1 Department of Microbiology, Montana State University, Bozeman, MT, USA
2 Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA
3 Department of Microbiology, Columbia University, New York, NY, USA
4 Department of Microbiology, University of Minnesota, Minneapolis, MN, USA
5 Research Institute for Children and Louisiana State University Health Sciences Center, Children's Hospital, New Orleans, LA, USA
Correspondence
Bruce L. Granger
bgranger{at}montana.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY656807, AY656808, AY660719 and AY661302.
Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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Adhesive biofilms of C. albicans may form on tissue surfaces, bioprostheses and catheters; such biofilms exhibit increased resistance to antifungal agents and may be reservoirs for reinfection (Douglas, 2003). Adhesion of C. albicans is mediated by the cell wall, the primary structural basis of which is cross-linked glucans that make up 5060 % of the wall weight (Kapteyn et al., 2000
); mannoproteins with adhesive and other functions (Chaffin et al., 1998
) make up another 3040 %, and small amounts of chitin add tensile strength (Munro et al., 2003
). Most wall proteins have structural features that result in replacement of their C termini with glycosylphosphatidylinositol (GPI) membrane anchors, which may themselves be removed prior to transfer of the polypeptide to
-1,6-glucans in the wall matrix (Kapteyn et al., 2000
). Wall proteins are also typically N- and O-glycosylated, with the N-glycans including long, branched chains of mannose linked with glycosidic and occasional phosphodiester bonds. Adhesion of C. albicans may involve specific wall glycoproteins, such as Hwp1p and products of the ALS gene family (Hoyer, 2001
; Sundstrom, 2002
), or glycans alone (Masuoka, 2004
); the regulation and mechanistic details of in vitro and in vivo cellular adhesion, however, remain poorly understood.
Thiol reagents, such as dithiothreitol (DTT) and 2-mercaptoethanol, solubilize a subset of extracellular mannoproteins from live cells, presumably as a result of reduction of proteinaceous disulfide bonds in the wall (Chattaway et al., 1974; de Nobel & Barnett, 1991
). Such extracts typically contain 510 % protein and 9095 % carbohydrate and, when administered to mice in certain vaccine formulations, afford protection against disseminated candidiasis (Han & Cutler, 1995
; Han et al., 1999
). Little is known about the identity of potential protective epitopes in these thiol extracts, other than
-1,2-di- and trimannose epitopes that are terminal elements of certain polysaccharide-chain branches (Han & Cutler, 1995
; Han et al., 1997
; Nitz et al., 2002
). We continue to examine the polypeptide component of such extracts for additional protective epitopes, as well as possible carriers for vaccines. Here, we describe a wall mannoprotein termed Ywp1p that has a complex post-translational itinerary and appears to inhibit both adhesion and biofilm formation by blastoconidia. Experimentally, Ywp1p may be useful as a marker of the yeast form of C. albicans and thus complement the growing panel of markers of hyphae, such as Hwp1p (Staab et al., 2003
).
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METHODS |
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YWP1, HIS1 and URA3 were amplified by PCR from C. albicans 3153A genomic DNA. Amplicons included 999 bp upstream (5') and 852 bp downstream (3') from the coding sequence of YWP1, 231 bp 5' and 51 bp 3' from the coding sequence of HIS1, and 435 bp 5' and 141 bp 3' from the coding sequence of URA3. HIS1 and YWP1 amplicons were cloned in the T vectors pGEM-T and pGEM-T Easy (Promega), respectively, in Escherichia coli TOP10 (Invitrogen). For combination of YWP1 and HIS1 in the same plasmid, a NotI restriction fragment containing YWP1 was cloned into a unique NotI site in the HIS1 plasmid after removal of a unique NsiI site from pGEM-T; this resulted in 50 bp vector sequence between HIS1 and YWP1. Prior to transformation, plasmids were linearized at a unique NruI site in HIS1 (121 bp upstream from the start codon; clones designated r) or a unique NsiI site in YWP1 (414 bp upstream from the start codon; clones designated s). The two alleles of YWP1 (YWP1-1 and YWP1-2) were distinguished readily by a HindIII site 920 bp upstream from the start codon of allele 1 only. Cloned C. albicans transformants had one of two independent HIS1 amplicons (HIS1-3 or HIS1-4) and were named as follows, where > indicates the relative orientation of the transcript (5'>3') and protein (N>C): strain 3L1, HIS1-3; strain 4L1, HIS1-4; strains 2r and 2s, HIS1-3<--YWP1-2>; strains 7r and 7s, HIS1-3<--YWP1-1>; strains 13r and 13s, HIS1-4>--YWP1-2<; strains 16r and 16s, HIS1-4>--YWP1-1<.
