Attachment Of Yeast Cells Research Articles

Enhanced ethanol production from lignocellulosic hydrolysate using Meyerozyma caribbica biofilm immobilized on modified epoxy foam

In comparison to planktonic cells, microbial biofilm immobilization is known to enhance ethanol production. To maintain this capability during long-term fermentation, the detachment of the biofilm must be prevented. In this study, the enhancement of the biofilm immobilization of the yeast Meyerozyma caribbica YLP01GX on bio-based epoxy foam (EF) was performed through plasma surface modification to improve the production of ethanol from oil palm empty fruit bunch hydrolysate. This newly synthesized EF serves as a suitable carrier due to its superior properties and cost-effectiveness. The modification could induce yeast cell attachment more rapidly with more extracellular polymeric substance (EPS) being produced in the presence of the yeast extract. Therefore, the immobilization period can be shortened. In comparison to untreated EF-immobilized cells (NEF-IC), the modified EF-immobilized cells (MEF-IC) showed an improved binding affinity for proteins in the EPS, which resulted in a higher adhesion force between the carrier and the yeast cell surface. The stable biofilm attachment on the modified EF can prevent biofilm detachment and improve the tolerance of the yeast to inhibitors in the hydrolysate, leading to enhanced ethanol production compared to NEF-IC. Additionally, the MEF-IC was reused several times with similar fermentation activity being observed. This approach offers a more efficient and economical fermentation process.

Isoconductivity method to study adhesion of yeast cells to gold electrode

Abstract In this paper, we used impedance spectroscopy and gold electrodes to detect the presence of yeast cells and monitor the attachment of these cells to the electrodes. We analyzed the effect of conductivity changes of the medium and the attachment on the electrode-electrolyte interface impedance. A three-electrode cell was designed to produce a uniform electric field distribution on the working electrode and to minimize the counter electrode impedance. Moreover, we used a small AC overpotential (10 mV) to keep the system within the linear impedance limits of the electrode-electrolyte interface. This study proposes a new method to differentiate the impedance changes due to the attachment of yeast cells from those due to conductivity changes of the medium. The experiments showed that when the difference between the cell suspension and base solution conductivities is within the experimental error, the impedance changes are only due to the attachment of yeast cells to the electrodes. The experiments also showed a strong dependence (decrease) of the parallel capacity of the electrode electrolyte interface with the yeast cell concentration of suspension. We suggest that this decrease is due to an asymmetrical redistribution of surface charges on both sides of cell, which can be modeled as a biologic capacity connected in series with the double layer capacity of the interface. Our results could help to explain the rate of biofilm formation through the determination of the rate of cell adhesion.

Time-lapse video microscopy and image analysis of adherence and growth patterns of Candida albicans strains

Digital image analysis of high time resolution video microscopy was used to investigate hyphal growth dynamics in different Candida albicans strains. The effects of the quorum sensing molecules tyrosol and farnesol, the deletion of the fungus specific protein phosphatase Z1 CaPPZ1), and the hypha-specific cyclin (HGC1) genes were analyzed by this method. Our system monitored cell growth in a CO2 incubator under near-physiological conditions and measured three major parameters under the following stringent conditions: (a) the time of yeast cell adherence, (b) the time of hyphal outgrowth, and (c) the rate of hyphal growth. This method showed that hyphal extension of wild-type SC5314 cells was accelerated by tyrosol and inhibited by farnesol. Hyphal growth rate was moderately lower in cappz1 and strongly reduced in hgc1 mutants. In addition, tyrosol treatment caused a firm adherence, while farnesol treatment and hgc1 mutation prevented the adherence of yeast cells to the surface of the culture flask. Transition from yeast-to-hyphal state was faster after tyrosol treatment, while it was reduced in farnesol-treated cells as well as in the cappz1 and hgc1 mutants. Our data confirm the notion that the attachment of yeast cells, the yeast-to-hyphal transition, and hyphal growth rate are closely related processes. Time-lapse video microscopy combined with image analysis offers a convenient and reliable method of testing chemicals, including potential drug candidates, and genetic manipulations on the dynamic morphological changes in C. albicans strains.

