Glabrata Cell Research Articles

Biochemical and metabolomic insights into antifungal mechanism of berberine against Candida glabrata.

An unprecedented expansion of antifungal therapy failure incidences in healthcare settings of Candida glabrata is the matter of global concern that needs to be addressed efficiently and effectively. In this pursuit, the present study has investigated the antifungal mechanism of benzylisoquinoline alkaloid berberine using biochemical, metabolic, and gene expression analysis, with the aim to delineate its therapeutic activity against C. glabrata and differentially fluconazole-responsive clinical isolates. Interestingly, the clinical isolates were found to be highly susceptible to berberine. Berberine was found to control the surface properties like hydrophobicity and charge of the cells. The cell membrane composition was altered by berberine, where the ergosterol and fatty acids were affected. The efflux pump activity was inhibited, and osmotic stress was generated in C. glabrata cells upon berberine exposure. The berberine has also generated oxidative stress and activated antioxidant system in C. glabrata cells. Furthermore, these observations were supported by the transcriptional expression study of C. glabrata cell genes (CDR1, RLM1, SLT2, SUR4, KRE1) and metabolomics analysis. Based on fold change analysis, the study identified 20 differential metabolites upon berberine treatment, which belong to central carbon, amino acids, and nucleotide pathways. The checkerboard analysis revealed the potentiation of some classically used antifungal drugs by berberine, thus suggesting it as a combinatorial nutraceutical adjuvant for the eradication of fungal infections. KEY POINTS: • Berberine exhibited better potency against azole-resistant clinical isolates • Berberine modulated metabolites of different pathways • Berberine generated oxidative stress and blocked efflux pump activity.

A novel class of Candida glabrata cell wall proteins with β-helix fold mediates adhesion in clinical isolates.

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed β-helix domain that is linked to a C-terminal β-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with β-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

In silico prediction and molecular docking study on the interaction of bioactive compounds of Adenanthera pavonina exploring the potential antifungal activity against Candida glabrata cell wall proteins

Candida glabrata infections being resistant to many azole antifungal agents, are difficult to treat. Various parts of Adenanthera pavonina have been used in traditional medicine. In the present study, an attempt was made to screen the bioefficacy of the identified phytoconstituents of A. pavonina on an in silico platform and identify some potential drug-like molecules that can impede important drug targets of C. glabrata using the molecular docking method. In a previous study related to the current research, the phytochemical profiling of the methanolic stem extract of A. pavonina was carried out using GC-MS to identify the phytoconstituents. The three-dimensional structure of the fungal receptors were derived by homology modeling using Modeller9v7 and the same for the ligands for which the structures were not available were drawn by ACD chemSketch. The docking of ligands and receptors were performed using PatchDock software. Druglikeliness and pharmacodynamics properties were evaluated using SWISS-ADME. GC-MS analysis of the A. pavonina extract revealed the presence of 17 phyto compounds, of which 2 heptyl 1,3dioxolane and methyl 4-o-methyl-d-arabinopyranoside best docked with the epithelial adhesion protein 6 receptor and cell wall transcription factor ACE2. Methyl 4-o-methyl-d-arabinopyranoside also best docked with the integral cell wall protein receptor. Although other compounds have shown good scores related to docking 2 heptyl 1, 3 dioxolane had an excellent binding affinity than the other ligands thus signifying its potent antifungal activity. 2 Heptyl 1, 3 Dioxolane was found to be BBB positive and 4-O-Methyl-D-arabinopyranoside is BBB negative. As the finding indicates, the two phyto compounds present in the methanolic stem extract of A. pavonina demonstrated good docking scores when docked with specific fungal cell wall receptors and thus can prove to be appropriated for the lead molecule.

Fluorescence-activated droplet sorting for enhanced pyruvic acid accumulation by Candida glabrata

One of the goals of metabolic engineering is to engineer strains that can optimally produce target metabolites. However, the current workflow for rational engineering of the metabolic pathway is sometimes time-consuming and labor-intensive. Here, we have established a cost-effective approach for screening for variants secreting metabolites. Different surface display systems were adopted and verified, which anchored pHluorin to the Candida glabrata cell surface to associate pyruvic acid detection with the read out of this reporter. A generalizable simulation approach based on computational fluid dynamics and regularity of generated droplet dimension was presented, which was found to be an efficient design tool to explore microfluidic characteristics or optimization. Finally, a microfluidic platform based on simulation coupled with surface display system was constructed. A mutant exhibiting a 73.6% increase in pyruvic acid production was identified. This ultrahigh-throughput screening pattern offers a practical guide for identifying microbial strains with many traits of interest.