For insertion of the green fluorescent protein (GFP) gene into C. albicans, yeast enhanced GFP (yEGFP; Cormack et al., 1997) was linked to the selectable markers HIS1 and URA3 to facilitate identification of transformants (Gerami-Nejad et al., 2001
). The coding sequence of GFP was amplified by PCR from pyGFP3 and spliced by overlap extension (Horton et al., 1989
) to the HIS1 and URA3 amplicons described above (GenBank accession nos AY656807 and AY656808, respectively); this presumably allows GFP to utilize the transcription-termination signals of the genes that are predicted to be immediately upstream and co-directional with HIS1 and URA3 in the C. albicans genome. Homologous recombination of transfected PCR amplicons was used to replace the coding sequence of one allele of YWP1 with GFP-HIS1, such that the start codon of YWP1 became the start codon of GFP and expression of GFP was driven by the YWP1 promoter (generating a soluble, cytosolic form of GFP). PCR analyses of genomic DNA from fluorescent transformants confirmed that GFP had indeed replaced YWP1. One of these strains (BJ3) was subcloned and screened for increased fluorescence; several subclones, including BJ3a (and its successive subclones, BJ3a1 and BJ3a1a) were found to have undergone a presumptive gene conversion or mitotic recombination event (Enloe et al., 2000
) that replaced the remaining YWP1 allele with GFP.
DNA vaccines.
All vaccine plasmids were derivatives of pBSA, created by removal of a G-418 resistance cassette (SalI fragment) from the expression vector pBGSA (Uthayakumar & Granger, 1995; GenBank accession no. AY660719). Inserted coding sequences were assembled from PCR amplicons or synthetic oligonucleotides (codon-optimized) that encoded one or more of the following elements: (i) various segments of Ywp1p, as indicated below; (ii) entire (140 aa) mouse interleukin 4 (mIL4, from a cloned cDNA in p2A-E3, ATCC 37561; Maecker et al., 1997
); (iii) entire (406 aa) mouse lysosome-associated membrane protein 1 (mLAMP-1 or lgp-A; Granger et al., 1990
; Rowell et al., 1995
); (iv) FISEAIIHVLHSR, an H-2-restricted T-cell epitope of sperm-whale myoglobin (SWM; Rothbard & Taylor, 1988
; Golvano et al., 1990
); and (v) FDTGAFDPDWPA, a peptide mimotope (S924) of group B streptococci capsular polysaccharide recognized by mAb S9 (Pincus et al., 1998
). For embedding of foreign coding sequences in the mLAMP-1 cDNA, the codon for proline 197 was changed to an alanine codon to create a unique NheI site; the vector harbouring this construct lacked the terminator of pBSA, instead utilizing the 3' untranslated region of the LAMP-1 cDNA. Sequences were confirmed by commercial, automated sequencing of plasmid DNA (Davis Sequencing).
Plasmids were delivered biolistically at 300 p.s.i. to the shaved abdomens of female BALB/c mice with a Helios gene gun (Bio-Rad) according to the manufacturer's instructions. Each shot nominally contained 1 µg DNA that had been precipitated and dried onto 0·5 mg 1·0 µm gold spheres, giving approximately 4x103 plasmids on each of 5x107 spheres. Histological examination showed that most of the particles that penetrated the skin were delivered to the basal layers of the epidermis. Typically, single shots were administered with a spacing of at least 4 weeks and blood was collected from tail veins 89 days after each boost. Unfractionated sera were assayed by immunofluorescence microscopy of COS-1 cells that had been transfected with one of the vaccine plasmids (described below).
Administered DNA vaccines encoded all 533 aa of Ywp1p or one of the following six chimeric polypeptides (some with tandemly repeated segments of Ywp1p): (i) mIL4/Ywp1 aa 51197/Ywp1 aa 51197; (ii) mLAMP-1 aa 1196/Ywp1 aa 51197/mLAMP-1 aa 198406; (iii) mIL4/SWM/Ywp1 aa 105161/S924/SWM; (iv) mIL4/SWM/Ywp1 aa 105161/Ywp1 aa 105161/S924/SWM; (v) mIL4/SWM/Ywp1 aa 21116/S924/SWM; (vi) mIL4/SWM/Ywp1 aa 21116/Ywp1 aa 21116/S924/SWM. Up to six extraneous codons were present as linkers between the above segments and contained NheI or AgeI sites that facilitated plasmid assembly (GenBank accession no. AY661302). Unexpectedly, segments encoding Ywp1 aa 21169 and 1334 could not be cloned in the desired orientation in the above expression plasmids, and most of the above YWP1-containing plasmids were difficult to create. Mice vaccinated with constructs (i), (iii), (iv) and the full-length YWP1 were challenged by intravenous injection of live C. albicans strain CA-1 in our standard protection assay (Han et al., 1999).
