Measuring the capacity of yeast for surface display of cell wall-anchored protein isoforms by using ß-lactamase as a reporter enzyme.

Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface-displayed ß-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use ß-lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface-displayed ß-lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real-time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high-throughput screening of potential ß-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

A core of cell wall proteins functions in wall integrity responses in Arabidopsis thaliana.

Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin (quasimodo2), cellulose (cellulose synthase3 je5), and xyloglucan (xyloglucan xylosyltransferase1 and 2), to probe whether changes in the expression of cell wall-related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin- and cellulose-deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan-deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load-bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

Exploring the Cell Wall and Secretory Proteins of Mycobacterium leprae as Biomarkers.

The bacterial cell wall is composed of a wide variety of intricate proteins in addition to lipids, glycolipids, and polymers. Given the diversity of cell wall proteins among bacterial species, they are a feasible target for biomarker identification and characterization in clinical research and diagnosis of the disease. The slow growth rate of Mycobacterium leprae poses a major hurdle in the accurate diagnosis of leprosy before the onset of peripheral neuropathy. The use of biomarker- based diagnostic methods can help in preventing the spread and manifestation of leprosy. Despite many advances in research methods and techniques, there remains a knowledge gap regarding the cell wall proteomes of M. leprae that can be used as biomarkers. The cell wall and secretory proteins of M. leprae are the major focus of this review article. This article enfolds the characteristics and functions of M. leprae cell wall proteins and gives an insight into those cell wall proteins that are yet to be established as biomarkers. Tools and techniques used in cell wall extraction and biomarker identification can also be explored in this article.

Candida parapsilosis Cell Wall Proteome Characterization and Effectiveness against Hematogenously Disseminated Candidiasis in a Murine Model.

Candida parapsilosis poses huge treatment challenges in the clinical settings of South Africa, and often causes infections among immunocompromised patients and underweight neonates. Cell wall proteins have been known to play vital roles in fungal pathogenesis, as these are the first points of contact toward environments, the host, and the immune system. This study characterized the cell wall immunodominant proteins of pathogenic yeast C. parapsilosis and evaluated their protective effects in mice, which could add value in vaccine development against the rising C. parapsilosis infections. Among different clinical strains, the most pathogenic and multidrug-resistant C. parapsilosis isolate was selected based on their susceptibility towards antifungal drugs, proteinase, and phospholipase secretions. Cell wall antigens were prepared by ß-mercaptoethanol/ammonium bicarbonate extraction from selected C. parapsilosis strains. Antigenic proteins were identified using LC-MS/MS, where 933 proteins were found, with 34 being immunodominant. The protective effect of the cell wall immunodominant proteins was observed by immunizing BALB/c mice with cell wall protein extracts. After the immunization and booster, the BALC/c mice were challenged with a lethal dose of C. parapsilosis. In vivo results demonstrated increased survival rates and lower fungal burden in vital organs in the immunized mice compared to the unimmunized mice, thereby confirming the immunogenic property of cell wall-associated proteins of C. parapsilosis. Therefore, these results advocated the potential of these cell wall proteins to act as biomarkers for the development of diagnostic assays and/or vaccines against infections caused by C. parapsilosis.

Multi-omics Data Integration in the Context of Plant Abiotic Stress Signaling.

In order to answer new biological questions, high-throughput data generated by new biotechnologies can be very meaningful but require specific and adapted statistical treatments. Thus, in the context of abiotic stress signaling studies, understanding the integration of cascading mechanisms from stress perception to biochemical and physiological adjustments necessarily entails efficient and valid analysis of multilevel and heterogeneous data. In this chapter, we propose examples to manage, analyze, and integrate multi-omics heterogeneous data. This workflow suggests and follows different general biological questions or issues answered with detailed code, data analysis, multiple visualizations, and always followed by brief interpretations. We illustrated this using the mixOmics package for the R software, as it specifically provides tools to address vertical and horizontal data integration issues. In order to illustrate this workflow, we used the usual omics datasets biologists can generate (phenomics, metabolomics, proteomics, and transcriptomics). These data were collected from two organs (leaf rosettes, floral stems) of five ecotypes of the model plant Arabidopsis thaliana exposed to two temperature growth conditions. They are available in the R package WallOmicsData. The workflow presented here is not limited to Arabidopsis thaliana and can be applied to any plant species. It can even be largely deployed to whatever the organisms of interest and the biological questions may be.

