Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

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

Msb2*-mediates protection of <i>C. albicans</i> and <i>E. coli</i> against histatin-5.

<p><i>C. albicans</i> strains CAF2-1 (wt), FCCa27 (<i>msb2</i>Δ<i>1</i>), REP18 (<i>msb2</i>Δ<i>0</i>) and <i>E. coli</i> DH5αF′ were allowed to react with the indicated amounts of histatin-5 for 1.5 h at 37°C, in the absence or presence of the affinity-purified secreted Msb2* protein. Colony-forming units were determined on YPD (<i>C. albicans</i> strains) or on LB medium (<i>E. coli</i>).</p

<i>C. albicans</i> strains.

<p><i>C. albicans</i> strains.</p

Secretion and processing of Msb2.

<p><i>C. albicans</i> strains grown in YPD medium to OD₆₀₀ = 6, centrifuged and cell extracts (50 µg protein derived from cells in 90 µl of medium) or medium (20 µl) were analyzed for epitope-tagged Msb2 protein. <b>A.</b> Immunoblot to detect HA-tagged Msb2. Proteins were separated by a 8% SDS-PAGE gel and immunoblots were reacted with rat anti-HA antibody. Strains tested included ESCa8 (<i>ACT1p</i>-<i>MSB2</i>^HA; lanes 3 and 8), ESCa3 (<i>ACT1p</i>-<i>MSB2</i>^HA-V5; lanes 4 and 9), ESCa9 (<i>MSB2p</i>-<i>MSB2</i>^HA; lanes 5 and 10) and ESCa10 (<i>MSB2p</i>-<i>MSB2</i>^HA-V5; lanes 6 and 11). Strains CAF2-1 (wt) and CIS23 (<i>PMT1</i>^HA) were used as negative and positive control strains, respectively. The migration of HA-tagged Msb2 and Pmt1 are indicated by the arrow and triangle, respectively. <b>B.</b> Immunoblot to detect V5-tagged Msb2. Proteins were separated by a 4–20% gradient SDS-PAGE gel and immunoblots were reacted with mouse monoclonal anti-V5 antibody. Identical strains and fractions as in (A) were tested. The migration of V5-tagged Msb2 and Pmt2 (strain CIS29) are indicated by the arrow and the triangle, respectively; a protein cross-reacting with the anti-V5 antibody is marked by the asterisk. <b>C.</b> Secretion of HA-tagged Msb2 protein during growth on agar. Cell suspensions were dropped on a membrane filter (pore diameter 0.45 µm) situated on a PVDF membrane, which had been placed on YPD agar (a). Colonies were allowed to grow for 15 h at 30°C (b). The membrane filter was removed and the PVDF membrane was probed by immunoblotting using rat anti-HA antibody (c). Strains tested were (1) CAF2-1 (wild-type), (2) CIS23 (<i>PMT1</i>^HA), (3) ESCa3 (<i>ACT1p</i>-<i>MSB2</i>^HA-V5) and ESCa10 (<i>MSB2p</i>-<i>MSB2</i>^HA-V5). <b>D.</b> Gel filtration chromatography of secreted Msb2. A Superdex 200 10/300 GL column was (a) calibrated using standard proteins of the indicated sizes (dotted lines) and (b) used to fractionate 500 µl of the medium of strain ESCa3 (Msb2^HA-V5), which had been grown at 30°C in SD medium to OD₆₀₀ = 10. The protein elution profiles were recorded by absorption at 280 nm. 200 µl fractions were collected and (c) tested by immunoblotting for the presence of HA-tagged Msb2. Fractions tested are placed at a position corresponding to the elution profile in b). <b>E.</b> Glycosylation of secreted Msb2. (a) Growth medium of strain ESCa3 (Msb2^HA-V5) was not treated (1) or treated with β-elimination reagent mixture over night (2,3); the sample in lane 3 was heated to 80°C before reagent addition in an attempt to increase deglycosylation. (b) The medium was not treated (1) or treated with TFMS (2). Samples were tested by immunoblotting as in A. The migration of glyosylated and deglycosylated Msb2* are indicated by the filled and open arrows, respectively. (c) Msb2 secreted by <i>pmt</i> mutants defective in protein-<i>O</i>-mannosyltransferases carrying carried pES11a (Msb2^HA-V5). Strains included ESca18 (<i>pmt1</i>), ESCa19 (<i>PMT2/pmt2</i>), ESCa20 (<i>pmt4</i>), ESCa21 (<i>pmt5</i>) and ESCa22 (<i>pmt6</i>) and were tested by immunblotting as in A.</p

Activity of Msb2 variants.