All expression plasmids were tested in cultured mammalian cells prior to administration to mice. Typically, transfection of COS-1 cells was followed 1 day later by formaldehyde or methanol fixation, then immunofluorescence microscopy utilizing the following antibodies specific for epitopes in the encoded polypeptides: mIL4 (rat mAb 11B11; Pharmingen), mLAMP-1 (rat mAb 1D4B; Chen et al., 1985), S924 (mouse mAb S9; Pincus et al., 1998
) or Ywp1p (the collection of antisera from vaccinated mice that developed during this study). Stability of the chimeric polypeptides was enhanced by adding the membrane-permeable protease inhibitor acetyl-leucyl-leucyl-norleucinal to the COS-1 cell medium at 20 µM for a few hours prior to fixation. The S924 peptide was included on the C-terminal side of some Ywp1p segments for two reasons: detection of the S9 epitope in transfected COS-1 cells demonstrated that the plasmid had directed synthesis of the chimeric polypeptide successfully and the presence of antibodies against S924 in vaccinated mice implied that their immune systems had also been exposed to the upstream Ywp1p segment. mIL4 was included in most constructs to foster a humoral rather than a cell-mediated immune response, as part of a wider vaccine effort to elucidate the contribution of circulating antibodies to immunoprotection (Cutler et al., 2002
). The mLAMP-1 chimeras tended to generate lower titres than the mIL4 chimeras, but were useful when expressed in COS-1 cells because their membrane anchorage made them less likely to be lost before or during fixation, and because they represented an alternative carrier unable to bind antibodies that might have been generated against the other carriers. Antiserum specificity was further demonstrated by Western blotting (described below), which showed that the anti-Ywp1p antisera gave strong signals that were absent from samples derived from ywp1/ywp1 knockout strains; sera from unvaccinated mice gave no such signals.
Virulence testing.
Prototrophic strains of C. albicans, with or without Ywp1p, were compared in a mouse model of disseminated candidiasis (Han et al., 1999). Cultures were grown in aerated liquid medium for 12 days at 30 °C; the cells were then washed and diluted in saline (155 mM NaCl) to an OD600 of 0·060 (nominally 2·5x106 cells ml1) and a 0·20 ml aliquot (
5x105 cells) was injected into the lateral tail vein of each mouse. Two trials utilized a single batch of female BALB/c mice from Charles River Laboratories.
Protein analysis.
PAGE of SDS-denatured proteins was performed as described by Laemmli (1970) (Figs 1 and 6b
) and Schägger & von Jagow (1987)
; acrylamide/bis concentrations in the resolving gels were 15/0·1 and 10/0·3 %, respectively. Isoelectric focusing (IEF) was performed in immobilized pH gradients (13 cm pH 310 Immobiline DryStrips from Amersham Biosciences) in the presence of saturated urea, 50 mM DTT or hydroxyethyl disulfide (Olsson et al., 2002
), and 1 % pH 310 ampholytes. Western blotting involved electrophoretic transfer to nitrocellulose or PVDF filters in the presence of 20 mM Tris/acetate (pH 8·3) and 20 % (v/v) methanol; antibody incubations and washes were performed in Tris-buffered saline (pH 7·5), 0·1 % gelatin and 0·1 % Tween 20. Secondary antibodies were conjugated to alkaline phosphatase and chromogenic detection utilized Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates. N-terminal seqencing by Edman degradation of polypeptides that had been alkylated with iodoacetamide, digested with peptide N-glycanase F (PNGase F; see below) and electroblotted from gels to PVDF filters was performed by Dr Laurey Steinke (Protein Structure Core Facility, University of Nebraska Medical Center, NE, USA). Mass-spectral analysis of in-gel-trypsinized Ywp1 propeptide involved MALDI-TOF mass spectrometry (MS) and post-source decay analysis of a C-terminal fragment (Mann et al., 2001
) and was performed by Dr John Leszyk (University of Massachusetts Medical School, MA, USA).
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Digestion with PNGase F (New England Biolabs) removes N-glycans from polypeptides and converts the linking asparagines to aspartates, a fact that was taken into account when theoretical pI values were calculated (http://www.expasy.org). Digestions were typically performed for 23 h at 37 °C in TEN8·5 buffer, with or without DTT at 2050 mM. Samples for Fig. 1 were also heated with 0·2 % SDS to first denature the proteins, and then mixed with 1 % NP-40 prior to digestion. For quantification of the Ywp1 propeptide, stationary-phase culture supernatants were not precipitated or concentrated, but digested directly with PNGase F (after adding 0·2 vol. 5x TEN8·5 buffer), resolved by SDS-PAGE and silver-stained; densitometry of the stained bands was followed by normalization to an unidentified band just above the propeptide.
Prior to digestion with recombinant, protease-free -1,3-glucanase (Quantazyme ylg; Quantum Biotechnologies), cells were extracted sequentially (all in TEN8·5 buffer) with 40 mM DTT, 1 % SDS at 0 °C, 1 % SDS at 95 °C and saturated urea containing 10 mM DTT at 25 °C. Residues were washed extensively and digested with Quantazyme at 25 °C in 50 mM Tris/HCl (pH 7·6), 1 mM EDTA, 10 mM DTT, 0·2 % Triton X-100. Extracts were precipitated with 2 vols ethanol after adding NaCl to 50 mM.
Phosphate determination.