Host Immune Responses to Surface S-Layer Proteins (SLPs) of Clostridioides difficile.

Clostridioides difficile, a nosocomial pathogen, is an emerging gut pathobiont causing antibiotic-associated diarrhea. C. difficile infection involves gut colonization and disruption of the gut epithelial barrier, leading to the induction of inflammatory/immune responses. The expression of two major exotoxins, TcdA and TcdB is the major cause of C. difficile pathogenicity. Attachment of bacterial abundant cell wall proteins or surface S-layer proteins (SLPs) such as SlpA with host epithelial cells is critical for virulence. In addition to being toxins, these surface components have been shown to be highly immunogenic. Recent studies indicate that C. difficile SLPs play important roles in the adhesion of the bacteria to the intestinal epithelial cells, disruption of tight junctions, and modulation of the immune response of the host cells. These proteins might serve as new targets for vaccines and new therapeutic agents. This review summarizes our current understanding of the immunological role of SLPs in inducing host immunity and their use in the development of vaccines and novel therapeutics to combat C. difficile infection.

Competitive metal-binding stoichiometry between calcium and strontium by cell wall proteins of Neurospora crassa.

Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca2+ /Sr2+ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca2+ /Sr2+ . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca2+ /Sr2+ fractions. Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca2+ and Sr2+ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca2+ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca2+ bound Sr2+ binding properties. Surprisingly, these proteins were able to bind Sr2+ ions (29 µmol) with equal affinity as to Ca2+ ions (42 µmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca2+ (an essential macro elemental metal ion) and Sr2+ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins. This stoichiometry could be due to similar atomic dimensions and metal-protein structure stabilizing properties of Sr2+ compared to Ca2+ .

Proteomic Analysis of Sporothrix schenckii Exposed to Oxidative Stress Induced by Hydrogen Peroxide.

Sporothrix schenckii modulates the expression of its cell wall proteins (CWPs) in response to reactive oxygen species (ROS) generated by the phagocytic cells of the human host, which allows it to evade and escape the immune system. In this study, we performed a comparative proteomic analysis of the CW of S. schenckii after exposure and nonexposure to H2O2. Several CWPs involved in CW remodeling and fungal pathogenesis that modulated their expression in response to this oxidizing agent were identified, as were a number of antioxidant enzymes and atypical CWPs, called moonlighting proteins, such as the Hsp70-5, lipase 1 (Lip1), enolase (Eno), and pyruvate kinase (Pk). Moreover, RT-qPCR assays demonstrated that the transcription of genes HSP70-5, LIP1, ENO, and PK is regulated in response to the oxidant. The results indicated that S. schenckii differentially expressed CWPs to confer protection against ROS upon this fungus. Furthermore, among these proteins, antioxidant enzymes and interestingly, moonlighting-like CWPs play a role in protecting the fungus from oxidative stress (OS), allowing it to infect human host cells.

Proteinous Components of Neutrophil Extracellular Traps Are Arrested by the Cell Wall Proteins of Candida albicans during Fungal Infection, and Can Be Used in the Host Invasion.

One of defense mechanisms of the human immune system to counteract infection by the opportunistic fungal pathogen Candida albicans is the recruitment of neutrophils to the site of invasion, and the subsequent production of neutrophil extracellular traps (NETs) that efficiently capture and kill the invader cells. In the current study, we demonstrate that within these structures composed of chromatin and proteins, the latter play a pivotal role in the entrapment of the fungal pathogen. The proteinous components of NETs, such as the granular enzymes elastase, myeloperoxidase and lactotransferrin, as well as histones and cathelicidin-derived peptide LL-37, are involved in contact with the surface of C. albicans cells. The fungal partners in these interactions are a typical adhesin of the agglutinin-like sequence protein family Als3, and several atypical surface-exposed proteins of cytoplasmic origin, including enolase, triosephosphate isomerase and phosphoglycerate mutase. Importantly, the adhesion of both the elastase itself and the mixture of proteins originating from NETs on the C. albicans cell surface considerably increased the pathogen potency of human epithelial cell destruction compared with fungal cells without human proteins attached. Such an implementation of adsorbed NET-derived proteins by invading C. albicans cells might alter the effectiveness of the fungal pathogen entrapment and affect the further host colonization.

Fungal Cell Wall Proteins and Signaling Pathways Form a Cytoprotective Network to Combat Stresses.