Physical Interactions between Yeast Pichia guilliermondii and Post-Harvest Fruit Pathogen Penicillium expansum

Attachment of yeast cells or bacteria on fungal hyphae have been observed in various antagonisms between microorganisms. Physical interactions between yeast Pichia guilliermondii and postharvest fruit pathogen Penicillium expansum in culture were studied in detail using light and transmission electron microscope to give better understanding on their mode of antagonism. Both organisms were co-cultured for 24-hr on potato dextrose agar. Light microscopy observations on the co-culture showed that the yeast cells attached firmly on the fungal hyphae. This attachment was inhibited by several substances such as enzymes degrading protein (protease or trypsin), a respiration inhibitor (sodium azide), an acid (hydrochloric acid) or an alkali (sodium hydroxide). Although autoclaved hyphae did not affect the attachment, but boiled enzymes and autoclaved yeast cells totally abolished the attachment. These evidences suggested that the attachment might be an active process mediated by certain protein from live yeast cells. Transmission electron micrographs on the ultrastructure of the co-culture revealed that the hyphae showed abnormalities in their structure and organelles, and a degree of obvious damage. Physical interactions observed in this study could be contributed to the mechanism of antagonism between P. guilliermondii and P. expansum. Key words: Pichia guilliermondii, Penicillium expansum, attachment, transmission electron microscope

In vitro attachment of phylloplane yeasts toBotrytis cinerea,Rhizoctonia solani, andSclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.

Yeast cell attachment: a tool modulating wall composition and resistance to 5-bromo-6-azauracil.

The attachment of Candida utilis, Kluyveromyces lactis, and Saccharomyces cerevisiae cells stimulates an increase in the content of cell wall polysaccharides and mannoproteins, accompanied by increased resistance to the inhibitory effect of 5-bromo-6-azauracil. The covalent attachment of viable yeasts was accomplished (via dialdehyde-amino spacers) by reaction of aldehyde groups of the carrier with reactive amino groups in accessible cell surface proteins. The employed technique enables the optimization of yeast sources of beta-1,3-, beta-1,6- glucans, mannan, and mannoprotein. The modulatory effect of the cell attachment is discussed.

Attachment of yeast to modified stainless steel wire spheres, growth of cells and ethanol production

The immobilization of yeast Saccharomyces cerevisiae, their growth and ethanol production were investigated using untreated and modified stainless steel wire spheres (WS) as carriers. The carrier surface was modified by oxidation, by treatment with titanium (IV) chloride (TiCl 4) or by γ-aminopropyltrietoxysilane (AS) in an attempt to raise the efficiency of the immobilization of the yeast cells. The influence of the cell fixation method on culture growth and ethanol synthesis was investigated. The immobilization of cells to carrier surface was checked by scanning electron microscopy (SEM). More closely attachment of yeast cells was seen on the aminated wire surface. It was established that during fermentation ethanol production by yeast was stimulated using immobilized cells in oxidized WS or treated with TiCl 4. Aminated WS surface stimulated the culture growth but decreased ethanol synthesis. Free yeast cells located in the pores of WS increased the biomass concentration and ethanol production only during the first cycle of batch fermentation. Stable cell growth and ethanol production was observed during subsequent 4–5 repeated fermentation cycles using washing out of free cells from WS before fermentation. The system productivity Q eth for ethanol synthesis was 0.92–1.25 g/l per h. Cell fixation in WS by lyophilization or convective dehydration improved cell attachment to wire surface but did not influence positively culture growth and ethanol synthesis. The conclusion was made that stainless steel WS filled with paste-like yeast biomass can be used as inoculum for repeated batch ethanol production. The modification method of the stainless steel wire surface significantly influences the immobilization efficiency of yeasts. Oxidized or modified by TiCl 4 wire surface and washing out free cells from WS can be recommended for ethanol production by immobilized yeasts.

Overexpression of the Candida albicans ALA1 gene in Saccharomyces cerevisiae results in aggregation following attachment of yeast cells to extracellular matrix proteins, adherence properties similar to those of Candida albicans.