Isolation, characterization and regulation of moonlighting proteases from Candida glabrata cell wall

Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10–50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.

Brazilian Copaifera Species: Antifungal Activity against Clinically Relevant Candida Species, Cellular Target, and In Vivo Toxicity.

Plants belonging to the genus Copaifera are widely used in Brazil due to their antimicrobial properties, among others. The re-emergence of classic fungal diseases as a consequence of antifungal resistance to available drugs has stimulated the search for plant-based compounds with antifungal activity, especially against Candida. The Candida-infected Caenorhabditis elegans model was used to evaluate the in vitro antifungal potential of Copaifera leaf extracts and trunk oleoresins against Candida species. The Copaifera leaf extracts exhibited good antifungal activity against all Candida species, with MIC values ranging from 5.86 to 93.75 µg/mL. Both the Copaifera paupera and Copaifera reticulata leaf extracts at 46.87 µg/mL inhibited Candida glabrata biofilm formation and showed no toxicity to C. elegans. The survival of C. glabrata-infected nematodes increased at all the tested extract concentrations. Exposure to Copaifera leaf extracts markedly increased C. glabrata cell vacuolization and cell membrane damage. Therefore, Copaifera leaf extracts are potential candidates for the development of new and safe antifungal agents.

Evaluation of the Molluscicidal, Artemicidal and Cytotoxic Activities of the Lectin from Opuntia ficus-indica Cladodes (OfiL)

Background: Biomphalaria glabrata is an intermediate host for the larvae of Schistosoma mansoni, which is distributed widely in the tropics. B. glabrata control is important to minimize the spread of schistosomiasis and natural compounds have been sought for use against this disease. The Artemia salina bench-top bioassay has been used to investigate the ecotoxicity of many natural compounds, and its results also correlate well with the in vitro cytotoxicity of natural compounds to tumor cells.
 Aims: To evaluate deleterious effects of the Opuntia ficus-indica lectin (OfiL) on B. glabrata, A. salina and human cancer cell lines.
 Methods: OfiL was isolated following a previously established protocol. The effects of OfiL on B. glabrata were investigated by determining survival of adults as well as development and hatching of embryos. The concentration required to kill 50% (LC50) of A. salina nauplii was determined. The cytotoxicity was determined using the human cell lines Hep-2 (human larynx epidermoid carcinoma), NCI-H292 (human lung mucoepidermoid carcinoma) and K562 (chronic myelocytic leukemia).
 Results: The development of most embryos (92.5–97.5%) treated with 1, 10 and 100 µg/mL of OfiL was found to be delayed, and dead (2.2–3.3%) and malformed (0.3–5.2%) embryos were also observed. OfiL did not kill B. glabrata adults, but a high percentage (30–45%) of the embryos generated by snails incubated with the lectin exhibited malformations. OfiL exhibited toxicity against A. salina (LC50: 61.02 µg/mL) but did not display cytotoxicity against the tumor cell lines evaluated.
 Conclusion: In conclusion, this study showed that OfiL can be a tool for schistosomiasis control that acts by impairing the viability of B. glabrata eggs and the fecundity of adult snails.

Cellular apoptosis: An alternative mechanism of action for caspofungin against Candida glabrata.

Background and Purpose:Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death.Materials and Methods:For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay.Results:According to the obtained MIC value (0.5 µg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 µg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 µg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (P<0.05). In addition, the rate of necrosis was significantly greater than that of apoptosis in response to caspofungin. Accordingly, necrosis occurred in 71.26%, 71.26%, and 61.26% of the cells at the caspofungin concentrations of 0.25, 0.5, and 1 µg/mL, respectively (P<0.05). The analysis of the data in the REST software demonstrated a significant increase in the expression of MCA1 and NUC1 genes (P<0.05).Conclusion:As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Remodeling of the Candida glabrata cell wall in the gastrointestinal tract affects the gut microbiota and the immune response

