Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

Abstract

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance.

Candida albicans Mucin Msb2 Is a Broad-Range Protectant against Antimicrobial Peptides

“…C. albicans strain ESCa3 produces a full-length hemagglutinin (HA) and V5 epitope-labeled Msb2 protein. Strain FCCa27 contains a partially deleted allele designated msb2⌬1 (encoding the 406 N-terminal residues of Msb2), which was found to be completely nonfunctional (10). YPD (1% yeast extract, 2% peptone, 2% glucose) or SD (0.67% yeast nitrogen base, 2% glucose) medium was used for growth of C. albicans strains on agar or in liquid cultures.…”

“…Strain ESCa3 produces full-length Msb2 doubly epitope tagged in its large extracellular and small cytoplasmic domains (HA and V5 epitopes, respectively); strain ESCa25 encodes Msb2 variant Msb2-⌬N, which lacks residues 33 to 481 of Msb2 (10). The extracellular domains of both Msb2 proteins (functional in AMP binding) were almost quantitatively released into the growth medium and are referred to as Msb2* and Msb2⌬N*, respectively.…”

“…The extracellular domains of both Msb2 proteins (functional in AMP binding) were almost quantitatively released into the growth medium and are referred to as Msb2* and Msb2⌬N*, respectively. The secreted HA-tagged Msb2* or Msb2-⌬N* protein fragments were purified by affinity chromatography and quantified immunologically, as previously described (10). Mucin from porcine stomach (type III, 0.5 to 1.5% bound sialic acid, partially purified powder; Sigma) was solubilized in water and autoclaved (20); the solubilized mucin was centrifuged (4,000 rpm, 30 min), and the supernatant was used in assays.…”

“…The Candida albicans Msb2 membrane protein has been designated a signaling mucin (8) because of its characteristic structure consisting of a short cytoplasmic C terminus, a transmembrane region, and a large, highly O-glycosylated exodomain, which is released in considerable amounts into the growth environment during planktonic and surface growth (9)(10)(11). Msb2 fulfills at least two functions to allow C. albicans survival and growth in the infected human host.…”

“…First, external stimuli, including surface growth and cell wall integrity, are signaled via Msb2 to trigger corresponding response or rescue pathways required for growth and hyphal morphogenesis (10,(12)(13)(14). Second, we recently found that the secreted Msb2 glycodomain (Msb2*) acts to protect C. albicans and Escherichia coli against toxicity of AMPs LL-37 and histatin-5 (10). The evidence suggested that Msb2* mediates its protective function by binding of AMPs and that this activity requires intact O-mannosylation by protein O-mannosyltransferases (Pmt proteins).…”