Beta-glucan Synthesis Research Articles

Optimizing the synthesis of yeast Beta-glucan via response surface methodology for nanotechnology application

BackgroundThe production of biopolymers from waste resources is a growing trend, especially in high-population countries like Egypt. Beta-glucan (β-glucan) belongs to natural polysaccharides that are derived from plant and microbial origins. In this study, following increasing demands for β-glucan owing to its bioactive properties, a statistical model to enhance microbial β-glucan production was evaluated for its usefulness to the food and pharmaceutical industries. In addition, a trial to convert β-glucan polymer to nanostructure form was done to increase its bioactivity.ResultsIngredients of low-cost media based on agro-industrial wastes were described using Plackett–Burman and central composite design of response surface methodology for optimizing yeast β-glucan. Minerals and vitamin concentrations significantly influenced β-glucan yield for Kluyveromyces lactis and nitrogen and phosphate sources for Meyerozyma guilliermondii. The maximum predicted yields of β-glucan recovered from K. lactis and M. guilliermondii after optimizing the medium ingredients were 407 and 1188 mg/100 ml; respectively. For the first time, yeast β-glucan nanoparticles (βGN) were synthesized from the β-glucan polymer using N-dimethylformamide as a stabilizer and characterized using UV–vis spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The average size of βGN was about 300 nm as determined by DLS. The quantitative variation of functional groups between β-glucan polymer and βGN was evaluated by FT-IR for explaining the difference in their biological activity against Normal Homo sapiens-Hela contaminant and Hepatic cancer cell lines.ConclusionsEnriching the low-cost media based on agro-industrial wastes with nutritional ingredients improves the yield of yeast β-glucan. The present study succeeds to form β-glucan nanoparticles by a simple method.

A Predicted Mannoprotein Participates in Cryptococcus gattii Capsular Structure.

The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C.gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C.gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C.gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C.gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C.gattii cell wall.

Using Aspergillus nidulans To Identify Antifungal Drug Resistance Mutations

Systemic fungal infections contribute to at least 10% of deaths in hospital settings. Most antifungal drugs target ergosterol (polyenes) or its biosynthetic pathway (azoles and allylamines), or beta-glucan synthesis (echinocandins). Antifungal drugs that target proteins are prone to the emergence of resistant strains. Identification of genes whose mutations lead to targeted resistance can provide new information on those pathways. We used Aspergillus nidulans as a model system to exploit its tractable sexual cycle and calcofluor white as a model antifungal agent to cross-reference our results with other studies. Within 2 weeks from inoculation on sublethal doses of calcofluor white, we isolated 24 A. nidulans adaptive strains from sectoring colonies. Meiotic analysis showed that these strains had single-gene mutations. In each case, the resistance was specific to calcofluor white, since there was no cross-resistance to caspofungin (echinocandin). Mutation sites were identified in two mutants by next-generation sequencing. These were confirmed by reengineering the mutation in a wild-type strain using a gene replacement strategy. One of these mutated genes was related to cell wall synthesis, and the other one was related to drug metabolism. Our strategy has wide application for many fungal species, for antifungal compounds used in agriculture as well as health care, and potentially during protracted drug therapy once drug resistance arises. We suggest that our strategy will be useful for keeping ahead in the drug resistance arms race.

Efficacy and Safety of Micafungin for Prophylaxis of Invasive Fungal Infections in Patients Undergoing Haplo-Identical Hematopoietic Stem Cell Transplant