The concentration of inorganic phosphate in culture supernatants was determined colorimetrically (Chen et al., 1956; Ames, 1966
). Final concentrations of solutes in each complete mixture were 0·5 M sulfuric acid (diluted from a 1 M stock), 5 mM ammonium molybdate (diluted from a 100 mM stock), 0·010·20 mM phosphate (diluted from a culture supernatant or a calibration solution) and 50 mM ascorbic acid (diluted from a 1 M stock). After 16 h at 2025 °C, A700 was determined in polystyrene cuvettes.
Flow cytometry.
Cultures of C. albicans were shaken at 200 r.p.m. in Erlenmeyer flasks that were 20 % full of medium 13 at 30 °C; such cultures were started with 100-fold dilutions of similar cultures that had an OD600 of 23. For analysis, aliquots of cell cultures were diluted in saline to an OD600 of 0·02 and analysed immediately in a Becton Dickinson FACScan equipped with a 488 nm argon laser. Detectors were set at E-1 (FSC), 220 (SSC) and 600 (FL1) in logarithmic mode, with a threshold of FSC 220. Data were recorded for 104 events per sample and analysed with WinMDI 2.7 software (http://facs.scripps.edu/software.html); gates of FSC 70-167, SSC 50-157 and FL1 80-230 excluded no more than 0·55 % of the events in any sample.
Adhesion assays.
Microcultures were grown in polystyrene plates that were tissue-culture grade (Greiner or Costar) or bacteriological grade (Fisher). Cultures were started with an OD600 of 0·1, using washed or unwashed cells from actively growing or stationary-phase cultures. After a period of growth, non-adherent cells were rinsed away by repeated flooding of the plate with water or other solution (as specified in Results) coupled with gentle rocking or swirling; adherent cells were stained for 10 min at 25 °C with 0·1 % crystal violet (CV) in water and destained with water. CV was extracted from stained cells with 50 % ethanol/1 % SDS and its A595 was measured. Stained cells on plates were imaged digitally with a flatbed scanner after drying. Confocal laser-scanning microscopy of GFP-expressing cells was performed by using a Leica TCS-SP2 AOBS microscope with a 63x, 0·9 NA, water-immersion objective and a 488 nm laser; image reconstruction was performed with Imaris software (Bitplane). Cells deep within a biofilm are imaged incompletely with this technique because of attenuation of laser excitation and fluorescence emission.
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RESULTS |
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DNA vaccines and the utility of anti-Ywp1p
Mice vaccinated with the full coding sequence of YWP1 (aa 1533) generated antibodies specific for Ywp1p, as did mice vaccinated with plasmids encoding shorter segments of Ywp1p (aa 21116, 51197 and 105161) linked to one or more carriers to form hybrid proteins, as outlined in Methods. These antisera were useful for identifying various forms and fragments of Ywp1p on Western blots (described below); specific binding of these antibodies to Ywp1p in intact C. albicans cells under a variety of conditions, however, was not evident by immunofluorescence microscopy, presumably because of epitope inaccessibility. Perhaps accordingly, when groups of mice that had the highest anti-Ywp1p titres were challenged by intravenous injection of live C. albicans, survival times were not found to be changed significantly (data not shown).
Secreted Ywp1p from which the N-glycan had been removed was analysed by silver staining and Western blotting (Fig. 4), revealing or confirming that: (i) the 11 kDa propeptide undergoes a mobility shift upon disulfide reduction and both forms are recognized by the antisera; (ii) PNGase F efficiently removes the N-glycan from the propeptide, even if the propeptide is not first reduced or denatured; (iii) a minor fraction of the propeptide is associated with high-molecular-mass (HMM) mannoprotein in the absence of reduction, but all of the propeptide immunoreactivity is present in the 11 kDa band upon reduction with DTT [note that the antibodies used in Fig. 4(b)
are specific for the propeptide only]; (iv) a minor fraction of the propeptide may also exist as a dimer in the absence of reduction; (v) mature Ywp1p exists in an HMM form, remaining in the stacking gel or barely entering the resolving gel; (vi) the HMM forms of Ywp1p migrate somewhat differently, depending on reduction and Kex2 status; and (vii) the kex2/kex2 samples show a small amount of mid-molecular-mass immunoreactivity that cannot be attributed to non-specific labelling (all other faintly labelled bands are also seen in samples from knockout lines that lack Ywp1p; data not shown). Interestingly, the antisera used in Fig. 4(c)
recognize the 12 kDa propeptide but not the 11 kDa propeptide, even though the vaccines encoded all but the N-terminal 3 kDa of the propeptides; an immunogenic epitope that includes the segment between the tribasic and dibasic sites must therefore exist.
