C.G. de Koster

Identification of cell wall-associated proteins from Phytophthora ramorum

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.

Mass spectrometric quantification of the adaptations in the wall proteome of Candida albicans in response to ambient pH

The mucosal layers colonized by the pathogenic fungus Candida albicans differ widely in ambient pH. Because the properties and functions of wall proteins are probably pH dependent, we hypothesized that C. albicans adapts its wall proteome to the external pH. We developed an in vitro system that mimics colonization of mucosal surfaces by growing biomats at pH 7 and 4 on semi-solid agarose containing mucin as the sole nitrogen source. The biomats expanded radially for at least 8 days at a rate of ~30 μm h(-1). At pH 7, hyphal growth predominated and growth was invasive, whereas at pH 4 only yeast and pseudohyphal cells were present and growth was noninvasive. Both qualitative mass spectrometric analysis of the wall proteome by tandem mass spectrometry and relative quantification of individual wall proteins (pH 7/pH 4), using Fourier transform mass spectrometry (FT-MS) and a reference mixture of (15)N-labelled yeast and hyphal walls, identified similar sets of >20 covalently linked wall proteins. The adhesion proteins Als1 and Als3, Hyr1, the transglucosidase Phr1, the detoxification enzyme Sod5 and the mammalian transglutaminase substrate Hwp1 (immunological detection) were only present at pH 7, whereas at pH 4 the level of the transglucosidase Phr2 was >35-fold higher than at pH 7. Sixteen out of the 22 proteins identified by FT-MS showed a greater than twofold change. These results demonstrate that ambient pH strongly affects the wall proteome of C. albicans, show that our quantitative approach can give detailed insights into the dynamics of the wall proteome, and point to potential vaccine targets.

High resolution end group determination of low molecular weight polymers by matrix-assisted laser desorption ionization on an external ion source fourier transform ion cyclotron resonance mass spectrometer

Matrix-assisted laser desorption ionization was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7-T superconducting magnet to analyze end groups of synthetic polymers in the mass range from 500 to 5000 u. Native, perdeutero methylated, propylated, and acetylated polyethylene glycol and polyvinyl pyrrolidone with unknown end-group elemental composition were investigated in the mass range up to 5000 u by using a 2,5-dihydroxybenzoic acid matrix. A small electrospray setup was used for the deposition of the samples. Two methods to process data were evaluated for the determination of end groups from the measured masses of the component molecules in the molecular weight ranges: a regression method and an averaging method. The averaging method is demonstrated to allow end-group mass determinations with an accuracy within 3 mu for the molecular weight range from 500 to 1400 and within 20 mu for the molecular weight range from 3400 to 5000. This is sufficient to identify the elemental composition of end groups in unknown polymer samples.

In Pursuit of Protein Targets: Proteomic Characterization of Bacterial Spore Outer Layers

Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two Bacilli and one Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.

A systematic study of the cell wall composition of Kluyveromyces lactis

In many ascomycetous yeasts, the cell wall is composed of two main types of macromolecules: (a) polysaccharides, with a high content of β‐1,6‐ and β‐1,3‐linked glucan chains and minor amounts of chitin; and (b) cell wall proteins of different types. Synthesis and maintenance of these macromolecules respond to environmental changes, which are sensed by the cell wall integrity (CWI) signal transduction pathway. We here present a first systematic analysis of the cell wall composition of the milk yeast, Kluyveromyces lactis. Electron microscopic analyses revealed that exponentially growing cells of K. lactis supplied with glucose as a carbon source have a wall thickness of 64 nm, as compared to 105 nm when growing on 3% ethanol. Despite their increased wall thickness, ethanol‐grown cells were more sensitive to the presence of zymolyase in the growth medium. Mass spectrometric analysis identified 22 covalently linked cell wall proteins, including 19 GPI‐modified proteins and two Pir wall proteins. Importantly, the composition of the cell wall glycoproteome depended on carbon source and growth phase. Our results clearly illustrate the dynamic nature of the cell wall of K. lactis and provide a firm base for studying its regulation. Copyright

Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in Candida glabrata Biofilms

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

Growth-dependent secretome of Candida utilis

Recently, the food yeast Candida utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. utilis. Cells were cultivated to the exponential or stationary growth phase, and the proteins in the medium were identified by MS. In parallel, a draft genome sequence of C. utilis strain DSM 2361 was determined by massively parallel sequencing. Comparisons of protein and coding sequences established that C. utilis is not a member of the CUG clade of Candida species. In total, we identified 37 proteins in the culture solution, 17 of which were exclusively present in the stationary phase, whereas three proteins were specific to the exponential growth phase. Identified proteins represented mostly carbohydrate-active enzymes associated with cell wall organization, while no proteolytic enzymes and only a few cytoplasmic proteins were detected. Remarkably, cultivation in xylose-based medium generated a protein pattern that diverged significantly from glucose-grown cells, containing the invertase Inv1 as the major extracellular protein, particularly in its highly glycosylated S-form (slow-migrating). Furthermore, cultivation without ammonium sulfate induced the secretion of the asparaginase Asp3. Comparisons of the secretome of C. utilis with those of Kluyveromyces lactis and Pichia pastoris, as well as with those of the human fungal pathogens Candida albicans and Candida glabrata, revealed a conserved set of 10 and six secretory proteins, respectively.

The Candida albicans cell wall protein Rhd3/Pga29 is abundant in the yeast form and contributes to virulence

The glycosylphosphatidylinositol‐modified protein Rhd3/Pga29 of the human pathogen Candida albicans belongs to a family of cell wall proteins that are widespread among Candida species but are not found in other fungi. Pga29 is covalently linked to the β‐1,3‐glucan framework of the cell wall via β‐1,6‐glucan. It is a small and abundant O‐glycosylated protein and requires the protein‐O‐mannosyl transferase Pmt1 for glycosylation. Furthermore, Pga29 is strongly expressed in yeast cells but is downregulated in hyphae. Removal of the PGA29 gene in C. albicans leads to a significant reduction of cell wall mannan; however, Pga29 does not seem to have a major role in maintaining cell wall integrity. In addition, adhesion capacity and hyphae formation appear normal in pga29 deletion mutants. Importantly, the pga29 deletion mutant is less virulent, and infection of reconstituted human epithelium with the pga29 mutant results in a diminished induction of proinflammatory cytokines, such as GM‐CSF, TNF, IL‐6 and IL‐8. We propose that the reduced virulence of the pga29 mutant is a consequence of altered surface properties, resulting in altered fungal recognition. Copyright

Iron restriction-induced adaptations in the wall proteome of Candida albicans

The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized glycosylphosphatidylinositol (GPI) proteins, it can exploit iron from host haemoglobin, ferritin and transferrin. Culturing C. albicans in rich medium supplemented with the ferrous iron chelator bathophenanthroline disulfonic acid or in the minimal medium yeast nitrogen base resulted in a strong decrease of the iron content of the cells. MS analysis of the changes in the wall proteome of C. albicans upon iron restriction showed a strong increase in the levels of the GPI-modified adhesin Als3, which also serves as a ferritin receptor, and of the GPI-modified CFEM (common in fungal extracellular membranes) domain-containing proteins Csa1, Pga7, Pga10, and Rbt5. The wall levels of the GPI-modified proteins Hyr1, the adhesin Als4 and the copper- and zinc-containing superoxide dismutase Sod4 also strongly increased, whereas the levels of Tos1 (a non-GPI protein) and the GPI-modified adhesin Als2 strongly decreased. Strikingly, peptides derived from the CFEM domain of the haem-binding proteins Csa1, Pga10 and Rbt5 were capable of forming iron adduct ions during MS analysis, consistent with a key role of this domain in haem binding.

Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.