<p><b>A.</b> Structure of Msb2 protein variants. The positions of signal sequence (SS), transmembrane region (TM) and HA- and V5-epitope tags are indicated. Plasmids encoding variants were chromosomally integrated into strain FCCa28, which produces the inactive Msb2-Δ1 variant by the <i>msb2</i>Δ<i>1</i> allele. Resulting transformants (encoded variants) were strains ESCa3 (Msb2^HA-V5), ESCa25 (Msb2-ΔN), ESCa38 (Msb2-ΔC) and ESCa39 (Msb2-ΔTM-C). Corresponding phenotypes are summarized in the table and are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002501#ppat.1002501.s001" target="_blank">Figure S1</a>. +, wild-type phenotype; −, <i>msb2</i> mutant phenotype with regard to Msb2* release, hypha formation, caspofungin resistance (Cas^R) and Cek1 phosphorylation (Cek1-P). <b>B.</b> Cek1 activation by strains producing variant Msb2 proteins. Cells were grown to stationary phase (st), diluted in fresh YPD medium, grown to OD₆₀₀ = 0.8 at 37°C and incubated further for 1 h in the presence (+) or absence (−) of tunicamycin (2 µg/ml). Cells in stationary phase (st) and after 1 h incubation were harvested and assayed for the activation of MAPK Cek1 by immunoblottings; the Hog1 MAPK protein signal was used as the loading control. Strains as in A., in addition strains ESCa37 encoding the Msb2-tail variant and strain ESCa7 carrying an empty vector (control) were tested.</p

Msb2*-mediates protection of <i>C. albicans</i> and <i>E. coli</i> against LL-37.

<p><b>A.</b> Purification of Msb2*. Msb2* in culture medium of strain ESCa3 (lanes 1, 3) was affinity-purified using an anti-HA column (lanes 2, 4) and samples were separated by SDS-PAGE (4–20% acrylamide gel). For silver staining 50 µl of medium/purified (med/pur) fractions (lanes 1, 2) and for immunoblotting using an anti-HA antibody 15 µl of medium/purified fractions (lanes 3, 4) was analyzed. <b>B.</b> Msb2 does not degrade LL-37. 3 µg of LL-37 were co-incubated without (lane 1) or with 10 µg of Msb2* (lane 2) for 1.5 h at 37°C. Samples were separated on a 18% SDS-PAGE gel; the migration of standard proteins is indicated. <b>C.</b> Immobilized Msb2* binds LL-37. 10 µg Msb2* or Msb2-ΔN* were allowed to attach to each well of polystyrene microtiter plates over night at 4°C. Wells were washed with PBST and unspecific binding sites were removed by incubation with skim milk solution. 5 µl (5 µg) of TAMRA-labelled LL-37 was allowed to bind for 1 h, wells were washed and TAMRA emission was recorded at 590 nm. As a control, coated wells were incubated first with 3 µg of unlabelled LL-37 for 1 h before addition of LL-37-TAMRA. <b>D.</b> Msb2*-mediated protection of <i>C. albicans</i> and <i>E. coli</i> against LL-37. Strain suspensions (5 µl) were co-incubated with LL-37 in the absence or presence of 10 µg Msb2* or 10 µg of its deleted variant Msb2*-ΔN for 1.5 h before determination of viable cell counts (CFUs). As a control, 10 µg BSA was used to replace Msb2*. <i>C. albicans</i> strains CAF2-1 (wt), FCCa27 (<i>msb2</i>Δ<i>1</i>), REP21 (<i>msb2</i>Δ<i>0 sho1</i>) and <i>E. coli</i> DH5αF′ were tested; means and standard deviations of triplicate assays are shown. <b>E.</b> Msb2* concentration dependence. <i>C. albicans</i> strain CAF2-1 was incubated with 3 µg LL-37 in the absence and presence of the indicated amounts of purified Msb2* and the deleted Msb2*-ΔN variant. In addition, the activity of HA peptides used for affinity purification of Msb2* was tested. <b>F.</b> Protection of <i>C. albicans</i> against LL-37 by medium proteins (secretome). 3 µg LL-37 was added to cells of strain CAF2-1 (5 µl; OD₆₀₀ = 0.3) in the absence or presence of culture medium (17 µl) of <i>C. albicans</i> strains grown to stationary phase. Following incubation for 1.5 h at 37°C cell viability (CFU) was tested. Secretome of control strain CAF2-1, FCCa27 (<i>msb2</i>Δ<i>1</i>) and SPCa2 (<i>pmt1</i>) was used. Means and standard deviations of triplicate assays are shown. Statistical significance was evaluated using a t-test (*, p<0.05; **, p<0.01; ***; p<0.001).</p

Model for Msb2 functions.