AbstractAbstract 4505Invasive fungal infections (IFI) such as candidiasis and mold infections cause significant morbidity and mortality among immunocompromised patients in recent years. Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis, with potent activity against most species of Candida and Aspergillus.The aim of this observational study was to investigate the efficacy and safety of Micafungin in prophylaxis of invasive fungal infections (IFI) in 20 high risk adult patients with various haematological diseases receiving haplo-identical allogeneic stem cell transplantation (Allo-SCT). Treatment success was defined as no proven, probable, or suspected IFI through therapy and the absence of proven or probable IFI through the end of 12 weeks after therapy. (EORTC criteria).These patients were monitored after Allo-SCT for galactomannan antigenemia at least once per week, and a computed tomography (CT) scan was performed in the case of persistent fever for at least 5 days after broad-spectrum empirical antibacterial therapy. Routine controls (e.g. detection of galactomannan antigenemia and CT scan) were performed systematically after 1 and 2 months and on day 100 after Allo-SCT.Only 2 patients had a history of possible aspergillosis before transplant treated by voriconazole. The patients received a median of 4 lines (2–7) of treatments before Allo-SCT. Ten patients (50%) received at least one auto-SCT; and 5 patients (25%) received a previous Allo-SCT. 18 patients (90%) received a reduced intensity conditioning regimen (RIC), with Fludarabine, Cyclophosphamide and Total body irradiation (TBI) 2 Gy based (75%), or Fludarabine, Busulfan, and Cyclophosphamide based (10%) or other (5%). while two patients (10%) received a myeloablative conditioning regimen with thiotepa. The patients and transplant details are shown in the table 1.Patients received a median of 26 infusions (range, 1–8) of Micafungin (50 mg/day i.v. as a 1h infusion). The treatment was initiated at the beginning of the transplant conditioning regimen until the hospital discharge. None of our patients discontinued the treatment for drug-related adverse events. Micafungin was not associated with any hepatotoxicity. Only one patient (5%) discontinued the treatment because an early disease progression.At last follow-up, there are 17 patients (85%) who are alive, with a median follow-up of 6 months (3–17). Three patients (15%) dead because disease progression.In all patients no candidas and/or Aspergillus species was documented after 3 and 6 months from transplant. None of our patients presented a positive galactomannan antigenemia >0.5. Seven patients (35%) presented a CMV reactivation. Only one patient presented an acute GvHD grade II.Micafungin has a good safety and tolerability profile, with an efficacy in preventing IFI in this high risk population. Our data provide support for an efficacy study in a prophylaxis setting, but prospective and comparative clinical trials using Micafungin are urgently needed to define the role of this drug in prophylaxis after haplo-identical Allo-SCT.Table 1Patient and transplantation characteristicsPatient and transplantation characteristicsn = 20 (%)Patients Age (median) [range]40 years (range, 20–60)Patients sexMale12 (60)Female8 (40)Disease typeHL6 (30)NHL5 (25)AML4 (20)MM2 (10)MF1 (5)MDS1 (5)CLL1 (5)Median number of prior chemotherapies before Allo-SCT [range]4 (2–7)Median number of prior Auto-SCT [range]1 (0–2)18 (40)22 (10)Status of disease at Allo-SCT>2 CR11 (55)PR6 (30)PD3 (15)Median interval between auto and Allo-SCT months [range]Donor typeBrother8 (40)Mother6 (30)Sister3 (15)Son2 (10)Father1 (5)Donor/recipient sex mis match9 (45)Conditioning regimenFlu 5+Cy 2+ TBI15 (75)Flu 5 +Cy 2+ Bu 22 (10)Flu 3+TT 2+ Bu 32 (10)Flu 6+ Cy 1+ ATG 41 (5)GvHD prophylaxisCSA+MMF+Cy20 (100)Stem cell sourcePeripheral Blood11 (55)Bone Marrow9 (45)Stem cell dose median [range]CD34+ × 106/kg4,25 (0,8–10,8)CD3+ × 106/kg140 (17–411)Days with ANC< 500 × 109/l21 (14–49)Days with platelets<20 × 109/l26 (14–140) Disclosures:No relevant conflicts of interest to declare.

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

Galactofuranose (Gal f) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Gal f from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Gal f residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Gal f transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. Af glfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Gal f, which was restored in the Af glfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.

Interaction of Candida albicans Biofilms with Antifungals: Transcriptional Response and Binding of Antifungals to Beta-Glucans

