Oral disease is linked to low nestling condition and brood size in a raptor species living in a highly modified environment.

Anthropogenic stressors can favor the occurrence of noninfectious disease that can be worsened by the impact of opportunistic pathogens, making the epizootiology of environmental diseases difficult to unravel. The incidence and impact of oral lesions in nestlings of a facultative scavenger species, the black kite Milvus migrans, were examined over seven breeding seasons in the highly degraded environment close to Madrid, Spain. We found an overall prevalence of 31% of nestlings with oral lesions, with no clear spatial pattern in nests with affected and unaffected individuals. The occurrence and number of oral lesions were negatively associated with nestling body condition and brood size. Broods, where all siblings had oral lesions, were smaller than those where some or all siblings were apparently healthy, suggesting that oral disease could be causing nestling mortality and, consequently, brood size reduction. In turn, nestling body condition was negatively affected by lesion occurrence, brood size, and laying date. Although these relationships were bidirectional, piecewise structural equation modeling analyses showed a greater negative effect of body condition on lesion occurrence than vice versa, indicating that nestlings in poorer body condition were more likely to develop oral lesions (which could contribute to aggravate their state of deterioration) than those in better condition. Nestlings from small broods were also more likely to have oral disease (directly or indirectly through their lower body condition) than nestlings from large broods. Nestlings that hatched last in the broods showed greater development stress than those that hatched first. Anthropogenic stressors could trigger poor body condition, and contribute to microbiota dysbiosis-related diseases. Although further research is needed to determine the consequences for the long-term fitness of individuals, actions should be taken to mitigate adverse conditions that may favor the appearance of environmental diseases associated with peri-urban areas, given their rapid expansion over natural areas.

Fungal signatures of oral disease reflect environmental degradation in a facultative avian scavenger.

Degradation of natural ecosystems increases the risk of infections in wildlife due to microbiota dysbiosis. However, little is known about its influence on the development of fungal communities in predators and facultative avian scavengers. We evaluated the incidence of oral disease in wild nestling black kites (Milvus migrans) under contrasting environmental degradation conditions, and explored their oral fungal patterns using molecular methods and multivariate analysis. Oral lesions were found in 36.8% of the 38 nestlings examined in an anthropogenically altered habitat (southeastern Madrid, Spain), but in none of the 105 nestlings examined in a well-conserved natural area (Doñana National Park, Spain). In a subsample of 48 black kites, the composition of the oral fungal community differed among symptomatic nestlings from Madrid (SM) and asymptomatic nestlings from Madrid (AM) and Doñana (AD). Opportunistic fungal pathogens (e.g., Fusarium incarnatum-equiseti species complex, Mucor spp., Rhizopus oryzae) were more prevalent in SM and AM than in AD. Hierarchical clustering and principal component analyses revealed that fungal patterns were distinct between both study areas, and that anthropogenic and natural environmental factors had a greater impact on them than oral disease. Fungal signatures associated with anthropogenic and natural stresses harbored some taxa that could be used to flag oral infection (F. incarnatum-equiseti species complex and Alternaria), indicate environmental degradation (Alternaria) or provide protective benefits in degraded environments (Trichoderma, Epicoccum nigrum and Sordaria). Co-occurrence associations between potentially beneficial and pathogenic fungi were typical of AM and AD, hinting at a possible role in host health. This study shows that early-life exposure to highly degraded environments induces a shift towards a higher prevalence of pathogenic species in the oral cavity of black kites, favoring oral disease. Furthermore, our findings suggest potential ecological applications of the monitoring of oral mycobiome as a bioindication of oral disease and environmental degradation.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis.

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis.

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

Vultures from different trophic guilds show distinct oral pathogenic yeast signatures and co-occurrence networks.