GFP as a reporter of YWP1 expression
The coding sequence of one or both alleles of YWP1 was replaced with GFP to create strains BJ3 and BJ3a1a. We screened various growth media and conditions and found that low phosphate concentrations increased GFP fluorescence of these strains by upregulating YWP1 expression. Quantitative analysis by flow cytometry revealed that, under normal phosphate conditions, GFP fluorescence reached a peak soon after the end of the exponential phase (OD600 1), well before the culture reached stationary phase (Fig. 5a
). This suggested that YWP1 expression peaked during the exponential phase, as there is a lag of
1 h between transcription of GFP and detection of GFP fluorescence under these growth conditions (based on our unpublished studies of GFP driven by the PHO3 promoter). Growth in low-phosphate medium resulted in greater fluorescence per cell, a delayed peak in fluorescence and greater fluorescence in stationary phase (Fig. 5b
). Epifluorescence microscopy of different morphological forms of C. albicans revealed that YWP1 expression was downregulated upon filamentation. Hyphal and pseudohyphal forms devoid of detectable GFP fluorescence arose in liquid filamentation media, such as the medium of Lee et al. (1975)
, at 37 °C, and in 1 % yeast extract/2 % peptone/0·1 % Tween 80 at 23 °C. Filaments that had invaded agar were seen to have little or no GFP fluorescence, but blastoconidia that arose at the distal tips of the filaments were fluorescent, having resumed GFP (YWP1) expression (data not shown).
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Fate of secreted Ywp1p
Aliquots of liquid cultures were analysed at different stages of growth (Fig. 6). For both the culture-medium and DTT extracts, silver staining of the deglycosylated Ywp1 propeptide (Fig. 6a
) showed that it was barely detectable just after exponential phase (14 h, OD600
1·8), but increased into early stationary phase (62 h, OD600
5·2). Similar increases were seen in the full complement of HMM mannoproteins, although not in the unidentified sharp bands in the DTT extracts. Western blotting of Ywp1p in these same fractions (without deglycosylation) showed a similar pattern (Fig. 6b
), with the HMM form of Ywp1p undetectable in the earliest samples, but increasing toward stationary phase. Cultures grown in low-phosphate medium showed greater expression of Ywp1p and earlier detection with the antibodies. Electrophoretic mobility was retarded noticeably for HMM Ywp1p from supernatants of low-phosphate cultures, but not from the DTT extracts of those cells. Finally,
-1,3-glucanase digests of cells that had been exhaustively pre-extracted to remove disulfide bonded and non-covalently linked proteins showed immunoreactive Ywp1p at all time points (Fig. 6c
). Attached
-1,6-glucans presumably contribute to the large size and exceptionally low mobility of these
-1,3-glucanase-digestion products.
YWP1 gene disruption and virulence testing
The two alleles of YWP1 were disrupted sequentially (and partially deleted) by homologous recombination. The only phenotypic change that we noted was increased adhesion and biofilm formation, as described below. The histidine auxotrophy of the original ywp1/ywp1 knockout strain (Ca#12) allowed YWP1 and prototrophy to be restored simultaneously by plasmid integration. For this purpose, YWP1 and HIS1 were combined in several plasmids that included both alleles of YWP1, as well as all possible orientations of YWP1 and HIS1 relative to each other. Half of these plasmids (see Methods) were then linearized at a unique restriction site in either HIS1 or YWP1 in order to create recombinogenic ends that would target integration to their respective loci. Two prototrophic clones from each transformation were analysed. All integrations that were targeted to the YWP1 locus resulted in lines that secreted amounts of Ywp1p similar to that of a heterozygous (YWP1/ywp1) line, whereas integrations that were targeted to the HIS1 locus resulted in lines that secreted variable amounts of Ywp1p, from none detectable to normal (wild-type) amounts. We have not fully analysed the latter transformants genetically, but it seems likely that the 999 bp upstream from the coding sequence of YWP1 that was included in the transforming DNA has promoter activity, but does not necessarily restore normal patterns of expression. Combined with transformants in which just HIS1 was reintegrated, a panel of prototrophic lines with different amounts of Ywp1p was thus generated. Production of Ywp1p was estimated by examining the quantity of propeptide in supernatants of stationary-phase cultures and revealed four groups, referred to below as having normal (wild-type), half, trace amounts or no Ywp1p. The relative propeptide quantities were: 100±5·8 % (ratio±SD) for SC5314, DAY185 and 16r1; 42±2·7 % for 2s1, 2s2, 7s1, 7s2, 13s1, 13s2, 16s1 and 16s2; 6·2±0·8 % for 2r1, 2r2, 7r1 and 7r2; and 1·5±1·5 % for 3L1, 4L1 and 16r2.
Prototrophic strains of C. albicans producing different amounts of Ywp1p (from none to normal) were tested in a mouse model of haematogenously disseminated candidiasis. We observed little or no difference in survival rates as a function of the presence or absence of Ywp1p (data included as supplementary material with the online version of this paper).