Candida species are part of the normal flora of humans, but once the immune system of the host is impaired and they escape from commensal niches, they shift from commensal to pathogen causing candidiasis. Candida albicans remains the primary cause of candidiasis, accounting for about 60% of the global candidiasis burden. The cell wall of C. albicans and related fungal pathogens forms the interface with the host, gives fungal cells their shape, and also provides protection against stresses. The cell wall is a dynamic organelle with great adaptive flexibility that allows remodeling, morphogenesis, and changes in its components in response to the environment. It is mainly composed of the inner polysaccharide rich layer (chitin, and ß-glucan) and the outer protein coat (mannoproteins). The highly glycosylated protein coat mediates interactions between C. albicans cells and their environment, including reprograming of wall architecture in response to several conditions, such as carbon source, pH, high temperature, and morphogenesis. The mannoproteins are also associated with C. albicans adherence, drug resistance, and virulence. Vitally, the mannoproteins contribute to cell wall construction and especially cell wall remodeling when cells encounter physical and chemical stresses. This review describes the interconnected cell wall integrity (CWI) and stress-activated pathways (e.g., Hog1, Cek1, and Mkc1 mediated pathways) that regulates cell wall remodeling and the expression of some of the mannoproteins in C. albicans and other species. The mannoproteins of the surface coat is of great importance to pathogen survival, growth, and virulence, thus understanding their structure and function as well as regulatory mechanisms can pave the way for better management of candidiasis.

Lysine Residues in the MK-Rich Region Are Not Required for Binding of the PbsP Protein From Group B Streptococci to Plasminogen.

Binding to plasminogen (Plg) enables bacteria to associate with and invade host tissues. The cell wall protein PbsP significantly contributes to the ability of group B streptococci, a frequent cause of invasive infection, to bind Plg. Here we sought to identify the molecular regions involved in the interactions between Plg and PbsP. The K4 Kringle domain of the Plg molecule was required for binding of Plg to whole PbsP and to a PbsP fragment encompassing a region rich in methionine and lysine (MK-rich domain). These interactions were inhibited by free L-lysine, indicating the involvement of lysine binding sites in the Plg molecule. However, mutation to alanine of all lysine residues in the MK-rich domain did not decrease its ability to bind Plg. Collectively, our data identify a novel bacterial sequence that can interact with lysine binding sites in the Plg molecule. Notably, such binding did not require the presence of lysine or other positively charged amino acids in the bacterial receptor. These data may be useful for developing alternative therapeutic strategies aimed at blocking interactions between group B streptococci and Plg.

New Players in the Interaction Between Beetle Polygalacturonases and Plant Polygalacturonase-Inhibiting Proteins: Insights From Proteomics and Gene Expression Analyses.

Plants possess various defense strategies to counter attacks from microorganisms or herbivores. For example, plants reduce the cell-wall-macerating activity of pathogen- or insect-derived polygalacturonases (PGs) by expressing PG-inhibiting proteins (PGIPs). PGs and PGIPs belong to multi-gene families believed to have been shaped by an evolutionary arms race. The mustard leaf beetle Phaedon cochleariae expresses both active PGs and catalytically inactive PG pseudoenzymes. Previous studies demonstrated that (i) PGIPs target beetle PGs and (ii) the role of PG pseudoenzymes remains elusive, despite having been linked to the pectin degradation pathway. For further insight into the interaction between plant PGIPs and beetle PG family members, we combined affinity purification with proteomics and gene expression analyses, and identified novel inhibitors of beetle PGs from Chinese cabbage (Brassica rapa ssp. pekinensis). A beetle PG pseudoenzyme was not targeted by PGIPs, but instead interacted with PGIP-like proteins. Phylogenetic analysis revealed that PGIP-like proteins clustered apart from "classical" PGIPs but together with proteins, which have been involved in developmental processes. Our results indicate that PGIP-like proteins represent not only interesting novel PG inhibitor candidates in addition to "classical" PGIPs, but also fascinating new players in the arms race between herbivorous beetles and plant defenses.

The Craterostigma plantagineum protein kinase CpWAK1 interacts with pectin and integrates different environmental signals in the cell wall.