Vultures have evolved adaptive mechanisms to prevent infections associated with their scavenging lifestyle. However, food-borne exposure to antimicrobial pharmaceuticals can promote opportunistic infections with adverse outcomes. Here, we used multivariate and network analyses to increase understanding of the behavior of the yeast communities causing oral mycosis outbreaks recently reported in wild nestling cinereous (Aegypius monachus), griffon (Gyps fulvus) and Egyptian (Neophron percnopterus) vultures (CV, GV and EV, respectively) exposed to antibiotics from livestock farming. Common and unique yeast signatures (of Candida, Debaromyces, Diutina, Meyerozyma, Naganishia, Pichia, Rhodotorula, Trichosporon and Yarrowia species) associated with oral mycoses were identified in the three vulture species. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) highlighted that oral lesions from CV and GV shared similar yeast signatures (of major causative pathogens of opportunistic mycoses, such as Candida albicans, Candida parapsilosis and Candida tropicalis), while EV had a distinct yeast signature (of uncommon pathogenic species, such as Candida dubliniensis, Candida zeylanoides, Pichia fermentans and Rhodotorula spp.). Synergistic interactions between yeast species from distinct fungal phyla were found in lesions from CV and GV, but not in EV. These formed co-occurrence subnetworks with partially or fully connected topology. This study reveals that the composition, assembly and co-occurrence patterns of the yeast communities causing oral mycoses differ between vulture species with distinct feeding habits and scavenging lifestyles. Yeast species widely pathogenic to humans and animals, and yeast co-occurrence relationships, are distinctive hallmarks of oral mycoses in CV and GV. These vulture species are more exposed to antibiotics from intensively medicated livestock carcasses provided in supplementary feeding stations and show higher incidence of thrush-like oral lesions than EV. These findings may be useful for development of new initiatives or changes in the conservation of these avian scavengers affected by anthropogenic activities.

Enrichment of ATP Binding Proteins Unveils Proteomic Alterations in Human Macrophage Cell Death, Inflammatory Response, and Protein Synthesis after Interaction with Candida albicans.

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.

Diagnosis of Invasive Candidiasis: From Gold Standard Methods to Promising Leading-edge Technologies.

Invasive Candidiasis (IC) poses a major public health problem worldwide. Despite the introduction of new antifungal agents and changes in clinical practices, its morbidity and mortality rates and healthcare costs remain persistently high. This is mainly because of the serious underlying conditions of infected patients (critically ill or severely immunocompromised patients) and the difficulties encountered in early diagnosing this opportunistic mycosis and initiating prompt and appropriate antifungal therapy. In the light of this great clinical challenge, the past decades have witnessed the development of diverse early detection and therapeutic intervention strategies aimed at minimizing the clinical impact and economic burden of this healthcare-associated infection caused by Candida species. Here, we review the currently available methods for IC diagnosis. These encompass (i) gold standard methods (fungal culture and tissue histopathology), (ii) pathogen-derived biomarker detection tests (PCR, protein antigens, mannan, ß-D-glucan and D-arabinitol-based assays), (iii) host-derived biomarker detection tests (Candida albicans germ tube antibodies or CAGTA, anti-mannan antibodies, other infection-specific antibodies, procalcitonin, serum amyloid A, interleukin 17, interleukin 23 and transforming growth factor ß-based assays), (iv) clinical prediction algorithms (Candida score, Candida colonization index and other prediction rules), and (v) leading-edge molecular, proteomic and immunomic technologies (such as peptide nucleic acid-fluorescent in situ hybridization or PNA-FISH, T2 magnetic resonance or T2Candida assay, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or MALDI-TOF MS, among others). Their strengths, utility, limitations as well as combined use to assist in the diagnosis of this life-threatening and costly fungal infection (including candidemia and deepseated candidiasis) are also discussed.

Oral mycoses in avian scavengers exposed to antibiotics from livestock farming.

The exposure to antimicrobial pharmaceuticals as environmental contaminants can exert direct and indirect detrimental effects on health of wildlife. Fungal infections pose a major threat to domestic, captive-housed wild and free-ranging wild animals worldwide. However, little is known about their role in disease in birds in the wild. Here, we evaluated the incidence of thrush-like lesions in the oral cavity of wild nestling cinereous vultures (Aegypius monachus), griffon vultures (Gyps fulvus), Egyptian vultures (Neophron percnopterus) and golden eagles (Aquila chrysaetos) exposed to veterinary antibiotics via the consumption of medicated livestock carcasses. Lesions, which varied in number, size and location, were more frequent in the cinereous (77.8%, n=9) and griffon vultures (66.7%, n=48) than in the Egyptian vultures (28.6%, n=21) and golden eagles (28.6%, n=7). In all individuals (100%, n=24) of a subsample of the affected nestlings, yeast species were isolated from thrush-like oral lesions and identified using a well-established system based on their carbohydrate assimilation profiles and other complementary tests. Fourteen yeast species from seven genera (Candida, Meyerozyma, Pichia, Yarrowia, Cryptococcus, Rhodotorula and Trichosporon) were isolated from the lesions of the four host species. We found differential infections and effects depending on host age-related exposure or susceptibility to different yeast species across the development of nestling griffon vultures. This unprecedented outbreak of oral mycoses is alarming because of the delicate conservation status of several of the affected species. The role of livestock antibiotics in the transition of yeast species from commensal to opportunistic pathogens should be evaluated in an attempt to avoid the detrimental effects of contamination and disease on host health, as well as on the transmission of fungal emerging pathogens among wildlife populations and species, and their dissemination across livestock and human populations.