YWP1 affects adhesion and biofilm formation
Ywp1p-dependent differences in adhesion and biofilm formation were visualized most easily by comparative growth of microcultures in single polystyrene dishes. After removal of non-adherent cells, adherent cells were apparent upon oblique lighting against a dark background and after staining with CV (Fig. 7a). A strong correlation between adhesion and the lack of Ywp1p was evident under conditions in which all of the cells remained in the yeast form. Strains possessing little or no Ywp1p developed an adhesive blastoconidial biofilm that covered most of the plastic and was up to several cells thick, as shown in optical cross-section (Fig. 7b
). Under the hemispherical droplets of culture medium, biofilm morphology varied across the diameter of the adhesive patch of cells, with thicker biofilms and discrete microcolonies more evident at the periphery (where conditions such as gas-exchange rates may be different). In contrast, cells with normal amounts of Ywp1p showed sparse, monolayer adhesion. Strains with half of the normal amount of Ywp1p showed intermediate amounts of adhesion, but were more similar to the Ywp1p-replete strains than Ywp1p-negative strains (Fig. 7a
). Scraping and dispersal of the adherent Ywp1p-negative cells revealed that they were predominantly single cells without or with buds; filamentous forms were not observed.
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Assays were run routinely in polystyrene tissue-culture plates with a chemically modified surface that is hydrophilic. Similar Ywp1p-dependent adhesion patterns were seen on alternative substrates, such as unmodified polystyrene (hydrophobic, bacteriological-grade plates), Tween 80-treated tissue-culture plates, glass coverslips that were untreated or cationized with Alcian Blue (Sommer, 1977), polyvinyl chloride coverslips and sheets of polyethylene and silicone elastomer. Non-adherent cells were routinely washed away with water, but similar adhesion patterns were seen upon washing with various concentrations of sodium chloride or with Tris-buffered saline containing 1 mM EDTA, 0·1 % Tween 20, 0·1 % Tween 80 or 0·1 % gelatin. The patches of adherent cells on the various substrates persisted for days or weeks at room temperature in these solutions. Growth of the microcultures was routinely allowed to proceed for 23 days to early stationary phase, but biofilm patterns were similar from less than 1 day to more than 10 days growth. Similar adhesive biofilms were seen in the Ywp1p-negative microdroplet cultures when the plates were inverted during the growth period, which allowed non-adherent cells to fall away from the substrate.
When grown at 24 °C, the adhesion patterns were qualitatively similar to those at 30 °C, but quantitatively, there was slightly less difference between the Ywp1p-containing and Ywp1p-lacking strains. At 30 °C, adhesion patterns were similar in a variety of growth media, such as yeast nitrogen base with 50 mM glucose, the medium of Lee et al. (1975) and medium 13, which had galactose substituted for glucose, low (0·2 mM) phosphate or the additives 0·1 % BSA or 100 mM NaMOPS at pH 7·4 (conditions under which the cultures remained blastoconidial). At 37 °C, however, the Ywp1p correlation with adhesion was usually much less or not evident when cultures became filamentous (data not shown).
When actively growing or stationary-phase blastoconidia were washed free of culture medium, suspended in 5 mM NaCl and allowed to settle onto tissue-culture plates at 0 or 30 °C (conditions that do not support growth), the same patterns of adhesion were observed (more adhesion of Ywp1p-negative strains). Furthermore, secretions of strains that possessed or lacked Ywp1p were found to be capable of inhibiting adhesion and biofilm formation, even after fractionation of the secretions by ethanol precipitation. Fig. 7(c) shows that such fractions, when added at their original concentration to fresh culture medium, inhibited adhesion of all strains in our microdroplet assay; fractions from cultures that contained Ywp1p, however, were more inhibitory than fractions from cultures that lacked Ywp1p.
