Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

Abstract

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

“…albicans Sap8 mutants are hypervirulent in OPC. Our previous work suggested a possible role for Saps, and in particular Sap8, in the processing of signaling mucin Msb2 in C. albicans in vitro (32). To directly assess the role of SAP8 in vivo, we examined the virulence of a ⌬sap8 strain in murine oral candidiasis.…”

“…However, only germinated cells exhibited this aggregation, suggesting that cell surface molecules that permit Sap6-mediated cell-cell aggregation are exposed upon germination. It is unclear from our experiments whether Sap proteinases have an enzymatic role in cell wall remodeling to expose these adhesins, either directly or through MAPK signaling (32,35), since experiments to epithelial cell monolayers were infected with the CAI4 and SAP deletion strains for 90 min. After extensive washing, the adherent cells were harvested using 0.1% Triton X-100 and plated on YPD agar plates, and the plates were incubated at 30°C for 24 h to determine the numbers of CFU of adherent fungal cells.…”

Interplay between Candida albicans and the Antimicrobial Peptide Armory

“…Extracellularly, it acts to protect the pathogen against AMPs. It is currently debated if secreted aspartic proteases (SAPs) are responsible for the cleavage and release of the secreted Msb2 glycodomain, referred to as Msb2* (55,59). Msb2* inactivates a wide range of AMPs, including the human cathelicidin LL-37, Hst 5, hNP-1, and hBD1 by tight binding (Fig.…”

Signaling Domains of Mucin Msb2 in Candida albicans

“…The precursor is cleaved and the exodomain released in substantial amounts into the environment during planktonic and surface growth (8,15,19). Previous studies suggested that the N-terminal, TM, and cytoplasmic domains of Msb2 convey different cellular functions (8).…”