Eulogio Valentín

Solubilization and Analysis of Mannoprotein Molecules from The Cell Wall of Saccharomyces cerevisiae

Purified walls from Saccharomyces cerevisiae were treated chemically to release intrinsic mannoproteins. Boiling in 2% SDS gave the best results, although treatment in 6 M-urea at room temperature also released significant amounts of mannoprotein radioactivity. Triton X-100, sodium deoxycholate and EDTA were poor solubilizers. Electrophoretic patterns of SDS- or urea-released mannoproteins in SDS-acrylamide gels indicated a great heterogeneity of molecular species, with more than 60 bands. Zymolyase, a glucan-digesting complex, released about half of the mannoproteins, but these species showed an altered mobility on SDS-acrylamide gels and had a lowered capacity for precipitation by ethanol. Action of the enzyme on isolated walls was favoured by dithiothreitol, as is the case with whole cells, and repeated treatments with SDS and Zymolyase released all of the mannoproteins from the wall. Solubilizing treatments other than SDS had a differential effect on recently or formerly incorporated mannoproteins in the wall. The results suggest an asymmetrical arrangement of molecules in the envelope and point to dynamic changes inside the wall as it thickens as a result of cell aging.

Structure of the Saccharomyces cerevisiae cell wall: Mannoproteins released by zymolyase and their contribution to wall architecture

Abstract Purified zymolyase containing β-glucanase activity preferentially released a 29 kDa mannoprotein from isolated yeast cell walls and a high-molecular-mass (greater than 120 kDa) material. Endo-β-N-acetylglucosaminidase H digestion indicated that the 29 kDa mannoprotein contains a unique core coligosaccharide N-glycosidically linked to a 26 kDa peptide moiety. Cells grown in the presence of tunicamycin incorporated the nonglycosylated 26 kDa peptide into the wall, but not the large mannoprotein molecules. Treatment of isolated walls with SDS solubilized more than 30 different mannoproteins, one of tehm being the 29 kDa species, but the large-size molecules were not affected. Regenerating protoplasts incorporated into the forming walls most of the SDS-solubilizable species seen in mature cell walls, but the zymolyase-solubilizable mannoproteins were absent. Wall mannoproteins have also been compared with those of the periplasmic space, most of the species being commonly present at both compartments. Turnover of individual species has been studied by pulse and chase experiments. While mannoproteins from the walls remain stable for long periods, periplasmic molecules exhibit a rapid turnover rate.

A study of the Candida albicans cell wall proteome

Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC‐MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride‐pyridine (HF‐pyridine), and whole SDS‐extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF‐pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 “atypical” cell wall proteins that lack the above‐mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found “atypical” wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.

Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall beta-1,3-glucan.

Identification of PIR/CIS3 gene was carried out by amino‐terminal sequencing of a protein band released by β‐mercaptoethanol (β‐ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β‐1,3‐glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys214‐12aa‐Cys227‐COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site‐directed mutation of two other cysteine amino acids (Cys130Ser or Cys197Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2‐unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Δ and gsc2/fks2Δ cells, defective in β‐1,3‐glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by β‐ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the β‐1,3‐glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2‐unprocessed Pir4 form. Pir4 failed to be incorporated in alkali‐sensitive linkages involving β‐1,3‐glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the β‐1,3‐glucan. Copyright

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.

A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector λgt11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N‐terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of β‐glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub‐apical locations, suggesting that it participates directly in the mycelial wall architecture.

Critical steps in fungal cell wall synthesis : strategies for their inhibition

Development of new effective antifungal drugs is limited by the absence of specific target sites in the fungal cells. Knowledge of the fungal cell wall structure and biosynthesis is of interest in searching for a potential target site for new chemotherapeutic agents. Our group has demonstrated that the fungal cell wall is a metabolically active structure where interaction between distinct components occurs to give rise to the mature cell wall structure. Mannoproteins play an essential role in the cell wall organization, and there is evidence for the formation of covalent bonds between these molecules and the structural polymers (glucans and chitin) outside the plasma membrane. Such interactions, which specifically occur at the fungal cell wall, are of great interest in defining target sites for potential new chemotherapeutic agents, which may inhibit the interactions and, thus, lead to a defective cell wall formation and cell death.

Pga13 in Candida albicans is localized in the cell wall and influences cell surface properties, morphogenesis and virulence.

The fungal cell wall is an essential organelle required for maintaining cell integrity and also plays an important role in the primary interactions between pathogenic fungi and their hosts. PGA13 encodes a GPI protein in the human pathogen Candida albicans, which is highly up-regulated during cell wall regeneration in protoplasts. The Pga13 protein contains a unique tandem repeat, which is present five times and is characterized by conserved spacing between the four cysteine residues. Furthermore, the mature protein contains 38% serine and threonine residues, and therefore probably is a highly glycosylated cell wall protein. Consistent with this, a chimeric Pga13-V5 protein could be localized to the cell wall, but only after deglycosylation was performed. Disruption of PGA13 led to increased sensitivity to Congo red, Calcofluor white, and zymolyase, and to a diminished ability of protoplasts to recover their cell wall. In addition, pga13Δ mutants exhibited delayed filamentation, a higher surface hydrophobicity, and increased adherence and flocculation (cell-cell interactions). Furthermore, transcript profiling showed that expression of four members of the ALS family (adhesin-encoding genes) is up-regulated in the pga13Δ null mutant. Altogether, these results indicate that Pga13 is a wall-localized protein that contributes to cell wall synthesis and is important for acquiring normal surface properties. The contribution of Pga13 to surface hydrophilicity may be important for cell dispersal during development of invasive infections, and possibly for morphological development. This is consistent with the observed reduced virulence of pga13Δ mutants in a mouse model of disseminated candidiasis.

Adhesive Properties and Hydrolytic Enzymes of Oral Candida albicans Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.

Cloning of cDNAs coding for Candida albicans cell surface proteins

Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1.5 kb for 11Y and 1.1 kb for 24M. Both transcripts were present in yeast cells as well as in germ-tubes. The whole genes were isolated from a C. albicans genomic library in the YRp7 vector by hybridization with the cDNA probes. Monospecific antibodies were purified from polyclonal antisera by affinity for the fusion proteins. Western blot analysis with 11Y-specific antibodies revealed a cross-reactivity with material found in the yeast cell wall as well as in other subcellular fractions, whereas clone 24M codes for a 30 kDa protein detected mainly in the membrane fraction and in the SDS-solubilized material from mycelial cell walls. Sequencing of the cDNA molecules and restriction map of the cloned genes demonstrate that clone 11Y is an enolase previously characterized in C. albicans, whereas clone 24M does not show significant homology with any other cloned gene.