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DISCUSSION |
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Upon disruption of both alleles of YWP1, the only phenotypic change that we noted was slightly increased adhesiveness of yeast cells, but no obvious tendency to flocculate in liquid suspension. We developed a simple method to demonstrate that cells lacking Ywp1p were intrinsically more adhesive toward plastic surfaces and formed thicker biofilms when allowed to grow at 30 °C on these surfaces. This tendency was less evident when cells were grown at 37 °C, a temperature that promotes filamentation in these strains, and rarely evident in filament-inducing media at 37 °C; this is presumably because YWP1 is downregulated under these conditions or because additional mechanisms of adhesion regulation become operative. A large number of independently engineered strains of C. albicans were compared in our studies, to help rule out the possibility that unanticipated genetic modifications or differences in the expression of selectable markers were responsible for the observed differences in adhesion (Bain et al., 2001). We were able to correlate adhesive biofilm formation inversely with the amount of Ywp1p that each strain possessed, as measured by the quantity of Ywp1 propeptide secreted. Micromanipulation of exclusively blastoconidial biofilms formed by strains lacking Ywp1p revealed the presence of an extracellular polymeric material holding the cells together (data not shown; Chandra et al., 2001
; Douglas, 2003
); such biofilms were dispersed readily to predominantly single cells and single cells with buds by repetitive pipetting, indicating that the extracellular material was relatively fragile and that the thickness of the biofilm was not a result of the failure of motherdaughter yeast pairs to separate physically from one another. Yeast cells appeared to be anchored more firmly to the substrate than to each other, defining at least two layers or types of adhesion, as has been described for other types of C. albicans biofilms (Chandra et al., 2001
; Douglas, 2003
). The blastoconidial nature of these biofilms has some resemblance to certain wild-type strains of Candida parapsilosis (Kuhn et al., 2002
). Only sparse adhesive monolayers were evident for strains possessing normal amounts of Ywp1p, indicating that Ywp1p must interfere with both adhesion to plastic and subsequent biofilm formation in the absence of filamentation. The nature of this interference is currently being explored, but potential clues have emerged from the current work. For example, strains with half of the normal amount of Ywp1p show less than half of the blastoconidial-biofilm accumulation of strains with no Ywp1p, suggesting a non-linear relationship between Ywp1p content and biofilm quantity. Secretions containing Ywp1p inhibit the adhesion of cells to plastic, as well as the subsequent development of biofilms, even after such secretions have been through an ethanol-precipitation cycle. Washed cells that have Ywp1p in their walls are intrinsically less adhesive to plastic. Whether these are direct or indirect effects of Ywp1p remains to be determined; a direct effect might be binding of Ywp1p to, and inactivation or blockage of, adhesins, whereas an indirect effect might be catalytic modification of the cell wall or extracellular polymeric material. Additional clues are starting to emerge from biochemical and structural descriptions of Ywp1p, as discussed below.
Transcription of YWP1 (also termed IPF5185 and CA1678) was recently reported to be greater in C. albicans biofilms than in planktonic cells (García-Sánchez et al., 2004). The structural similarity of Ywp1p to known adhesins suggested that this might facilitate the cohesion of cells within the biofilm. Our data support the opposite interpretation, i.e. that overexpression of YWP1 may result in detachment and dissemination of blastoconidial daughter cells from mature biofilms. Comprehensive transcript profiling has indicated that YWP1 transcripts do not show a change of more than twofold during the white/opaque phenotypic switch (Lan et al., 2002
), or more than threefold when receptive cells are exposed to
-factor mating pheromone (Bennett et al., 2003
); however, such changes in Ywp1p levels can have significant effects on adhesion and biofilm formation, leaving open the possibility that Ywp1p plays some role in phenotypic switching and/or mating.
Low phosphate upregulates YWP1
YWP1 expression is upregulated when the extracellular phosphate concentration is low, suggesting that Candida may avoid adhesion and biofilm formation in environments where phosphate is sensed as limiting. This does not appear to be because biofilms require more phosphate than do planktonic cells, however, as is the case for Pseudomonas fluorescens (Kemner et al., 2004); our preliminary comparative measurements of total phosphate by chemical methods (Ames, 1966
) and total phosphorus by scanning electron microscopy/energy-dispersive X-ray spectroscopy have shown no enrichment in biofilm blastoconidia (data not shown). Our suspension cultures of C. albicans showed uniform uptake of phosphate from the culture medium (independent of the external concentration, as long as there was a surplus) prior to stationary phase and no uptake thereafter, indicating a limited capacity for phosphate storage. In the absence of measurable external phosphate, one to three additional doublings occurred, presumably through utilization of stored phosphate (cf. Lillie & Pringle, 1980
). Upregulation of YWP1 by low phosphate does not appear to be part of a general starvation response, as no upregulation was noted when essential arginine or uridine was limited in cultures of the auxotrophic BJ3a1a, and only a slight upregulation (
10 %) was observed upon glucose limitation (data not shown). By using whole-genome expression analysis, many S. cerevisiae genes involved in the acquisition and storage of phosphate were found by Ogawa et al. (2000)
to be upregulated by low phosphate, but no gene that encodes a GPI protein was upregulated consistently under the conditions of their experiment (three to four exponential-phase doublings in up to approx. 0·1 mM inorganic phosphate). As S. cerevisiae has no obvious sequence homologue of Ywp1p, there is currently no indication that S. cerevisiae has any protein with an analogous role.
YWP1 expression patterns and virulence
As reported by GFP fluorescence, expression of YWP1 in phosphate-replete, batch cultures of blastoconidia appears to peak soon after the exponential phase of growth, when the OD600 is 3050 % of its ultimate stationary-phase value. The decline in fluorescence per cell thereafter is presumably due to dilution of GFP into daughter cells, followed by slow degradation without replenishment in non-dividing cells. This suggests that the expression of YWP1 is greatest during maximal growth rates of yeast, but declines or shuts off as the growth rate declines, and is low or non-existent in stationary-phase cells. Ywp1p may thus foster dispersal of blastoconidia when growth conditions are most favourable. Expression of YWP1 shuts down upon filamentation, as shown by GFP fluorescence (this study) and transcript analysis (Nantel et al., 2002; Sohn et al., 2003
), perhaps to promote initial adhesion and biofilm formation. YWP1 expression appears to be under complex regulatory control (Sohn et al., 2003
; Doedt et al., 2004
), but our studies of protein levels have not indicated that disruption of one allele of YWP1 results in a compensatory upregulation of the other allele or restoration of normal amounts of Ywp1p.
