Presence Of N-acetylglucosamine Research Articles

The NagY regulator: A member of the BglG/SacY antiterminator family conserved in Enterococcus faecalis and involved in virulence.

Enterococcus faecalis is a commensal bacterium of the gastrointestinal tract but also a major nosocomial pathogen. This bacterium uses regulators like BglG/SacY family of transcriptional antiterminators to adapt its metabolism during host colonization. In this report, we investigated the role of the BglG/SacY family antiterminator NagY in the regulation of the nagY-nagE operon in presence of N-acetylglucosamine, with nagE encoding a transporter of this carbohydrate, as well as the expression of the virulence factor HylA. We showed that this last protein is involved in biofilm formation and glycosaminoglycans degradation that are important features in bacterial infection, confirmed in the Galleria mellonella model. In order to elucidate the evolution of these actors, we performed phylogenomic analyses on E. faecalis and Enterococcaceae genomes, identified orthologous sequences of NagY, NagE, and HylA, and we report their taxonomic distribution. The study of the conservation of the upstream region of nagY and hylA genes showed that the molecular mechanism of NagY regulation involves ribonucleic antiterminator sequence overlapping a rho-independent terminator, suggesting a regulation conforming to the canonical model of BglG/SacY family antiterminators. In the perspective of opportunism understanding, we offer new insights into the mechanism of host sensing thanks to the NagY antiterminator and its targets expression.

The Involvement of Cell-Type-Specific Glycans in Hydra Temporary Adhesion Revealed by a Lectin Screen

Hydra is a freshwater solitary polyp, capable of temporary adhesion to underwater surfaces. The reversible attachment is based on an adhesive material that is secreted from its basal disc cells and left behind on the substrate as a footprint. Despite Hydra constituting a standard model system in stem cell biology and tissue regeneration, few studies have addressed its bioadhesion. This project aimed to characterize the glycan composition of the Hydra adhesive, using a set of 23 commercially available lectins to label Hydra cells and footprints. The results indicated the presence of N-acetylglucosamine, N-acetylgalactosamine, fucose, and mannose in the adhesive material. The labeling revealed a meshwork-like substructure in the footprints, implying that the adhesive is mainly formed by fibers. Furthermore, lectins might serve as a marker for Hydra cells and structures, e.g., many labeled as glycan-rich nematocytes. Additionally, some unexpected patterns were uncovered, such as structures associated with radial muscle fibers and endodermal gland cells in the hypostome of developing buds.

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans.

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein.

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

The general transcriptional repressor Tup1 governs filamentous development in Candida tropicalis.

Filamentous development is associated with the ability to cause infections and colonize the host in pathogenic Candida species. Candida tropicalis is one of the major fungal pathogens of humans. The conserved transcriptional repressor Tup1 plays a critical role in the regulation of transcription and filamentation in yeast species. Despite its central role, the full coding sequence of TUP1 has not been found in the reported genome sequence of C. tropicalis to date. In this study, we report the identification of Tup1 and characterize its role in filamentous growth in C. tropicalis. As expected, C. tropicalis Tup1 exhibits general conserved features to the orthologs of other fungi in terms of its structure and function. Deletion of TUP1 in C. tropicalis leads to increased filamentation under several culture conditions. However, Tup1 indeed exhibits species-specific roles in the regulation of filamentous development in C. tropicalis. For example, unlike the tup1/tup1 mutant of Candida albicans, the tup1/tup1 mutant of C. tropicalis is able to exist in the yeast form at low temperatures or in the presence of N-acetylglucosamine (GlcNAc). Acidic pH conditions also favor the yeast form of the tup1/tup1 mutant of C. tropicalis. Quantitative real-time PCR (qRT-PCR) assays indicate that Tup1 may regulate filamentous development through the transcriptional control of key filamentation regulators in C. tropicalis, such as Ume6, Brg1, Wor1, Sfl2, Ahr1, and Zcf3. Taken together, our findings demonstrate both conserved and species-specific roles of Tup1 in the regulation of filamentation and provide novel insights into the biology of C. tropicalis.

N-acetylglucosamine-Mediated Expression of nagA and nagB in Streptococcus pneumoniae.

