In the present study we examined multiple virulence factors such as extracellular secreted hydrolytic enzymes and biofilm development which contribute to the ability of Malassezia species to colonize host tissues, and cause disease. However, a few number of studies focused on virulence factors’ expression and pathogenesis of the Malassezia species.
Our results demonstrated that all Malassezia species included in our study, like many other fungal and bacterial species [15] produced biofilms through a discrete sequence of events, including fungal surface adhesion, microcolony formation, and biofilm maturation.
A better knowledge of the mechanisms of antifungal drug resistance may lead to the development of novel therapies for biofilm-based diseases. Multiple mechanisms have been proposed for the biofilm resistance phenomenon [16]. Metabolic quiescence has been proposed as a mechanism of antimicrobial resistance in biofilm bacteria [17] and fungi [16]. However, our data revealed that CVS activity showed biofilm cells to be metabolically active in our Malassezia species, in line with data previously reported for Candida albicans and C. parapsilosis [18]. It is therefore unlikely that biofilm production is a major factor promoting antifungal resistance of Malassezia species [16].
Biofilm production was studied only for M. furfur [19, 20] and M. pachydermatis [21]. Angiolella et al, demonstrated that biofilm adherence and hydrophobicity are considered as virulence factors in Malassezia furfur species [20].
Also, these studies showed that M. pachydermatis strains from dogs with or without skin lesions are capable to form biofilm with variable quantity and structures which are likely to be strain-dependent [21].
In our study, we showed that all Malassezia species produced lipase and 95% of M. globosa showed a very high enzymatic activity. In fact, lipases catalyze the hydrolysis of ester bonds of triacylglycerols, resulting in the release of fatty acids. In almost all organisms, lipases play essential roles in lipid metabolism, including digestion, transport, and the processing of dietary lipids. Lipases also play important roles in the virulence of skin-associated lipophilic fungal pathogens of the Malassezia spp. The gene that encodes for lipases in M. furfur , M. pachydermatis , and M. globosa has been identified, and recent genome sequencing efforts have revealed at least 14 lipase-encoding genes in M. globosa [21].
Weerapong et al suggested that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth [12].
Interestingly, we showed a high lipase activity from M. globosa strains isolated from pityriasis versicolor. In previous study, M. globosa, one of the most frequently isolated Malassezia spp. from patients with dandruff and seborrheic dermatitis, displayed extracellular lipase activity [9, 22, 23].
Phospholipase activity is another virulence factor exhibited by pathogenic microorganisms which permits to hydrolyze one or more ester linkages in glycerophospholipids, resulting in the release of free fatty acids.
In this study, all strains had the ability to express phospholipase expect for M. restricta with only 70% were phospholipase producers. Additionally, we noted a high phospholipase activity for Malassezia isolates recovered from Neonates group samples.
This virulence factor intrinsic to Malassezia yeasts has been discussed in association with the pathogenesis of seborrheic dermatitis. The increased level of production of phospholipase has been shown only for pathogenic M. pachydermatis strains [10].
Current information on phospholipases in Malassezia spp. is limited. Juntachai et al. detected extracellular phospholipase activities in M. furfur, M. pachydermatis, M. slooffiae, M. sympodialis, M. globosa, M. restricta, and M. obtusa. However, enzyme activity was higher only in M. pachydermatis [24].
The ability to express enzymes not only varies among different species of Malassezia but also differs among the strains of same species isolated from different clinical manifestations. Interestingly, we showed that strains isolated from different groups patients produced phospholipase. The production of Malassezia phospholipases on the skin could result in the removal of epidermal lipids, disruption of the epidermal barrier function, and the development of seborrheic dermatitis when sebum production is constitutionally decreased [10].
The keratinases are proteolytic enzymes in nature. They mainly attack the disulfide bond of the keratin substrate [25, 26].
As previously reported the dermatophytes and non-dermatophytes species were both keratinase producers and they are capable of damaging the keratinized structure of the skin [26-28]. In our study, all M. slooffiae isolates were keratinase producers. However, only the half of M. furfur has a positive enzymatic activity. Peyton et al found that M. furfur is capable of degrading keratin [26, 29]. But, Muhsin et al reported negative keratinase activity in M. furfur [26].
In our study we found a positive correlation between the secretion of keratinase and phospholipase. These virulence factors appear to act synergistically to contribute to the virulence of Malassezia strains.
The genome sequencing data revealed that M. globosa possesses a total of 14 lipases and 9 phospholipases. Among them, four lipases (MGL_3878, MGL_3507 MGL_0799, and MGL_0798) and two phospholipases (MGL_4252 and MGL_3326) were expressed on human scalps [30]. Few studies were carried out to analyze expression of lipases and phospholipases of the fungus [31]. However, to our knowledge no information is currently available for expression of keratinase. In our study, the RT-qPCR analysis of expression and copy number of the 3 genes responsible for the virulence of 20 M. globosa, showed the overexpression of one or more genes in 5/5 of folliculitis strains and 7/8 in pityriasis versicolor isolates. The phospholipase highest expression level was 12.4 folds shown in isolates collected from folliculitis. Moreover, we noted a statistical significant difference of the lipase gene expression (P = 0.072) was associated with the strains collected from patient with folliculitis vs group control. These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus. The concurrently overproduction of the three genes was observed in a five strains isolated from patients with folliculitis and pityriasis versicolor. However, overexpression was noted in only 2 cases of control group suggesting that theses enzymes play an important role in the pathogenicity of Malassezia disorder inducing a transition from colonization to infection. Interestingly, further studies evaluated the expression of lipases and phospholipases of Malassezia restricta in patients with seborrheic dermatitis and suggested that majority of the patients displayed expression of these enzymes [32].
Moreover, Patino-Uzcategui et al. found a significant higher expression of MGL 0797, MGL 0798, and Mflip1 virulence genes in Seborrheic Dermatitis HIV patients. They concluded that lipases may be related to the development of Seborrheic Dermatitis and could be considered as virulence factors [8].
In the study of Gharehbolagh et al, the Real-time PCR was applied to investigate the contribution of the MGL_3741 gene to pathogenicity of Malassezia globosa in pityriasis versicolor. These authors revealed that this gene can be related to the pathogenicity of this yeast and it can be a candidate for new antifungal investigation with better action to treatment of pityriasis versicolor [33]. However, further studies are steel needed to determine this role.