Several of our engineered prototrophic strains, with or without Ywp1p, were compared in a mouse model of disseminated candidiasis. Little or no correlation between Ywp1p levels and survival times was evident. In this model, the infecting cells grew at 37 °C in a filamentation-promoting environment, conditions under which the expression of YWP1 and effects of Ywp1p on adhesion may have been minimal. Thus, alternative pathogenesis models, such as infection of epithelia at lower temperatures or dissemination from one site of infection to another, might be more likely to reveal a link between YWP1 and virulence.
Post-translational itinerary of Ywp1p
Our data suggest that Ywp1p has a role in inhibiting adhesion as an integral cell-wall component, as well as after being shed or secreted into the extracellular milieu. We observed a considerable lag between the period of peak YWP1 expression and the phases at which Ywp1p could be extracted from cells with DTT or found free in the surrounding medium. The expected fate of GPI proteins with structures similar to Ywp1p is covalent incorporation into the cell wall through -1,6-glucan (Kapteyn et al., 2000
); indeed, covalent attachment of Ywp1p to glucan is supported by our antibody detection of Ywp1p that was liberated from wall residues of proliferating and stationary-phase cells by
-1,3-glucanase. Similarly, de Groot et al. (2004)
demonstrated the covalent linkage of Ywp1p (termed Pga24p) to this matrix by MS analysis of tryptic peptides after digestion of wall residues with glucanase or HF-pyridine, which cleaves phosphodiester bonds of GPI anchors. Conceivably, the Ywp1p that we found in the surrounding medium was liberated from the wall during early stationary-phase remodelling or was never incorporated covalently into the wall; perhaps an intermediary form that was liberated from its GPI anchor, making it effectively soluble in the periplasm or wall itself (de Nobel & Lipke, 1994
), diffused (or was released) slowly into the medium. Formation of intermolecular disulfide bonds might slow the egress of such forms through the cell wall and allow a fraction of the Ywp1p to accumulate in a DTT-extractable form as the culture matures; this would parallel the observed six- to sevenfold increase in disulfide-bond content of S. cerevisiae cell walls during the stationary phase (de Nobel et al., 1990
). Accurate quantification of the relative proportion of each form of Ywp1p at each destination and at each growth stage may eventually reveal functional correlations.
The propeptide of Ywp1
The large, persistent propeptide of Ywp1 was the first form of Ywp1 to be identified, and it subsequently proved to be a useful surrogate marker for the presence and abundance of Ywp1. We have not noted any extracellular form of Ywp1p with uncleaved propeptide, consistent with the usual cleavage of propeptides from pro-proteins in a late Golgi compartment prior to secretion (Redding et al., 1991). Propeptides often act as intramolecular chaperones, specifically catalysing the proper folding and, in some cases, proper disulfide-bond formation of the collinear polypeptide (Eder & Fersht, 1995
). Cleaved propeptides may be degraded after this function or they may persist to serve as inhibitors, anchors or independent signalling molecules (e.g. Annes et al., 2003
). Our data show that the Ywp1 propeptide remains associated with the cell wall and appears in the culture medium only when the HMM form of Ywp1p also appears there. The contribution of the Ywp1 propeptide to early protein maturational events, as well as later adhesion-related events, remains to be determined.
Variability in the C terminus of the Ywp1p propeptide suggested that more than one proteinase could be involved in its cleavage and maturation. Cleavage at the tribasic site of Ywp1p generated the 11 kDa form seen in Kex2p+ strains, whereas cleavage at the dibasic site generated the 12 kDa form seen in Kex2p strains. This was surprising, because Kex2p is thought to be primarily responsible for cleavage at consensus dibasic (-KR-) sites; however, in C. albicans, dibasic sites may be cleaved by proteinases other than Kex2p, albeit less efficiently (Newport & Agabian, 1997). Perhaps Kex2p is essential for activation of proteinase(s) that trim the Ywp1 propeptide from 12 to 11 kDa, or Kex2p is itself responsible for cleavage at the tribasic site. Regardless of which proteinases cleave Ywp1p in the tribasicdibasic region, the N terminus of mature HMM Ywp1p is conceivably the same in wild-type and Kex2p strains, and both it and the cleaved propeptide may function normally in a Kex2p strain. In our assay of blastoconidial adhesion, Kex2p strain CNA3-1 showed no increase in adhesion or biofilm formation over Ywp1p-replete strains (data not shown). Additional Kex2p substrates, which include Hwp1p and other adhesin-like proteins, have been tentatively identified in C. albicans (Newport et al., 2003
), raising the possibility of a more expansive role for propeptides in regulating cellular adhesion.
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ACKNOWLEDGEMENTS |
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Received 29 September 2004;
revised 6 January 2005;
accepted 3 February 2005.