Candida albicans maintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions. We show that adherence due to the C. albicans gene ALA1 when transformed into Saccharomyces cerevisiae, is comprised of two sequential steps. Initially, C. albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase). This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase). Neither of these phases is observed in S. cerevisiae. However, expression of the C. albicans ALA1 gene from a low-copy vector causes S. cerevisiae transformants to attach to ECM-coated magnetic beads without appreciable aggregation. Expression of ALA1 from a high-copy vector results in both attachment and aggregation. Moreover, transcriptional fusion of ALA1 with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter. The adherence of C. albicans and S. cerevisiae overexpressing ALA1 to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The similarities in the adherence and aggregation properties of C. albicans and S. cerevisiae overexpressing ALA1 suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C. albicans.

Antibody response that protects against disseminated candidiasis

We previously showed that surface mannans of Candida albicans function as adhesins during yeast cell attachment to mouse splenic marginal zone macrophages. The mannan adhesin fraction was encapsulated into liposomes and used to vaccinate mice over a 5- to 6-week period. Circulating agglutinins specific for the fraction correlated with increased resistance to disseminated candidiasis. Antiserum from vaccinated animals protected naive BALB/cByJ mice against C. albicans serotype A and B strains and Candida tropicalis. Antiserum also protected SCID mice against disseminated disease. The serum protective ability was stable at 56 degrees C, but this ability was adsorbed by C. albicans cells. The antiserum was divided into three fractions after separation by high-performance liquid chromatography. One fraction contained all of the agglutinin activity and transferred resistance to naive mice. A second fraction also transferred resistance. Two monoclonal antibodies (MAbs) specific for candidal surface determinants were obtained. MAb B6.1 is specific for a mannan epitope in the adhesin fraction, and MAb B6 is specific for a different epitope in the fraction. Both MAbs are immunoglobulin M, and both strongly agglutinate candidal cells, but only MAb B6.1 protected both normal and SCID mice against disseminated candidiasis. In one experiment, 10 normal mice were given MAb B6.1 and challenged with yeast cells. Six mice survived the 67-day observation period; 4 of the survivors were cured as evidenced by the lack of CFU in the kidney and spleen. Our studies show that antibodies against certain cell surface antigens of C. albicans help the host resist disseminated candidiasis.

Evidence for adhesin activity in the acid-stable moiety of the phosphomannoprotein cell wall complex of Candida albicans

Previously, we showed that Candida albicans hydrophilic yeast cells adhere specifically to mouse splenic marginal-zone macrophages. The adhesins are part of the yeast cell wall phosphomannoprotein complex, and one adhesin site, which reacts with the monoclonal antibody 10G, was identified as a beta-1,2-linked tetramannose in the acid-labile portion of the complex. We report here that the acid-stable part of the complex, which has not been reported previously to have adhesin activity, is in large part responsible for yeast cell binding to the splenic marginal zone. The phosphomannoprotein complex, termed Fr.II, was isolated from C. albicans serotype B yeast cells by beta-mercaptoethanol extraction and concanavalin A-agarose affinity chromatography. Fr.II is devoid of the serotype A-specific antigen factor 6, which functions in yeast cell attachment to epithelial cells. The acid-stable part of Fr.II (i.e., Fr.IIS) was obtained by mild acid hydrolysis and size exclusion fractionation. Fr.IIS was further fractionated into four fractions, Fr.IIS1, Fr.IIS2, Fr.IIS3, and Fr.IIS4, by concanavalin A-agarose column chromatography and elution with a linear gradient of alpha-methyl-D-mannopyranoside. Adhesin activity of these fractions was determined by their ability to block yeast cell binding to the splenic marginal zone. Fr.IIS1 and Fr.IIS2 yielded more material and stronger adhesin activity than either Fr.IIS3 or Fr.IIS4. Only Fr.IIS1 did not react with antibodies (anti-factor 5 and monoclonal antibody 10G) specific for the acid-labile beta-1,2-linked oligosaccharides. Fr.IIS1-coated latex beads attached specifically to the marginal zone in a pattern identical to that of yeast cell binding. Furthermore, Fr.IIS1-latex bead attachment was inhibited by soluble Fr.II or Fr.IIS. Initial chemical analyses indicate that the adhesin site on Fr.IIS1 is a carbohydrate because adhesin activity was destroyed by periodate oxidation but not by proteinase K digestion.