In this study, we have explored the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylglucosamine (NAG). Transcriptome comparison of S. pneumoniae D39 wild-type grown in chemically defined medium (CDM) in the presence of 0.5% NAG to that grown in the presence of 0.5% glucose revealed elevated expression of many genes/operons, including nagA, nagB, manLMN, and nanP. We have further confirmed the NAG-dependent expression of nagA, nagB, manLMN, and nanP by β-galactosidase assays. nagA, nagB and glmS are putatively regulated by a transcriptional regulator NagR. We predicted the operator site of NagR (dre site) in PnagA, PnagB, and PglmS, which was further confirmed by mutating the predicted dre site in the respective promoters (nagA, nagB, and glmS). Growth comparison of ΔnagA, ΔnagB, and ΔglmS with the D39 wild-type demonstrates that nagA and nagB are essential for S. pneumoniae D39 to grow in the presence of NAG as a sole carbon source. Furthermore, deletion of ccpA shows that CcpA has no effect on the expression of nagA, nagB, and glmS in the presence of NAG in S. pneumoniae.

Effect of light emutting diode (LED) lamps and N-acetylglucosamine (NAG) on organogenesis in protocorm-like bodies (PLBs) of a Cymbidium hybrid cultured in vitro

Abstract not available
 Plant Tissue Cult. & Biotech. 24(2): 273-277, 2014 (December)

Biosynthesis of hexa- and pentameric chitooligosaccharides using N-acetyl-glucoseaminyl transferase from rhizobial bacteria

Chitooligosaccharides are widely used, which provides considerable interest in their biosynthesis. Enzymatic synthesis of hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose using N-acetylglucoseaminyl transferase possessing the unique properties from rhizobial bacteria Rhizobium sp. GRH2 and M. loti has been performed in E. coli cells. Culturing of E. coli expressing the appropriate recombinant enzymes in the presence of N-acetylglucosamine as a substrate resulted in the synthesis of significant amounts of the desired chitooligosaccharides via high performance liquid chromatography and mass-spectrometry confirmed their conformity to standards.

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.

Sugar Binding Effects on the Enzymatic Reaction and Conformation Near the Active Site of Pokeweed Antiviral Protein Revealed by Fluorescence Spectroscopy

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N-acetyl glucosamine residues of lipopolysaccharides.

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.

Community shifts of actively growing lake bacteria after N-acetyl-glucosamine addition: improving the BrdU-FACS method.

In aquatic environments, community dynamics of bacteria, especially actively growing bacteria (AGB), are tightly linked with dissolved organic matter (DOM) quantity and quality. We analyzed the community dynamics of DNA-synthesizing and accordingly AGB by linking an improved bromodeoxyuridine immunocytochemistry approach with fluorescence-activated cell sorting (BrdU-FACS). FACS-sorted cells of even oligotrophic ecosystems in winter were characterized by 16S rRNA gene analysis. In incubation experiments, we examined community shifts of AGB in response to the addition of N-acetyl-glucosamine (NAG), one of the most abundant aminosugars in aquatic systems. Our improved BrdU-FACS analysis revealed that AGB winter communities of oligotrophic Lake Stechlin (northeastern Germany) substantially differ from those of total bacteria and consist of Alpha-, Beta-, Gamma-, Deltaproteobacteria, Actinobacteria, Candidatus OP10 and Chloroflexi. AGB populations with different BrdU-fluorescence intensities and cell sizes represented different phylotypes suggesting that single-cell growth potential varies at the taxon level. NAG incubation experiments demonstrated that a variety of widespread taxa related to Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Planctomycetes, Spirochaetes, Verrucomicrobia and Chloroflexi actively grow in the presence of NAG. The BrdU-FACS approach enables detailed phylogenetic studies of AGB and, thus, to identify those phylotypes which are potential key players in aquatic DOM cycling.

Study of trans–trans farnesol effect on hyphae formation by Yarrowia lipolytica

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.