The gastrointestinal (GI) microbiota acts a natural barrier to the proliferation of opportunistic pathogens. Candida glabrata is an opportunistic yeast pathogen that has adapted to colonize all segments of the human GI tract. We observed an increase in Escherichia coli, Enterococcus faecalis, and Bacteroides vulgatus populations, and a decrease in Lactobacillus johnsonii, Bacteroides thetaiotaomicron, and Bifidobacterium animalis in mice with DSS-induced colitis. This reduction was more pronounced for L. johnsonii during C. glabrata overgrowth. In addition, C. glabrata overgrowth increased mouse mortality and inflammatory parameters, and modulated the expression of intestinal receptors and signaling pathways. The C. glabrata cell wall underwent various changes during the course of C. glabrata colonization, and showed a significant increase in chitin. C. glabrata deficient in chitin synthase-3 induced fewer inflammatory parameters than the parental strain during intestinal inflammation. Oral administration of chitin attenuated the impact of colitis, and reduced the number of aerobic bacteria and C. glabrata overgrowth, while chitinase-3-like protein-1 increased. This study provides evidence that inflammation of the gut alters the microbial balance and leads to C. glabrata cell wall remodeling through an increase in chitin, which is involved in promoting persistence of C. glabrata in the gut.

H+ channels in embryonic Biomphalaria glabrata cell membranes: Putative roles in snail host-schistosome interactions.

The human blood fluke Schistosoma mansoni causes intestinal schistosomiasis, a widespread neglected tropical disease. Infection of freshwater snails Biomphalaria spp. is an essential step in the transmission of S. mansoni to humans, although the physiological interactions between the parasite and its obligate snail host that determine success or failure are still poorly understood. In the present study, the B. glabrata embryonic (Bge) cell line, a widely used in vitro model for hemocyte-like activity, was used to investigate membrane properties, and assess the impact of larval transformation proteins (LTP) on identified ion channels. Whole-cell patch clamp recordings from Bge cells demonstrated that a Zn2+-sensitive H+ channel serves as the dominant plasma membrane conductance. Moreover, treatment of Bge cells with Zn2+ significantly inhibited an otherwise robust production of reactive oxygen species (ROS), thus implicating H+ channels in the regulation of this immune function. A heat-sensitive component of LTP appears to target H+ channels, enhancing Bge cell H+ current over 2-fold. Both Bge cells and B. glabrata hemocytes express mRNA encoding a hydrogen voltage-gated channel 1 (HVCN1)-like protein, although its function in hemocytes remains to be determined. This study is the first to identify and characterize an H+ channel in non-neuronal cells of freshwater molluscs. Importantly, the involvement of these channels in ROS production and their modulation by LTP suggest that these channels may function in immune defense responses against larval S. mansoni.

Nanoscopic characterization of immunogenic b-glucan exposure on Candida species pathogens

Abstract The cell walls of Candida spp. are composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell wall polysaccharide whose surface exposure is masked from the innate immunoreceptor Dectin-1 to evade immunity. Previous research suggested that the physical presentation geometry of β-glucan may determine whether or not it can be recognized by Dectin-1. We used super resolution imaging to measure the fine structure of glucan exposure on C. albicans cell walls before and after glucan unmasking with the antimycotic drug caspofungin. Most exposed glucan on C. albicans yeast and hyphae exists as single Dectin-1 binding sites. Caspofungin-mediated glucan unmasking results in nanoscale remodeling of glucan exposures, resulting in increased density and size of glucan exposure sites. The less studied pathogen C. glabrata exhibits significantly greater glucan exposure than C. albicans in type strains, but recent clinical isolates of C. glabrata have lower exposure, suggesting that C. glabrata also masks its glucan. We used C. glabrata cell wall mutants to define the role of N-mannan structure in maintaining glucan masking. The less complex N-mannan structures of mutant strains provided poorer glucan masking, increasing the density of nanoscale glucan exposure sites. a-(1,6)-mannan backbone length was a major structural determinant of glucan masking, while a-(1,2;1,3)-mannan side chain density was less critical to masking function. This work provides the first characterization of changing glucan exposure nanostructure at the pathogenic cell wall surface that participates in host-Candida interaction. Further studies will examine the functional impact of glucan nanostructure on Dectin-1 activation.

Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis.

Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

A novel Candida glabrata cell wall associated serine protease

We set out to identify the Candida glabrata cell wall attached proteases which may play a role as virulence factors in candidosis, particularly in the immunocompromized host. We studied a clinical C. glabrata strain T-1639, which was isolated from a patient from the Helsinki University Central Hospital. With non-reducing 2-D electrophoresis using parallel fluorogenic gels and mass spectrometry we identified a novel appr. 25 kDa (192 aa in length) cell wall located protease with an estimated pI of 7.6. The LC-MS/MS peptides matched with the ORF of predicted C. glabrata CBS138 cell wall protein Cwp1.2p/pI 7.7/212 aa (http://cbi.labri.fr/Génolevures/[NCBI access 49525604, UniProt access Q6FTZ7]), which is an ortholog to Saccharomyces cerevisiae cell wall protein Cwp1p (UniProt access P28319). The novel serine protease was released by β-1,3-glucanase treatment from the cell wall. In contrast to previous predictions this protease has an enzymatic function instead of being merely a structural cell wall protein. The protease showed gelatinolytic activity and was inhibited by PMSF, a known serine protease inhibitor. Further characterization of the protease may give insight to its role in infections caused by C. glabrata and possibly aid in the development of new kinds of antifungal drugs.

Two non-exclusive strategies employed to protect Torulopsis glabrata against hyperosmotic stress

Several recent reports described an apoptosis-like programmed cell death (PCD) process in yeast in response to different environmental challenges. In this study, hyperosmotic stress caused by high NaCl concentration in culture medium induced cell death in the haploid yeast Torulopsis glabrata. Propidium iodide (PI) and PI/rhodamine-123 (Rh123) dual staining with flow cytometry showed that high salinity decreased intact cells by 16.5 %, increased necrotic cells by nearly twofold, and altered fermentative parameters appreciably. Morphological and biochemical indicators of apoptosis were apparent, specifically a decrease in mitochondrial membrane potential (∆Ψm), translocation of phosphatidylserine (PS) from the inner to the outer side of the plasma membrane, generation of reactive oxygen species (ROS), and involvement of caspase all while plasma membrane integrity was maintained. Additionally, it was found that overexpression of YCA1 drastically stimulated cell death, indicating that activation of metacaspase might lead to cell death. However, T. glabrata growth under hyperosmotic stress was enhanced when FIS1, HOG1, and GPD2 were overexpressed, or when exogenous proline or glutathione (GSH) were added into the cultures, both of which could repress caspase-3 activity. Thus, in these concrete cases of overexpression of anti-apoptotic or anti-necrotic factors and pharmacological manipulations, it decreased T. glabrata cell death that might help to achieve higher fermentative efficiency.

Histatin 5 Resistance of Candida glabrata Can Be Reversed by Insertion of Candida albicans Polyamine Transporter-Encoding Genes DUR3 and DUR31

Candida albicans and Candida glabrata are predominant fungi associated with oral candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with high fungicidal activity against C. albicans, however many strains of C. glabrata are resistant. Since Hst 5 requires fungal binding to cell wall components prior to intracellular translocation, reduced Hst 5 binding to C. glabrata may be the reason for its insensitivity. C. glabrata has higher surface levels of β-1,3-glucans as compared with C. albicans; however these differences did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly, the biofilm matrix of C. glabrata contained significantly higher levels of β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell through polyamine transporters Dur3p and Dur31p that are uncharacterized in C. glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate limiting step is reduced uptake that can be overcome by expression of C. albicans Dur proteins in C. glabrata.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Identification and Differential Gene Expression of Adhesin-Like Wall Proteins in Candida glabrata Biofilms

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

Chemokinetic Effect of Interleukin-1β on Cultured Biomphalaria glabrata Embryonic Cells

Previous studies have indicated that a molecule with cytokine activity, possibly an interleukin-1-like (IL-1) molecule, plays a role in the killing of larval stages of the blood fluke Schistosoma mansoni in the snail host Biomphalaria glabrata. The purpose of the present experiment was to test the effects of recombinant-human IL-1beta (rhIL-1beta) on embryonic B. glabrata (Bge) cell motility to determine whether the cells respond to the cytokine. Response was measured using a variation of a chemokinetic assay in which cells in culture were separated from variable concentrations of rhIL-1beta by a semi-permeable membrane containing pores to allow migration. A double staining technique was developed to ascertain cell movement across the membrane. The number of cells moving across the membrane significantly increased in a concentration-dependent manner relative to the presence of increasing amounts of rhIL-1beta below the membrane. The number of cells that moved across the membrane increased until a threshold was reached, after which migration decreased. Further, the rhIL-1beta-mediated increase in Bge cell migration across the membrane was abrogated by the addition of IL-1 receptor antagonist protein. These data indicate that Bge cells respond specifically to rhIL-1beta. As such, these data also indicate that Bge cells may serve as a useful model for elucidation of the role of cytokines or cytokine-like molecules in the snail/schistosome relationship.