Candida albicans can form biofilms that exhibit elevated intrinsic resistance to various antifungal agents, in particular azoles and polyenes. The molecular mechanisms involved in the antifungal resistance of biofilms remain poorly understood. We have used transcript profiling to explore the early transcriptional responses of mature C. albicans biofilms exposed to various antifungal agents. Mature C. albicans biofilms grown under continuous flow were exposed for as long as 2 h to concentrations of fluconazole (FLU), amphotericin B (AMB), and caspofungin (CAS) that, while lethal for planktonic cells, were not lethal for biofilms. Interestingly, FLU-exposed biofilms showed no significant changes in gene expression over the course of the experiment. In AMB-exposed biofilms, 2.7% of the genes showed altered expression, while in CAS-exposed biofilms, 13.0% of the genes had their expression modified. In particular, exposure to CAS resulted in the upregulation of hypha-specific genes known to play a role in biofilm formation, such as ALS3 and HWP1. There was little overlap between AMB- or CAS-responsive genes in biofilms and those that have been identified as AMB, FLU, or CAS responsive in C. albicans planktonic cultures. These results suggested that the resistance of C. albicans biofilms to azoles or polyenes was due not to the activation of specific mechanisms in response to exposure to these antifungals but rather to the intrinsic properties of the mature biofilms. In this regard, our study led us to observe that AMB physically bound C. albicans biofilms and beta-glucans, which have been proposed to be major constituents of the biofilm extracellular matrix and to prevent azoles from reaching biofilm cells. Thus, enhanced extracellular matrix or beta-glucan synthesis during biofilm growth might prevent antifungals, such as azoles and polyenes, from reaching biofilm cells, thus limiting their toxicity to these cells and the associated transcriptional responses.

Synthesis of Cyclic β-Glucan Using Laminarinase 16A Glycosynthase Mutant from the Basidiomycete Phanerochaete chrysosporium

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-beta-D-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with alpha-laminariheptaosyl fluoride (alphaL7F) revealed how alphaL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with alphaL7F the major product was a cyclic beta-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely beta-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic beta-glucan.

Analysis of Candida albicans plasma membrane proteome

The opportunistic human fungal pathogen Candida albicans causes a wide variety of infections including deep systemic syndromes. The C. albicans plasma membrane is an important interface in the host-pathogen relationship. The plasma membrane proteins mediate a variety of functions, including sensing and signalling to the external environment, in which the glycosylphosphatidylinositol (GPI)-anchored membrane proteins play a crucial role. A subproteomic approach to obtain a global picture of the protein composition of the C. albicans plasma membrane was developed, and different strategies were tested in order to extract the largest number of yeast plasma membrane proteins and GPI-anchored membrane proteins. These methods involved: (i) protoplast generation, (ii) mechanical disruption, (iii) ultracentrifugation in sucrose gradients, and (iv) Na(2)CO(3) treatments. To isolate GPI-anchored proteins two additional steps were performed: two-phase separation and phosphatidylinositol-phospholipase C treatment. After LC-MS/MS analysis using both a MALDI-TOF/TOF and a linear ion trap quadrupole, a total of 214 membrane proteins were identified, including 41 already described as plasma membrane proteins, 20 plasma membrane associated proteins, and 22 proteins with unknown membrane localisation. Bioinformatic analysis revealed that this set of C. albicans membrane proteins is highly enriched in proteins involved in biopolymer biosynthesis or transport processes. Furthermore, after phosphatidylinositol-phospholipase C treatment, 12 GPI-anchored membrane proteins were released and identified; most of them are associated with cell wall beta-glucan synthesis and maintenance or are virulence factors, such as phospholipases or aspartyl proteinases.

Time course global gene expression analysis of an in vivo Candida biofilm.

Candida infection of devices is common and invariably associated with biofilm growth. Exploratory microarray studies were undertaken to identify target genes associated with biofilm formation from an in vivo catheter model over time. We compared messenger RNA levels from Candida albicans grown in an in vivo central venous catheter biofilm model at 12 h (intermediate growth) and 24 h (mature) to in vitro planktonic cells without a biofilm substrate, using C. albicans oligo arrays. A total of 124 transcripts were similarly up-regulated at the 12- and 24-h time points. Ontology categories most highly represented included energy/metabolism (12%), carbohydrate (10%), and protein (13%) synthesis and modification, and transport (6%). Numerous genes were previously identified from in vitro biofilm studies. These genes included those associated with hyphal growth, amino acid metabolism, adherence, drug resistance, ergosterol biosynthesis, and beta-glucan synthesis. In the current data set, adherence genes were unique to those from the earlier time point. Differences between the current in vivo biofilm expression data and that previously reported from in vitro models, including alterations in metabolism and carbohydrate processing, may be due to the continuous availability of nutrients from host serum and the incorporation of the host-pathogen interaction.

A barley cellulose synthase-like CSLH gene mediates (1,3;1,4)-beta-D-glucan synthesis in transgenic Arabidopsis.