Mechanistic Aspects and Novel Biomarkers of Responder and Non-Responder Phenotypes in Galactosamine-Induced Hepatitis

The amino sugar galactosamine (galN) induces alterations in the hepatic uridine nucleotide pool and has been widely used as a model of human viral hepatitis. Histopathological and clinical chemistry analyses of a cohort of rats following administration of galN revealed extreme interindividual variability in the extent of the toxic response which enabled classification of 'responder' and 'non-responder' phenotypes. An integrative metabolic profiling approach was applied to characterize biomarkers of exposure to galN in urine, serum, feces and liver from responders and non-responders. The presence of N-acetylglucosamine and galN in the urine correlated with the occurrence and extent of toxic response. Conversely, the novel identification of galN-pyrazines in the feces of non-responders and their virtual absence in the feces of responders suggests an alternative means of distribution and metabolism of galN in non-responders. The absence of the UDP-hexosamines in the liver of non-responders further supports differential metabolism of galN and suggests an ability of non-responders to avoid UDP-glucose depletion. An observed disturbance of gut microbial derived metabolites in the urine and feces of non-responders may suggest a role of the microflora in reducing the effective dose of galN. This systems level metabonomic approach has provided new mechanistic insights into differential response to galN and is widely applicable to the study of interindividual variation in metabolism for any xenobiotic intervention.

Changes in peritoneal mesothelial cells phenotype after chronic exposure to glucose or N-acetylglucosamine

Glucose is commonly used as an osmotic solute in peritoneal dialysis fluids despite vast knowledge about deleterious peritoneal and systemic effects of that solute. N-acetylglucosamine (NAG) is a solute of the comparable size to glucose, with strong anti-inflammatory properties. We compared the chronic in vitro effect of both solutes on phenotype of peritoneal mesothelial cells. Experiments were performed of primary cultures of human peritoneal mesothelial cells, which were cultured over 4 weeks in medium supplemented either with glucose 45 mmol/L (GLU) or with NAG 45 mmol/L (NAG). Generation of reactive oxygen species (ROS) in cells was studied, as well as their ability to proliferate, synthesis of cytokines, fibronectin, and factors regulating peritoneal fibrinolysis. Cells cultured in the presence of glucose 45 mmol/L generated more ROS (+73% vs control, P < 0.01), whereas NAG did not stimulate generation of ROS. GLU caused hypertrophy of mesothelial cells (+53% vs control, P < 0.001) and prolonged their population doubling time (+16% vs control, P < 0.01); NAG did not cause significant changes in these parameters. Healing of mesothelial monolayer after mechanical injury was impaired in GLU treated cells: (-48% vs control, P < 0.001 and -40% vs NAG, P < 0.05). Synthesis of Il-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and fibronectin was higher in GLU group as compared with control: + 86%, P < 0.001, +38%, P < 0.05, +51%, P < 0.001, +38%, P < 0.05, respectively. In the presence of NAG, these parameters were comparable with the control group, but at the same time NAG stimulated synthesis of hyaluronan: +116% versus control, P < 0.001 and + 96% versus GLU, P < 0.01. Treatment with GLU resulted in decline of tissue plasminogen activator/plasminogen activator inhibitor-1 (t-PA/PAI-1) ratio by 23% versus control, P < 0.001, whereas NAG increased that parameter by 43%, P < 0.01 versus control. Glucose, contrary to NAG, induces oxidative stress and proinflammatory and profibrotic changes in mesothelial cells. NAG seems to be more biocompatible osmotic solute than glucose.

Characterization of Esophageal Submucosal Glands in Pig Tissue and Cultures

The submucosal glands (SMGs) of the pig esophagus, like the human, secrete mucin and bicarbonate, which help in luminal acid clearance and epithelial protection. The aim of this study was to characterize histochemically the esophageal SMGs and a primary culture obtained from these glands. Tissues and cultures were stained with hematoxylin and eosin, periodic acid Schiff, Alcian blue, lectins, or cytokeratins. In the perfused esophagus, addition of carbachol increased mucin secretion by approximately 2-fold. The results indicate that [1] a method for culturing SMG cells was developed; [2] conventional staining indicates the presence of sulfated, acidic, and neutral mucopolysaccharides in glands and cultures; [3] lectin binding indicates the presence of N-acetyl glucosamine, N-acetyl neuraminic acid, N-acetyl galactosamine, and alpha-L: -fucose in mucous cells and cultures; [4] cytokeratin and lectin staining indicated similarities with Barrett epithelium (columnar metaplasia of the esophagus); and [5] cholinergic agonists enhance mucin secretion and this could play a significant role in esophageal protection.