MAIN CONCLUSION: The cell wall protein CpWAK1 interacts with pectin, participates in decoding cell wall signals, and induces different downstream responses. Cell wall-associated protein kinases (WAKs) are transmembrane receptor kinases. In the desiccation-tolerant resurrection plant Craterostigma plantagineum, CpWAK1 has been shown to be involved in stress responses and cell expansion by forming a complex with the C. plantagineum glycine-rich protein1 (CpGRP1). This prompted us to extend the studies of WAK genes in C. plantagineum. The phylogenetic analyses of WAKs from C. plantagineum and from other species suggest that these genes have been duplicated after species divergence. Expression profiles indicate that CpWAKs are involved in various biological processes, including dehydration-induced responses and SA- and JA-related reactions to pathogens and wounding. CpWAK1 shows a high affinity for "egg-box" pectin structures. ELISA assays revealed that the binding of CpWAKs to pectins is modulated by CpGRP1 and it depends on the apoplastic pH. The formation of CpWAK multimers is the prerequisite for the CpWAK-pectin binding. Different pectin extracts lead to opposite trends of CpWAK-pectin binding in the presence of Ca2+ at pH 8. These observations demonstrate that CpWAKs can potentially discriminate and integrate cell wall signals generated by diverse stimuli, in concert with other elements, such as CpGRP1, pHapo, Ca2+[apo], and via the formation of CpWAK multimers.

High Biofilm Formation of Non-Smooth Candida parapsilosis Correlates with Increased Incorporation of GPI-Modified Wall Adhesins.

Candida parapsilosis is among the most frequent causes of candidiasis. Clinical isolates of this species show large variations in colony morphotype, ranging from round and smooth to a variety of non-smooth irregular colony shapes. A non-smooth appearance is related to increased formation of pseudohyphae, higher capacity to form biofilms on abiotic surfaces, and invading agar. Here, we present a comprehensive study of the cell wall proteome of C. parapsilosis reference strain CDC317 and seven clinical isolates under planktonic and sessile conditions. This analysis resulted in the identification of 40 wall proteins, most of them homologs of known Candida albicans cell wall proteins, such as Gas, Crh, Bgl2, Cht2, Ecm33, Sap, Sod, Plb, Pir, Pga30, Pga59, and adhesin family members. Comparative analysis of exponentially growing and stationary phase planktonic cultures of CDC317 at 30 °C and 37 °C revealed only minor variations. However, comparison of smooth isolates to non-smooth isolates with high biofilm formation capacity showed an increase in abundance and diversity of putative wall adhesins from Als, Iff/Hyr, and Hwp families in the latter. This difference depended more strongly on strain phenotype than on the growth conditions, as it was observed in planktonic as well as biofilm cells. Thus, in the set of isolates analyzed, the high biofilm formation capacity of non-smooth C. parapsilosis isolates with elongated cellular phenotypes correlates with the increased surface expression of putative wall adhesins in accordance with their proposed cellular function.

Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris.

Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

Systematic Comparison of Cell Wall-Related Proteins of Different Yeasts.

Yeast cell walls have two major roles, to preserve physical integrity of the cell, and to ensure communication with surrounding molecules and cells. While the first function requires evolutionary conserved polysaccharide network synthesis, the second needs to be flexible and provide adaptability to different habitats and lifestyles. In this study, the comparative in silico analysis of proteins required for cell wall biosynthesis and functions containing 187 proteins of 92 different yeasts was performed in order to assess which proteins were broadly conserved among yeasts and which were more species specific. Proteins were divided into several groups according to their role and localization. As expected, many Saccharomyces cerevisiae proteins involved in protein glycosylation, glycosylphosphatidylinositol (GPI) synthesis and the synthesis of wall polysaccharides had orthologues in most other yeasts. Similarly, a group of GPI anchored proteins involved in cell wall biosynthesis (Gas proteins and Dfg5p/Dcw1p) and other non-GPI anchored cell wall proteins involved in the wall synthesis and remodeling were highly conserved. However, GPI anchored proteins involved in flocculation, aggregation, cell separation, and those of still unknown functions were not highly conserved. The proteins localized in the cell walls of various yeast species were also analyzed by protein biotinylation and blotting. Pronounced differences were found both in the patterns, as well as in the overall amounts of different groups of proteins. The amount of GPI-anchored proteins correlated with the mannan to glucan ratio of the wall. Changes of the wall proteome upon temperature shift to 42 °C were detected.

Interacting with Hemoglobin: Paracoccidioides spp. Recruits hsp30 on Its Cell Surface for Enhanced Ability to Use This Iron Source.

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.

Scalar nanostructure of the Candida albicans cell wall; a molecular, cellular and ultrastructural analysis and interpretation.

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of ß(1,3)- and ß(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or ß-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure-function relationships that underpin the pathobiology of this fungal pathogen.