 mycelia cm-2, a ratio of 1:0.21. In addition, McGinley et al. found that the corneocyte count on lesions was three times higher than that on normal skin, and this provided increased space for the organisms to colonize. They concluded that Malassezia was the causative organism and that the mycelial form of the organism was "instrumental in creating the lesion" of PV.
In 1979, Faergemann and Bernander (132) collected samples from 30 patients with PV, using curettes to scrape both lesional and normal skin. They recovered P. orbiculare from the lesions of all patients and from normal skin of 24 patients but did not isolate P. ovale from any sample. They suggested that P. ovale might not be common in the Swedish population where the study was carried out.
Ashbee et al. (27) carried out a study of the microbiology of PV and took samples from sites on the trunk and head of 10 patients. They measured the total amount of Malassezia and the amount of each serovar (A, B, and C) at each site in an attempt to determine if any particular serovar was associated with the lesions of the disease. They found that although serovar A predominated on the trunk, this was the same in both patients and controls and did not reflect association with the disease. No one serovar predominated on the head sites. Overall, there was no difference in the total population density of Malassezia or the distribution of serovars on lesional skin compared to control skin at the same sites. These findings appear to conflict with those of McGinley et al., but whereas McGinley et al. recorded total counts by microscopy, the Ashbee study used viable counts on culture; hence, the results are not directly comparable.
Since the differentiation of the new species of Malassezia, several groups have published studies examining the mycology of PV (94, 95, 172, 300). The results of these studies are summarized in Table 6. Overall, M. globosa has been isolated from between 25 and 97% of the patients, and some of the authors have suggested that this species may be implicated in the pathogenesis of PV. However, because these studies used qualitative samplin