Systematic identification in silico of covalently bound cell wall proteins and analysis of protein-polysaccharide linkages of the human pathogen Candida glabrata.

Candida glabrata is an important cause of systemic candidiasis in humans. This paper reports a systematic analysis of the putative glycosylphosphatidylinositol-modified (GPI) proteins of C. glabrata, a large part of which are covalently bound to the cell wall glucan network and the remainder of which are retained in the plasma membrane, and of cell wall proteins (CWPs) which are covalently bound in a mild-alkali-sensitive manner. In silico genomic analysis revealed 106 putative GPI proteins. Fifty-one of these GPI proteins could be categorized as adhesive proteins, potentially implicated in fungus-host interactions or biofilm formation during the development of fungal infections. Eleven proteins belonged to well-known GPI protein families of glycoside hydrolases, probably involved in cell wall expansion and remodelling during growth. Other identified GPI proteins included phospholipases, aspartic proteases, homologues of ScEcm33p and ScKre1p, and structural CWPs. Interestingly, the GPI algorithm predicted three orthologues of an abundant CWP in S. cerevisiae, Cwp1p, which is absent in Candida albicans. To evaluate the in silico predictions, isolated cell walls were extracted using HF-pyridine, which specifically cleaves phosphodiester bonds, to release GPI-CWPs. Immunological analysis of the extract using one-dimensional SDS-PAGE and anti-ScCwp1p antiserum indicated the presence of a Cwp1p homologue in C. glabrata cell walls. Further analysis by two-dimensional gel electrophoresis and electrospray ionization tandem mass spectrometry (ESI-MS/MS) confirmed the presence of two of the predicted Cwp1p proteins, Cwp1.1p and Cwp1.2p. Crh1p, a putative 1,3-beta-glucan remodelling enzyme, was also identified. In silico genomic analysis further revealed five putative Pir proteins (Pir1-5p) and five members of the Bgl2 glycoside hydrolase family 17, belonging to a class of putative CWPs that can be extracted with NaOH. Immunological analysis of mild-alkali-extracted CWPs showed the presence of a ScPir2p homologue. Together, these experimental data and in silico predictions represent the first systematic analysis of the C. glabrata cell wall proteome.

Surface Membrane Polypeptides Associated with Hemocytes from Schistosoma mansoni-Susceptible and -Resistant Strains of Biomphalaria glabrata (Gastropoda)

Previous studies have shown that hemocytes from inbred susceptible and resistant strains of the snail, Biomphalaria glabrata, differentially react to primary sporocysts of the blood fluke, Schistosoma mansoni. It has been hypothesized that this differential reactivity may be due to differences in the expression of hemocyte surface determinants serving as parasite recognition and/or hemocyte activating factors. In order to begin addressing this hypothesis the hemodynamics and surface polypeptides of circulating hemocytes from susceptible and resistant B. glabrata strains were compared in two morphologically distinct cellular subpopulations that differed in their substrate adherence properties. When compared to a S. mansoni-resistant snail strain, two susceptible strains exhibited overall lower total circulating cell numbers and a consistently lower proportion of nonadherent hemocytes. Exposure of snails to S. mansoni miracidia induced significant increases in total circulating cell number in all strains by 2 days postexposure (PE). However, the proportion of adherent and nonadherent hemocytes remained constant in each snail strain at 2 and 4 days PE. A sensitive technique involving biotin labeling of polypeptides at the surface of living hemocytes, followed by nonreducing SDS-PAGE separation and electrotransfer to nitrocellulose, revealed a similar pattern of hemocyte surface polypeptides ranging in apparent molecular weights from 210 to 30 kDa in the three B. glabrata strains. One exception was the presence of 66-kDa polypeptide expressed at the surface of both adherent and nonadherent hemocytes in the two susceptible snail strains, which was only weakly expressed or absent from hemocytes of the resistant 13-16-R1 B. glabrata strain. In addition, exposure of snails to S. mansoni miracidia appeared to modulate (enhance) expression of the 66-kDa polypeptide in susceptible B. glabrata cell populations. Although the functional importance of this membrane polypeptide is unknown, the differential enhancement of expression in susceptible vs resistant snail hemocytes may reflect potentially important biochemical changes related to previously observed strain-specific reactivities to invading larval S. mansoni. Finally, a complex of high-molecular-weight surface polypeptides (approx 210 kDa) associated with adherent hemocytes was consistently reduced or absent in nonadherent cell preparations suggesting a possible role in substrate adhesion.