Evidence that Candida albicans binds via a unique adhesion system on phagocytic cells in the marginal zone of the mouse spleen.

We recently demonstrated by using an ex vivo adhesion assay that Candida albicans yeast cells exhibit a unique binding affinity for the marginal zone of the spleen. This binding event provides a working model for studying mechanisms of organ dissemination of the fungus from the blood. By using the ex vivo assays reported here, we showed by bright-field and electron microscopic techniques that mouse spleen marginal zone cells capable of ingesting India ink particles are also involved in yeast cell attachment. During splenic clearance of yeast cells from the circulation in vivo, C. albicans is also associated exclusively with marginal zone cells capable of ingesting India ink. The ability to ingest the ink particles is not necessarily related to yeast cell adherence, because the fungal cells did not bind to phagocytic cells in the splenic red pulp. In fact, the marginal zone phagocytic cells appear to have a unique binding system, because yeast cells also did not bind to phagocytes in other tissues, such as the thymus and peritoneum, or to seven different myeloid cell lines. In addition, antibodies to a number of well-characterized murine adhesion molecules, such as leukocyte integrins, LECAM-1, and CD44, had no effect on binding. On the basis of these results, we propose that splenic marginal zone phagocytes express a novel adhesion system that involves either a unique adhesion molecule or previously described adhesion molecules with unique binding activities.

Role of fibronectin in the pathogenesis of candidal infections.

Adherence of Candida albicans yeast cells to mammalian cells in vitro is promoted by a cell-surface adhesin that is probably a mannan or a mannoprotein. This observation is based on results of studies that utilized either lectins or antibodies to Candida that block attachment. Selective enzymatic degradation of cell wall constituents has been used to define the surface ligand of the candidal cell. Recently, nonadhering, spontaneous mutants of C. albicans, from both yeast and mycelial forms, that lack specific mannoproteins have been described. In addition, such mutants are avirulent, an indication that attachment plays a role in the disease process. The mammalian receptor for Candida has not been identified, but in endocarditis it could be fibronectin, both because fibronectin is present at the endocardial lesion and because those Candida species that bind to fibronectin in vitro exhibit a high disease potential; i.e., Candida species that bind to fibronectin in vitro also cause endocarditis, while those that do not bind in vitro do not cause disease. Attachment of yeast cells both to human buccal and vaginal epithelial cells is reduced significantly if yeast cells are preincubated with fibronectin, a finding that indicates a possible role for fibronectin as the epithelial surface receptor for Candida.

Pathogenesis of vaginal candidiasis: studies with a mutant which has reduced ability to adhere in vitro.

A spontaneous, cerulenin-resistant mutant of Candida albicans (strain 4918-10) was found to adhere less readily to human vaginal mucosal cells in vitro than a wild type C. albicans (strain 4918). In a murine model of vaginal infection, strain 4918-10 was found to be less virulent than wild type C. albicans, i.e., the infection rate caused by 4918-10 was only 31% of that observed with wild type, 4918. A chitin-soluble extract (CSE) prepared from 4918 blocked attachment of yeast cells to human vaginal epithelial cells, while CSE from 4918-10 did not significantly reduce the attachment of yeasts to vaginal cells. Both 4918 and 4918-10 produced hyphae in vitro and in vivo, were negative for proteinase production and grew equally well at 28 degrees C and 37 degrees C. The data suggest that adherence to vaginal mucosa may be an important determinant in the pathogenesis of vaginal infection caused by C. albicans.