<p>The dual function of <i>C. albicans</i> Msb2 protein is shown. The precursor protein (A) is cleaved during growth and the extracellular domain, which is highly O-glycosylated (indicated by white dots), is shed into the medium (B). Msb2 has an intracellular function in activating the Cek1 MAP kinase and the secreted exodomain is able to protect cells against AMPs (triangles).</p

Structure and activity of epitope-tagged Msb2.

<p> <b>A.</b> Structure of <i>MSB2</i> alleles encoding Msb2 variants. The <i>MSB2</i> coding region with sequences encoding the signal sequence (SS), the transmembrane region (TM), HA- /V5-epitopes and relevant restriction sites used for the construction of <i>MSB2</i> variant alleles are shown. <b>B.</b> Tagged <i>MSB2</i> alleles confer inhibitor resistance. Sensitivity of strains to caspofungin (125 ng/ml), tunicamycin (2 µg/ml) and Pmt1 inhibitor (12 µM) was tested by a drop dilution test. <b>C.</b> Tagged <i>MSB2</i> alleles reconstitute formation of hyphae on YPM agar. Colonies of strains were photographed following growth for 2 d at 37°C. Strains CAF2-1 (wt), FCCa27 (<i>msb2</i>Δ<i>1 URA3</i>) and FCCa28 (<i>msb2</i>Δ<i>1 ura3</i>) were compared to FCCa28 transformants. Transformants contained empty plasmid pDS1044-1 (ESCa7; <i>msb2</i>Δ<i>1</i>[empty]), pES10 (ESCa8; <i>msb2</i>Δ<i>1</i>[<i>ACT1p</i>-<i>MSB2</i>^HA]) or pES11a (ESCa3; <i>msb2</i>Δ<i>1</i>[<i>ACT1p</i>-<i>MSB2</i>^HA-V5]) integrated in the <i>LEU2</i> locus. The tagged <i>MSB2</i> allele was also placed under transcriptional control of the authentic <i>MSB2</i> promoter by directing integration of <i>Hpa</i>I-cut pES11a into the <i>msb2</i>Δ<i>1</i> allele of FCCa28 (ESCa10; <i>msb2</i>Δ<i>1</i> [<i>MSB2p</i>-<i>MSB2</i>^HA-V5)]). Strain CAP4-2164 (<i>pmt4</i>) was used as a supersensitive control strain <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002501#ppat.1002501-Prill1" target="_blank">[27]</a>.</p

Msb2 synthesis protects <i>C. albicans</i> against LL-37.

<p><b>A.</b> Basal LL-37-resistance of <i>C. albicans</i> depends on Msb2. The indicated strains were incubated with different LL-37 amounts for 1.5 h at 37°C before plating of cells to determine colony-forming units (CFUs). Standard deviations of triplicate measurements are indicated; statistical differences of mutant versus control strain cfu values were evaluated by a t-test. <b>B.</b> Staining of <i>C. albicans</i> by TAMRA-labelled LL-37. 50 µl of cells were resuspended in PBS were incubated for 5 min with 5 µg LL-37-TAMRA before visualization using phase contrast and fluorescence microscopy. CAF2-1. +/+; FCCa27, −/−; ESCa3, −/−[+]. <b>C, D.</b> LL-37 sensitivity of <i>C. albicans</i> strains producing Msb2 variants (C) and of <i>pmt</i> mutants producing undeleted Msb2 (D). Transformants producing variant Msb2 proteins are described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002501#ppat-1002501-g002" target="_blank">Figures 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002501#ppat-1002501-g003" target="_blank">3</a>. Strain suspensions (5 µl) were co-incubated with 2 µg LL-37 for 1.5 h before CFU determination. Means and standard deviations of triplicate assays are shown. Statistical significance using a t-test is indicated by * (p<0.05), ** (p<0.01) and *** (p<0.001).</p