The walls of grasses and related members of the Poales are characterized by the presence of the polysaccharide (1,3, 1,4)-beta-D-glucan (beta-glucan). To date, only members of the grass-specific cellulose synthase-like F (CSLF) gene family have been implicated in its synthesis. Assuming that other grass-specific CSL genes also might encode synthases for this polysaccharide, we cloned HvCSLH1, a CSLH gene from barley (Hordeum vulgare L.), and expressed an epitope-tagged version of the cDNA in Arabidopsis, a species with no CSLH genes and no beta-glucan in its walls. Transgenic Arabidopsis lines that had detectable amounts of the epitope-tagged HvCSLH1 protein accumulated beta-glucan in their walls. The presence of beta-glucan was confirmed by immunoelectron microscopy (immuno-EM) of sectioned tissues and chemical analysis of wall extracts. In the chemical analysis, characteristic tri- and tetra-saccharides were identified by high-performance anion-exchange chromatography and MALDI-TOF MS following their release from transgenic Arabidopsis walls by a specific beta-glucan hydrolase. Immuno-EM also was used to show that the epitope-tagged HvCSLH1 protein was in the endoplasmic reticulum and Golgi-associated vesicles, but not in the plasma membrane. In barley, HvCSLH1 was expressed at very low levels in leaf, floral tissues, and the developing grain. In leaf, expression was highest in xylem and interfascicular fiber cells that have walls with secondary thickenings containing beta-glucan. Thus both the CSLH and CSLF families contribute to beta-glucan synthesis in grasses and probably do so independently of each other, because there is no significant transcriptional correlation between these genes in the barley tissues surveyed.

Antifongiques : cibles cellulaires et mécanismes de résistance

Antifungals of systemic use for the treatment of invasive fungal infections belong to four main chemical families which have globally three cellular targets in fungal cells: fluorinated pyrimidines act on deoxyribonucleic acid (DNA) replication and protein synthesis; polyenes and azoles are toxic for ergosterol and its biosynthetic pathway; lipopeptides inhibit the synthesis of cell wall beta glucans. The resistance mechanisms that are developed by some fungi begin to be well understood particularly in Candida yeasts. The underlying bases of these mechanisms are either mutations that modify the antifungal target, or that block access to the target, and, on the other hand, the overexpression of genes encoding the target, or some membrane proteins involved in the active efflux of antifungal drugs.

Combined action of micafungin, a new echinocandin, and human phagocytes for antifungal activity against Aspergillus fumigatus

Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis. We postulated micafungin and host phagocytic cells could act together in damaging fungi. Using the metabolic XTT assay, micafungin alone (0.01 and 0.10 μg/ml) inhibited Aspergillus fumigatus germlings by 48% and 61%, respectively. Polymorphonuclear neutrophils (PMNs) inhibited germlings by 53%. Micafungin at 0.01 or 0.10 μg/ml and PMNs resulted in additive inhibition, 82% and 99%, respectively. Monocyte-derived macrophage (MDM) monolayers inhibited germling growth by 66%; micafungin (0.01 or 0.10 μg/ml) alone inhibited by 32% and 42%, respectively. MDMs and micafungin (0.01 or 0.10 μg/ml) caused an additive inhibition of growth, 85% and 95%, respectively. Hyphae were generated by incubation of conidia for 24 h with or without micafungin. PMNs alone, added to hyphae, inhibited growth by 19% in the subsequent 20 h. Hyphae generated in the presence of micafungin (0.10 μg/ml) and subsequently cultured with micafungin for 24 h inhibited growth by 64%. PMNs plus micafungin resulted in 82% inhibition. Monocytes alone inhibited hyphal growth by only 5%. Hyphae produced in the presence of micafungin (0.01 μg/ml) and incubated again with micafungin for 24 h inhibited growth by 47%; combination with monocytes resulted in 62% inhibition. These data indicate that micafungin inhibits growth of tissue forms of A. fumigatus, and phagocytes and micafungin together have an additive effect. These findings support the thesis that the greater efficacy of micafungin in vivo compared with in vitro could be due to combined effect of phagocytic cells and micafungin.

