Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae , which consists of an outer dense layer of mannan, a middle lucent layer of β−1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall P. carinii , as well as the cell wall of S. cerevisiae , can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β,3-gIucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae . These observations indicate that a major component of the cyst wall of P. carinii is β-1,3-glucan.     

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.     

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.     

Analysis of Pneumocystis carinii cyst wall. I. Evidence for an outer surface membrane

It has long been thought that the cyst form of Pneumocystis carinii, which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii. This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a beta-1-3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Zymolyase Treatment Exposes p55 Antigen of Pneumocystis carinii

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminai 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IIFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with β-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.     

Analysis of Pneumocystis carinii Cyst Wall I. Evidence for an Outer Surface Membrane

ABSTRACT. It has long been thought that the cyst form of Pneumocystis carinii , which can resist host defenses and antimicrobial drugs, is responsible for relapses of P. carinii pneumonia. The thick wall of the cyst is immunogenic and rich in glucosyl/mannosyl and N-acetyl-D-glucosamine residues. In this study we have demonstrated the presence of a hitherto unreported outer membrane in the cyst wall of P. carinii . This membrane was detected by a combination of techniques, including transmission electron microscopy, freeze-fracture electron microscopy, and membrane labeling with fluorescent lipid analogs following treatment of P. carinii cysts from infected rats for 30 min with Zymolyase, a β-1–3 glucanase. As in gram-negative bacteria and blue-green algae, this 2nd membrane may have an important role in osmoregulation and nutrient utilization; it may also mediate the interaction of P. carinii with its host and serve as a target for drug therapy.     

Analysis of Pneumocystis carinii Cyst Wall. II. Sugar Composition

ABSTRACT. Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii , might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii -host interaction and play an important protective role by creating a permeability barrier around the cyst.     

Analysis of Pneumocystis carinii cyst wall. II. Sugar composition

Pneumocystis carinii cysts are capable of resisting host defenses and antimicrobial drugs and are therefore thought to be responsible for relapses of P. carinii pneumonia in AIDS and other immunocompromised patients. The interaction of P. carinii with its host, and other P. carinii, might be mediated by molecules which form the outer surfaces of this organism. Carbohydrates are known to play many roles in cell-cell adhesion, and have been detected on the surface of P. carinii by lectin labeling experiments. In this study P. carinii cyst wall material was obtained from Zymolyase treatment. Alditol acetate derivatives of neutral and amino sugars or trimethylsilyl derivatives of methyl glycosides were prepared from the monosaccharides released from the sample by acid hydrolysis. Analyses were done by a combination of gas chromatography and mass spectrometry. Glucose was found to be the major sugar constituent. Mannose and galactose were present in equal ratios. A lesser amount of N-acetyl-D-glucosamine, and trace amounts of ribose and sialic acid were present in the cyst wall samples analyzed. These sugars may mediate P. carinii-host interaction and play an important protective role by creating a permeability barrier around the cyst.     

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls.

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.     

KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties

The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.     

A Method for Isolation of RNA from Pneumocystis carinii

Toial RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and weli behind the prokaryotic large rRNA species.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.     

Zymolyase removes callose from germinating pollen and pollen tube walls

The impermeable callosic wall that surrounds germinating pollen grains and pollen tubes is removed by brief treatment with Zymolyase, an enzyme preparation that hydrolyzes linear glucose polymers with beta-1,3 linkages. Zymolyase treatment does not interfere with pollen tube growth or cylosis in the vegetative cell.     

Cellulase Localization in Hyphae of Achlya ambisexualis

Cellulase (EC 3.2.1.4; beta-1, 4-glucan glucanohydrolase) was localized at the ultrastructural level and found to occur in dictyosomes and vesicles, around the periphery of unidentified storage bodies, between the plasmalemma and the cell wall, and on the outer surface of the cell wall in the male strain (E87) of Achlya ambisexualis after treatment with the sex hormone antheridiol.     

A method for isolation of RNA from Pneumocystis carinii

Total RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and well behind the prokaryotic large rRNA species.     

beta-1,6-Glucan synthesis in Saccharomyces cerevisiae

beta-1,6-Glucan is an essential fungal-specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to beta-1,3-glucan and chitin. Genetic approaches have identified genes that upon disruption result in beta-1,6-glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of beta-1,3-glucan and chitin synthesis, it is likely that the bulk of beta-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.     

Chemical analysis of the hyphal wall of Schizophyllum commune.

1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.