N-Acetylglucosamine Reduces Inflammatory Response during Acute Peritonitis in Uremic Rats

Background: Peritoneal dialysis (PD) induces intraperitoneal inflammation and that process may be uremia. The goal of this study is to evaluate the effect of ure- mia on the kinetics of peritonitis and furthermore test the anti-inflammatory potential of N-acetylglucosamine (NAG) in a uremic environment. Methods: Experiments were performed on healthy Wistar rats and on animals with impaired renal function. Acute PD was performed in all animals with dialysis fluid containing either glucose (GLU) or NAG as osmotic solutes. Peritonitis was induced by addition of lipopolysaccharide from Escherichia coli (LPS) to the dialysis fluid. Transperitoneal transport of water and solutes as well as intraperitoneal and systemic inflammation were evaluated. Results: Uremia reduces peritoneal permeability to total protein during peritonitis (–33% vs. control, p < 0.001) and increases net ultrafiltration (+2.5 ± 2.2 vs. –2.7 ± 3.2 ml in control, p < 0.001). In uremic rats with peritonitis, reduced dialysate levels of the following inflammatory mediators were detected as compared to healthy animals: MCP-1 (–15%, p < 0.01); IL-1β (–53%, p < 0.001), and elastase (–28%, p < 0.02). In the serum of uremic rats, the increase in TNFα and MCP-1 concentrations was smaller than in control rats: –44% (p < 0.02) and –39% (p < 0.001), respectively. NAG used as an osmotic solute in rats with preserved renal function decreases intraperitoneal and systemic inflammation during acute peritonitis. Drained dialysate volume was increased in the NAG group by 32% (p < 0.001) and transperitoneal loss of protein was reduced by 21% (p < 0.002). When NAG was used as the osmotic solute instead of GLU, intraperitoneal inflammation in uremic animals was further reduced: TNFα (–40%, p < 0.05); IL-1β (–49%, p < 0.005); MCP-1 (–21%, p < 0.005). The presence of NAG also reduced the increased blood level of IL-1β (–47%,p < 0.02) and MCP-1 (–36%, p < 0.02). Conclusions: Intensity of acute peritonitis is reduced during uremia. NAG exerts a systemic and peritoneal anti-inflammatory action under conditions of uremia that confirms the potential use of this compound as an osmotic agent in the PD fluids that also decreases inflammation.

KRE5Gene Null Mutant Strains ofCandida albicansAre 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.

The gate controlling cell wall synthesis in Staphylococcus aureus.

Glucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-N-acetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from N-acetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N-acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N-acetylglucosamine. This and the preferential incorporation of N-acetylglucosamine over glucose into cell wall material showed that N-acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.

Cloning and characterization of PRB1, a Candida albicans gene encoding a putative novel endoprotease B and factors affecting its expression

Several cDNA fragments corresponding to transcripts differentially expressed under conditions that favor mycelial growth of Candida albicans were identified by the “differential display” technique. One of these was cloned and used as a probe to rescue the full gene from a genomic library of the fungus. The sequence identified a single, uninterrupted open reading frame of 1395 nucleotides encoding a putative protein of 465 residues and a theoretical molecular weight of 50.3 kDa, present in the genome as a single copy located at chromosome 2 in different strains. The gene product showed high homology with subtilisin-like proteases, mainly PRB1, the vacuolar B protease from Saccharomyces cerevisiae, and for this reason it was designated as a putative CaPRB1. Expression of the gene was not directly related to fungal morphogenesis, but to the initial response to inducers: Heat shock and the presence of N-acetyl glucosamine. It was also subject to nitrogen, but not to carbon catabolite repression, although glucose inhibited the GlcNAc stimulatory effect. The gene was, in our hands, unable to complement PRB1 mutation in S. cerevisiae. C. albicans prb null mutants did not show any distinct alteration in the phenotype. CaPRB1 is the first gene coding for a putative vacuolar serine protease cloned from C. albicans.