Aspartyl protease MfSAP2 is a key virulence factor in mycelial form of skin fungi Malassezia furfur

Abstract

Malassezia is the dominant genus of fungi residing on human skin and is associated with both healthy skin and many dermatological conditions. Among these skin diseases, pityriasis versicolor (PV) has strong etiological connections with Malassezia. In the hyper- or hypo-pigmented scales of PV patients, Malassezia is enriched in its mycelial form, which is rarely present on healthy skin. How these Malassezia hyphae contribute to disease pathology in PV is unknown. In this study, we observed a distinct shift in the extracellular proteolytic activity when Malassezia furfur transitions from yeast to hyphae. We identified that the expression of the aspartyl protease MfSAP2 is dramatically up-regulated at both the mRNA and protein level when M. furfur is in the mycelial form. We determined the protease substrate specificity and observed that MfSAP2 can degrade corneodesmosome proteins, which are intercellular adhesive proteins between corneocytes in the stratum corneum. In a 3D human skin model with MfSAP2 treatment, we observed clear degradation of corneodesmosin, a component of the corneodesmosome. Taken together, our study demonstrates that a secreted protease is a key virulence factor associated with M. furfur mycelium and is potentially involved in the disease pathogenesis of PV.

Keywords: protease; skin barrier; skin microbiome; virulence factor.

Figure 1. Optimization of culture conditions to induce M. furfur hyphae formation in vitro.

(A) Scanning electron microscopy images of M. furfur CBS 6001 cultures on 1.5% mDixon (left), minimal media (center) or minimal media supplemented with kojic acid and L-DOPA (minimal media ++, right). All cultures were kept in microaerobic conditions. Hyhae are indicated by arrows. Scale bar represents 1 µm. (B) Light microscopy images of M. furfur CBS 7019 culture in 0.5% agar containing four different media. The upper panel shows cultures kept at atmospheric conditions and the lower panels show culture in microaerobic conditions. Scale bar represents 10 µm. (C) Picture of each M. furfur CBS 7019 agar culture plate kept at atmospheric conditions.

Figure 2. Extracellular protease activity in hyphae-containing cultures is distinct from yeast culture.

(A) Proteolytic activity of culture supernatant isolated from M. furfur CBS 7019 agar cultures grown in 4 different media in microaerobic condition. Protease activity was assessed using internally quenched fluorescent substrates. (B) Heatmap of extracellular protease activity of M. furfur CBS 7019 cultures. The cultures were kept at either atmospheric or microaerobic conditions, either 0.5% or 1.5% agar in four different media. Protease activity of each substrate is normalized to S5 activity. Heatmap and hierarchical clustering are performed using R studio version 1.2.5042. (C) Correlation between protease cleavage of substrate S6 and percentage of hyphae in M. furfur CBS 7019 cultured in minimal broth media and incubated at 32°C at atmospheric conditions.

Figure 3. Aspartyl proteases are involved in hyphae formation.

(A) Inhibition of extracellular protease activity using class-specific protease inhibitors. Culture supernatant from M. furfur CBS 7019 cultured in 0.5% agar, MM ++ media at microaerobic condition was treated with either DMSO, aspartyl protease inhibitor pepstatin A (50 µM), serine protease inhibitor AEBSF (2 mM) or metalloprotease inhibitor EDTA (10 mM) and the residual proteolytic activity was assayed using the indicated fluorescent substrates. N = 3 technical replicates. (B) Effect on % hyphae formation when control, DMSO or pepstatin A (5 and 50 µM) were added to broth M. furfur CBS 7019 cultures. The extracellular protease activities of these cultures were determined using fluorescent substrates in (C). P>0.05 for all substrates when comparing control with DMSO, and P<0.05 when comparing all substrates at 5 uM and 50 uM treatment to control (using Mann-Whitney test). N = 3 biological replicates. Error bars represent S.D.

Figure 4. Expression of M. furfur aspartyl protease FUN_000222 (MfSAP2) is highly up-regulated during mycelial formation.

(A) mRNA expression of the 5 extracellular aspartyl proteases in M. furfur CBS 7019 cultured in different conditions. Expression of each protease is first normalized to the housekeeping gene (actin) and then normalized to the control culture condition (mDixon). N = 3 biological replicates for each condition. Error bars represent S.D. (B) Aspartyl protease expression of the mycelial-forming CBS 7019 and the non-mycelial forming CBS 14141. Aspartyl proteases are first enriched from each culture media using pepstatin-agarose and the isolated proteins are ran on SDS-PAGE. (C) Comparison of the enriched aspartyl proteases in LNA media for CBS 14141 and CBS 6001 (mycelial-forming). Each of the protein bands are subjected to in-gel digestion followed by mass spectrometry and the identified proteases are indicated.

Figure 5. Recombinant expression of MfSAP2 reveals its role in degradation of corneodesmosome proteins.

(A) Recombinant expression and purification of MfSAP2 in Pichia pastoris. Culture supernatant (input) was concentrated and purified first on cation exchange column (IEX) followed by size exclusion chromatography (SEC). (B) Cleavages detected at each amino acid position when recombinant MfSAP2 (rMfSAP2) was incubated with a pool of synthetic 14-mer peptides. Cleavages were monitored on LC-MS. (C) Substrate cleavage profile represented by iceLogo of the recombinant enzyme. The site of protease cleavage is indicated by the red arrow. (D, E, and F) Degradation of human skin epidermal proteins in vitro. Whole human skin epidermis (20 µg) was lysed and treated with different concentrations of rMfSAP2. The skin proteins were detected using western blot. β-actin was used as the loading control. (G) MfSAP2 degradation of cornedesmosin in 3D human skin equivalents. rMfSAP2 was applied on the skin cultures at three concentrations for 24 hr and harvested. Histology sections were obtained and immunohistochemistry was performed using antibodies directed against CDSN. Scale bar represents 20 µm.