Serum Antibody Profile during Colonization of the Mouse Gut by Candida albicans: Relevance for Protection during Systemic Infection.

Candida albicans is a commensal microorganism in the oral cavity and gastrointestinal and urogenital tracts of most individuals that acts as an opportunistic pathogen when the host immune response is reduced. Here, we established different immunocompetent murine models to analyze the antibody responses to the C. albicans proteome during commensalism, commensalism followed by infection, and infection (C, C+I, and I models, respectively). Serum anti-C. albicans IgG antibody levels were higher in colonized mice than in infected mice. The antibody responses during gut commensalism (up to 55 days of colonization) mainly focused on C. albicans proteins involved in stress response and metabolism and differed in both models of commensalism. Different serum IgG antibody-reactivity profiles were also found over time among the three murine models. C. albicans gut colonization protected mice from an intravenous lethal fungal challenge, emphasizing the benefits of fungal gut colonization. This work highlights the importance of fungal gut colonization for future immune prophylactic therapies.

Top-down characterization data on the speciation of the Candida albicans immunome in candidemia.

The characterization of pathogen-specific antigenic proteins at the protein species level is crucial in the development and molecular optimization of novel immunodiagnostics, vaccines or immunotherapeutics for infectious diseases. The major requirements to achieve this molecular level are to obtain 100% sequence coverage and identify all post-translational modifications of each antigenic protein species. In this article, we show nearly complete sequence information for five discrete antigenic species of Candida albicans Tdh3 (glyceraldehyde-3-phosphate dehydrogenase), which have been reported to be differentially recognized both among candidemia patients and between candidemia and control patients. A comprehensive description of the top-down immunoproteomic strategy used for seroprofiling at the C. albicans protein species level in candidemia as well as for the chemical characterization of this immunogenic protein (based on high-resolution 2-DE, Western blotting, peptide mass fingerprinting, tandem mass spectrometry and de novo peptide sequencing) is also provided. The top-down characterization data on the speciation of the C. albicans immunome in candidemia presented here are related to our research article entitled "Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia" (Pitarch et al., J. Proteomics, 2015, http://dx.doi.org/10.1016/j.jprot.2015.10.022).

Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia.

Serum antibodies to specific Candida proteins have been reported as potential diagnostic biomarkers for candidemia. However, their diagnostic usefulness at the protein species level has hardly been examined. Using serological proteome analysis, we explored the IgG-antibody responses to Candida albicans protein species in candidemia and control patients. We found that 87 discrete protein species derived from 34 unique proteins were IgG-targets, although only 43 of them were differentially recognized by candidemia and control sera. An increase in the speciation of the immunome, connectivity and modularity of antigenic species co-recognition networks, and heterogeneity of antigenic species recognition patterns was associated with candidemia. IgG antibodies to certain discrete protein species were better predictors of candidemia than those to their corresponding proteins. A molecular discriminator delineated from the combined fingerprints of IgG antibodies to two distinct species of phosphoglycerate kinase and enolase accurately classified candidemia and control patients. These results provide new insight into the anti-Candida IgG-antibody response development in candidemia, and demonstrate that an immunoproteomic signature at the molecular level may be useful for its diagnosis. Our study further highlights the importance of defining pathogen-specific antigens at the chemical and molecular level for their potential application as immunodiagnostic reagents or even vaccine candidates.

Serum antibody signature directed against Candida albicans Hsp90 and enolase detects invasive candidiasis in non-neutropenic patients.