Pneumocystis jiroveci Pneumonia in Adult Patients with AIDS

Pneumocystis jiroveci (P. carinii) is an opportunistic pathogen that has gained particular prominence since the onset of the AIDS epidemic. Among several important advances in diagnosis and management, appropriately targeting chemoprophylaxis to HIV-infected patients at high clinical risk for P. jiroveci pneumonia and the introduction of effective combination anti-retroviral therapy (including highly active antiretroviral therapy [HAART]) have contributed to the reduced incidence of P. jiroveci pneumonia. Despite the success of these clinical interventions, P. jiroveci pneumonia remains the most common opportunistic pneumonia and the most common life-threatening infectious complication in HIV-infected patients. Trimethoprim/sulfamethoxazole (cotrimoxazole) remains the first-line agent for effective therapy and chemoprophylaxis, and corticosteroids represent an important adjunctive agent in the treatment of moderate-to-severe P. jiroveci pneumonia. However, problems of chemoprophylaxis and treatment failures, high rates of adverse drug reactions and drug intolerance to first-line antimicrobials, high rates of relapse or recurrence with second-line agents, and newer concerns about the development of P. jiroveci drug resistance represent formidable challenges to the management and treatment of AIDS-related P. jiroveci pneumonia. With the expanding global problem of HIV infection, the intolerance or unavailability of HAART to many individuals and limited access to healthcare for HIV-infected patients, P. jiroveci pneumonia will remain a major worldwide problem in the HIV-infected population. New drugs under development as anti-Pneumocystis agents such as echinocandins and pneumocandins, which inhibit beta-glucan synthesis, or sordarins, which inhibit fungal protein synthesis, show promise as effective agents. Continued basic research into the biology and genetics of P. jiroveci and host defense response to P. jiroveci will allow the development of newer antimicrobials and immunomodulatory therapeutic agents to more effectively treat life-threatening pneumonia caused by this organism.

Characterisation of Mesorhizobium huakuii cyclic beta-glucan.

Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy. It was shown that M. huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents. The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28. The most abundant glucan molecules contained 22 glucose residues. Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages. The cyclic glucan produced by M. huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera. The synthesis of beta-glucan in M. huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.

Characterisation of Mesorhizobium huakuii cyclic beta-glucan.

Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy. It was shown that M. huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents. The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28. The most abundant glucan molecules contained 22 glucose residues. Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages. The cyclic glucan produced by M. huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera. The synthesis of beta-glucan in M. huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.

Potential drug targets in cyst-wall biosynthesis by intestinal protozoa.

Given that resistance to antiprotozoal drugs exists and is likely to increase and given that currently no reliable treatments exist for some of these infections, efforts to find new targets for chemotherapy must be continued. In the case of cyst-forming pathogenic protozoa, one such target might be encystment pathways and cyst-wall assembly. Information is increasing on protozoan encystment and, as it does, we can begin to answer the question of whether targeting it for chemotherapy is a viable drug strategy. Currently, there are significant efforts to understand encystment in Giardia and Entamoeba, and potential targets are being discovered as work on their encystment mechanisms progress. We know with certainty now that Giardia and Entamoeba cyst walls contain unique proteins and polysaccharides which differ from those of their hosts and thus make them potentially interesting targets for a variety of chemotherapeutic attacks. Although we lack detailed information about the other protozoan cyst formers, enough evidence exists for Giardia and Entamoeba that it seems prudent to screen them with some of the antifungal drugs, especially those that target mannoproteins, chitin synthesis, and beta (1, 3) glucan synthesis to ascertain if they target elements in these protozoan pathways that are similar to those found in fungi.

Characterization of ndvD, the third gene involved in the synthesis of cyclic beta-(1 --> 3),(1 --> 6)-D-glucans in Bradyrhizobium japonicum.

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic beta-(1 --> 3),(1 --> 6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for beta-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo beta-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral beta-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for beta-glucan synthesis. Our hypothesis for the mechanism of beta-(1 --> 3),(1 --> 6)-D-glucan synthesis is presented.

Involvement of β-Glucans in the Wide-Spectrum Antimicrobial Activity of Williopsis saturnus var. mrakii MUCL 41968 Killer Toxin

Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.

Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall.

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.

Bradyrhizobium japonicum mutants defective in cyclic beta-glucan synthesis show enhanced sensitivity to plant defense responses.

Susceptibility of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum to inducible plant defense metabolites such as phytoalexin and H2O2, was investigated. On the wild-type strain USDA 110 the soybean phytoalexin, glyceollin, showed bacteriostatic activity. Viable bacteria isolated from intact nodules were adapted to glyceollin. H2O2 in physiological concentrations did not affect wild-type bacteria. B. japonicum mutants defective in the biosynthesis of cyclic beta-(1-->3)-(1-->6)-glucans showed higher susceptibility to both phytoalexin and H2O2.