The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for ␤(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized ␤(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H ؉ -ATPase, and a 160-kDa protein which was labeled by an anti-␤(1-3) glucan antibody and was homologous to ABC bacterial ␤(1-2) glucan transporters.The fungal cell wall, which is specific and essential to fungal life, is mainly constituted of polysaccharides. Among all polysaccharides identified to date in the cell wall, ␤(1-3) glucans are the most prevalent, and they are present in all yeast and filamentous fungi investigated to date (14). Although ␤(1-3) glucan biosynthesis has been the subject of intensive research efforts for the last 30 years, the ␤(1-3) glucan biosynthetic pathway is not fully understood. It has been known since the early studies of Cabib and coworkers (22,31,35,36) that ␤(1-3) glucans are synthesized from UDP glucose by a membrane protein complex, ␤(1-3) glucan synthase (EC 2.4.1.34; UDP-glucose/liter 1,3-␤-D-glucan-3-␤-D-glucosyltransferase). Synthesis occurs on the cytoplasmic side of the plasma membrane, and ␤(1-3) glucan chains are extruded towards the periplasmic space (15,35). The glucan synthase complex has been characterized at the molecular level almost exclusively in the yeast Saccharomyces cerevisiae (5,7,12,19,29) and has been shown to be composed of two proteins: (i) the putative catalytic subunit Fksp, a large-molecular-size (Ͼ200 kDa) polypeptide with 16 transmembrane domains (12,29,30), and (ii) the regulatory subunit Rho1p, a small-molecular-size GTPase, which stimulates ␤(1-3) glucan synthase activity in its prenylated form (1,11,17,18,24,28,33).If the ␤(1-3) glucan synthase has been extensively analyzed in yeast, then this enzymatic complex has been poorly studied in filamentous fungi. Only one FKS gene had been cloned and sequenced to date in Aspergillus nidulans (23), and neither has a regulatory partner been identified nor has the cellular localization of the glucan synthase complex been investigated.This study was centered on the char...

The peptidoglycan structure of in vitro selected ampicillin-resistant mutant Enterococcus faecium D344M512 and of the susceptible parental strain D344S was determined by reverse phase high performance liquid chromatography and mass spectrometry. The muropeptide monomers were almost identical in the two strains The peptidoglycan of Escherichia coli is generated by polymerization of a precursor composed of N-acetylglucosamine (GlcNAc) 1 and N-acetylmuramic. The final steps of peptidoglycan synthesis involve polymerization of the glycan strands by glycosyltransferases and cross-linking of the peptide stems by DDtranspeptidases. The latter enzymes catalyze formation of a peptide bond between the ␣-carboxyl of D-Ala at the fourth position of a donor stem and the ⑀ amino group of meso-A2 pm at the third position of an acceptor stem generating a D-Ala 4 3 meso-A2 pm 3 cross-link (2).The first step of the transpeptidation reaction leads to the release of the C-terminal D-Ala 5 of the donor peptide stem and to the formation of a covalent adduct between the penultimate residue (D-Ala 4 ) and a conserved catalytic serine residue of the DD-transpeptidases (2, 3). Antibiotics of the ␤-lactam class, such as penicillin and ampicillin, are structural analogs of the C-terminal D-Ala 4 -D-Ala 5 end of peptidoglycan precursors and act as suicide substrates in a similar acylation reaction (4). The second step of the transpeptidation reaction results in a crosslinking and release of the DD-transpeptidases. In contrast, acylation of the DD-transpeptidases by ␤-lactams is nearly irreversible. The DD-transpeptidases are the killing target of the ␤-lactams, because transpeptidation is essential to maintain the integrity of the cell wall (2).The D-Ala 3 meso-A2 pm 3 cross-links generated by the DDtranspeptidases are prevalent in the peptidoglycan of E. coli although minor meso-A2 pm 3 3 meso-A2 pm 3 cross-links have been detected in the exponential (ϳ2%) and stationary (ϳ4%) phases of growth (5-7). The enzymes generating the minor meso-A2 pm 3 3 meso-A2 pm 3 cross-links have not been identified. By analogy with DD-transpeptidases, these putative LDtranspeptidases are thought to cleave the C-terminal D-Ala 4 of a donor tetrapeptide stem peptide before linking the ␣-carboxyl of meso-A2 pm 3 to the ⑀-amino group of meso-A2 pm 3 of an acceptor stem peptide (8, 9). The ␤-lactam ring does not contain any LD-peptide bond indicating that LD-transpeptidases do not belong to the family of penicillin-binding proteins (4, 10). In agreement with this notion, LD-carboxypeptidases, which cleave the C-terminal D-Ala 4 residue of tetrapeptide stems, are not acylated by ␤-lactams (11).The overall structure and mode of synthesis of peptidoglycan is conserved in eubacteria, although variations have been detected, in particular in the sequence of the peptide stem. In the Gram-positive bacteria Enterococcus faecium and Lactobacillus casei, D-iGlu at the second position is amidated, meso-A2 pm at the third position is replaced by L-Lys, and the ⑀-amino group...

The synthesis of bacterial cell wall peptidoglycan is a two-stage process. First, the disaccharide peptide monomer unit is assembled in a series of cytoplasmic and membrane reactions (1). In Enterococcus faecium, the resulting unit is composed of N-acetylglucosamine (GlcNAc) 1 and N-acetylmuramic acid (Fig. 1A). In clinical isolates, acquired high-level resistance to these antibiotics is generally associated with increased production of PBP5 or with amino acid substitutions near the conserved motifs of this protein (9 -13). Recently, we searched for other resistance mechanisms and obtained after five selection steps a highly ampicillin-resistant mutant, designated D344M512, or briefly M512, from the hypersusceptible E. faecium D344S that does not harbor the pbp5 gene. Analysis of the peptidoglycan structure by reverse-phase HPLC (RP-HPLC) coupled to mass spectrometry revealed substitution of D-Ala 4 3

Previous studies in Aspergillus fumigatus (Mouyna I., Fontaine T., Vai M., Monod M., Fonzi W. A., Diaquin M., Popolo L., Hartland R. P., Latgé J.-P, J. Biol. Chem. 2000, 275, 14882-14889) have shown that a glucanosyltransferase playing an important role in fungal cell wall biosynthesis is glycosylphosphatidylinositol (GPI) anchored to the membrane. To identify other GPI-anchored proteins putatively involved in cell wall biogenesis, a proteomic analysis has been undertaken in A. fumigatus and the protein data were matched with the yeast genomic data. GPI-anchored proteins of A. fumigatus were released from membrane preparation by an endogenous GPI-phospholipase C, purified by liquid chromatography and separated by two-dimensional electrophoresis. They were characterized by their peptide mass fingerprint through matrix-assisted laser desorption/ionization-time of flight-(MALDI-TOF)-mass spectrometry and by internal amino acid sequencing. Nine GPI-anchored proteins were identified in A. fumigatus. Five of them were homologs of putatively GPI-anchored yeast proteins (Csa1p, Crh1p, Crh2p, Ecm33p, Gas1p) of unknown function but shown by gene disruption analysis to play a role in cell wall morphogenesis. In addition, a comparative study performed with chitin synthase and glucanosyl transferase mutants of A. fumigatus showed that a modification of the growth phenotype seen in these mutants was associated to an alteration of the pattern of GPI-anchored proteins. These results suggest that GPI-anchored proteins identified in this study are involved in A. fumigatus cell wall organization.

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