Invasive candidiasis (IC) adds significantly to the morbidity and mortality of non-neutropenic patients if not diagnosed and treated early. To uncover serologic biomarkers that alone or in combination could reliably detect IC in this population, IgG antibody-reactivity profiles to the Candida albicans intracellular proteome were examined by serological proteome analysis (SERPA) and data mining procedures in a training set of 24 non-neutropenic patients. Despite the high interindividual molecular heterogeneity, unsupervised clustering analyses revealed that serum 22-IgG antibody-reactivity patterns differentiated IC from non-IC patients. Univariate analyses further highlighted that 15 out of the 22 SERPA-identified IgG antibodies could be useful candidate IC biomarkers. The diagnostic performance of one of these candidates (anti-Hsp90 IgG antibodies) was validated using an ELISA prototype in a test set of 59 non-neutropenic patients. We then formulated an IC discriminator based on the combined immunoproteomic fingerprints of this and another SERPA-detected and previously validated IC biomarker (anti-Eno1 IgG antibodies) in the training set. Its consistency was substantiated using their ELISA prototypes in the test set. Receiver-operating-characteristic curve analyses showed that this two-biomarker signature accurately identified IC in non-neutropenic patients and provided better IC diagnostic accuracy than the individual biomarkers alone. We conclude that this serum IgG antibody signature directed against C. albicans Hsp90 and Eno1, if confirmed prospectively, may be useful for IC diagnosis in non-neutropenic patients.

Prediction of the clinical outcome in invasive candidiasis patients based on molecular fingerprints of five anti-Candida antibodies in serum.

Better prognostic predictors for invasive candidiasis (IC) are needed to tailor and individualize therapeutic decision-making and minimize its high morbidity and mortality. We investigated whether molecular profiling of IgG-antibody response to the whole soluble Candida proteome could reveal a prognostic signature that may serve to devise a clinical-outcome prediction model for IC and contribute to known IC prognostic factors. By serological proteome analysis and data-mining procedures, serum 31-IgG antibody-reactivity patterns were examined in 45 IC patients randomly split into training and test sets. Within the training cohort, unsupervised two-way hierarchical clustering and principal-component analyses segregated IC patients into two antibody-reactivity subgroups with distinct prognoses that were unbiased by traditional IC prognostic factors and other patients-related variables. Supervised discriminant analysis with leave-one-out cross-validation identified a five-IgG antibody-reactivity signature as the most simplified and accurate IC clinical-outcome predictor, from which an IC prognosis score (ICPS) was derived. Its robustness was confirmed in the test set. Multivariate logistic-regression and receiver-operating-characteristic curve analyses demonstrated that the ICPS was able to accurately discriminate IC patients at high risk for death from those at low risk and outperformed conventional IC prognostic factors. Further validation of the five-IgG antibody-reactivity signature on a multiplexed immunoassay supported the serological proteome analysis results. The five IgG antibodies incorporated in the ICPS made biologic sense and were associated either with good-prognosis and protective patterns (those to Met6p, Hsp90p, and Pgk1p, putative Candida virulence factors and antiapoptotic mediators) or with poor-prognosis and risk patterns (those to Ssb1p and Gap1p/Tdh3p, potential Candida proapoptotic mediators). We conclude that the ICPS, with additional refinement in future larger prospective cohorts, could be applicable to reliably predict patient clinical-outcome for individualized therapy of IC. Our data further provide insights into molecular mechanisms that may influence clinical outcome in IC and uncover potential targets for vaccine design and immunotherapy against IC.

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition.

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

Identification of the Candida albicans immunome during systemic infection by mass spectrometry.

Over the past two decades, mass spectrometry (MS) has ceased to be a fairly exotic technique banished from the protein scientists' mind to become a seminal tool for deciphering the information encoded in the genomes of many biological species. Clues to this shift in the modus operandi for characterizing their proteomes stem from the progressive availability of full genome sequences and well-annotated protein databases of many model (micro)organisms, the development both of soft ionization methods for large biomolecules (peptides and proteins) and of innovative instrumentation designs, and the introduction of sophisticated search algorithms able to correlate MS information with sequence databases, to name but a few. Here we integrate the typical MS-based strategy for identifying proteins of Candida albicans, an opportunistic fungal pathogen of humans, which have proved to be present during systemic infection and targeted by the immune system as a consequence of its interaction with the host (i.e., the C. albicans immunome).

Proteomic profiling of serologic response to Candida albicans during host-commensal and host-pathogen interactions.

Candida albicans is a commensal inhabitant of the normal human microflora that can become pathogenic and invade almost all body sites and organs in response to both host-mediated and fungus-mediated mechanisms. Serologic responses to C. albicans that underlie its dichotomist relationship with the host (host-commensal and host-pathogen interactions) display a high degree of heterogeneity, resulting in distinct serum anti-Candida antibody signatures (molecular fingerprints of anti-Candida antibodies in serum) that can be used to discriminate commensal colonization from invasive disease. We describe the typical proteomic strategy to globally and integratively profile these host antibody responses and determine serum antibody signatures. This approach is based on the combination of classic immunoproteomics or serologic proteome analysis (two-dimensional electrophoresis followed by quantitative Western blotting and mass spectrometry) with data mining procedures. This global proteomic stratagem is a useful tool not only for obtaining an overview of different anti-Candida antibodies that are being elicited during the host-fungus interaction and, consequently, of the complex C. albicans immunome (the subset of the C. albicans proteome targeted by the immune system), but also for evaluating how this pathogen organism interacts with its host to trigger infection. In contrast with genomics and transcriptomics, this proteomic technology has the potential to detect antigenicity associated with posttranslational modification, subcellular localization, and other functional aspects that can be relevant in the host immune response. Furthermore, this strategy to define molecular fingerprints of serum anti-Candida antibodies may hopefully bring to light potential candidates for diagnosis, prognosis, risk stratification, clinical follow-up, therapeutic monitoring, and/or immunotherapy of candidiasis, especially of its life-threatening systemic forms.

Cell wall fractionation for yeast and fungal proteomics.

The cell wall is an external envelope shared by yeasts and filamentous fungi that defines the interface between the microorganism and its environment. It is an extremely complex structure consisting of an elastic framework of microfibrillar polysaccharides (glucans and chitin) that surrounds the plasma membrane and to which a wide array of different proteins, often heavily glycosylated, are anchored in various ways. Intriguingly, these cell wall proteins (CWPs) play a key role in morphogenesis, adhesion, pathogenicity, antigenicity, and as a promising target for antifungal drug design. However, the CWPs are difficult to analyze because of their low abundance, low solubility, hydrophobic nature, extensive glycosylation, covalent attachment to the wall polysaccharide skeleton, and high heterogeneity. We describe a typical procedure of cell wall fractionation to isolate and solubilize different CWP species from yeasts and filamentous fungi according to the type of linkages that they establish with other wall components and under suitable conditions for following reproducible proteomic analyses. CWPs retained noncovalently or by disulfide bonds are first extracted from isolated yeast or fungal cell walls by detergents and reducing agents. Subsequently, CWPs covalently linked to or closely entrapped within the internal glucan-chitin network are sequentially released either by mild alkali conditions or by enzymatic treatments first with glucanases and then with chitinases. This strategy is a powerful tool not only for obtaining an overview of the sophisticated cell wall proteome of yeasts and filamentous fungi, but also for characterizing mechanisms of incorporation, assembly and retention of CWPs into this intricate cellular compartment and their interactions with structural wall polysaccharides.

Collection of proteins secreted from yeast protoplasts in active cell wall regeneration.

The yeast cell wall is a dynamic and complex matrix of polysaccharides (glucans, mannans, and chitin), proteins and minor amounts of lipids that isolate the yeast from the extracellular medium, protecting it against osmotic and physical injuries. Removal of this essential structure for cell integrity and viability by controlled enzymatic digestion in an iso-osmotic medium brings about protoplast formation. When yeast protoplasts are incubated in an osmotically stabilized liquid nutrient medium, cell wall precursors are secreted into the culture medium to de novosynthesize the cell wall. During the early stages of the regeneration process of protoplast walls, many wall protein precursors (presumably structural proteins along with remodeling and cross-linking enzymes) are shed into the extracellular medium but not covalently incorporated into the nascent cell wall, intriguingly enabling their easy isolation and solubilization. We have developed a method to collect proteins secreted from yeast protoplasts in active cell wall regeneration under conditions that are suitable for subsequent proteomic analyses. This procedure circumvents some of the troubles intrinsically related to other extraction protocols of cell wall proteins, such as chemical or enzymatic modifications, and poor quality in protein resolution and identification because of linkages to glucan/chitin residues. It further offers a valuable model system to understand how the de novocell wall biosynthesis occurs in the yeast cell or how the yeast cell wall participates in morphogenesis.