Adherence of Candida to corneal surface

In the pathogenesis of mycotic infections, adherence of the microbes to surface structures prior to invasion appears to be the initial and essential step in a susceptible host. Adherence and inhibition of adherence of Candida albicans to rabbit corneal surface was investigated in vitro by light and scanning electron microscopic examinations. The results indicate that blastospores of Candida albicans rarely bind to intact corneal epithelium, but consistently adhere to stroma denuded of epithelium. Such adherence was inhibited by concanavalin A. With its strong affinity for the yeast cell wall carbohydrate mannan, concanavalin A may block the site of attachment of yeast cells to the corneal surface.

Lectin-mediated attachment and ingestion of yeast cells and erythrocytes by hamster fibroblasts

Yeast cell attachment to Concanavalin A (ConA)-coated fibroblasts depends on the degree of saturation of ConA-binding sites on the fibroblast. Under comparable conditions, fresh mouse erythrocytes fail to establish stable contacts with ConA-coated fibroblasts. The interaction of ConA-coated erythrocytes with fibroblasts and of non-coated erythrocytes with wheat germ agglutinin (WGA)-coated fibroblasts is remarkably less efficient than that of yeast cells interacting with ConA-coated fibroblasts. Ingestion of attached cells was not observed in any of the above lectin-mediated cell-cell interactions. Yeast cells coated with ConA show a high extent of attachment to fibroblasts (three-fold that of yeast cell attachment to ConA-coated fibroblasts). The attachment is highly temperature sensitive, being 3 times more at 37 °C than at 14 °C. A significant fraction of attached yeast cells (~46%) is ingested by the fibroblasts during the 60 min incubation at 37 °C. The ingestion exhibits a strong temperature dependence, being nil at 14 °C and amounting to 150 and 600 ingested yeast cells per 100 fibroblasts at 24 °C and 37 °C, respectively. Transmission and scanning electron microscopy of ConA-mediated yeast cell-fibroblast interaction indicates a tighter interaction when the yeast cells are coated with ConA than when the fibroblasts are coated with ConA. Thus spreading of the plasma membrane around the attached yeast cell as well as transduction of attachment to ingestion could be triggered only under conditions of a very extensive multibridge interaction between the two apposing surfaces. Such an interaction is not achieved when the mobility of ConA-receptors within the fibroblast membrane plane is restricted as a result of crosslinking with ConA.

Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis.

The effects of cryptococcal polysaccharide and selected serum factors on (i) the attachment of Cryptococcus neoformans to macrophages and (ii) the subsequent ingestion of yeast cells by the macrophages were investigated. Percent attachment was measured after incubation of yeast cells with macrophages at 4 C. Percent engulfment was determined after incubation of yeast cells and macrophages at 37 C. Nonencapsulated yeast cells readily attached to macrophages at the low temperature and were engulfed at a high rate at 37 C, whereas encapsulated yeast cells attached to macrophages at low rates and were engulfed at low rates. Addition of varying doses of purified cryptococcal polysaccharide to nonencapsulated yeast cells inhibited attachment at approximately the same concentration of polysaccharide required for inhibition of engulfment. Nonencapsulated yeast cells that attached to macrophages at 4 C were eluted from the macrophages by addition of purified cryptococcal polysaccharide to the incubation medium. Heat-labile opsonins were not required for attachment of yeast cells to macrophages, but they were necessary for maximal initial rates of phagocytosis. Heat-stable components of serum facilitated attachment of cryptococci, but their most important function appeared to be triggering the ingestion of attached yeast. Specific antiserum had no effect on the attachment and engulfment of nonencapsulated cryptococci, and the antiserum produced only a small enhancement of the engulfment of encapsulated cryptococci.

Concanavalin A-mediated attachment and ingestion of yeast cells by macrophages

The ConA-mediated interaction of yeast cells with macrophages was brought about in two steps. The first step involved the interaction of either macrophages or yeast cells with ConA, MConA or YConA in brief, respectively. The second step consisted of interacting the ConA-coated cells with their non-coated counterpart, yielding MConA-Y or M-YConA. The extent of yeast cell attachment to macrophages depends on the degree of saturation of ConA binding sites on the cell coated with ConA in the first step and on the temperature at which the two cell types interact. The temperature dependence in the range of 10–25 °C implies that cell-cell attachment is sensitive to the physical state of membrane lipids as reflected in increased lateral mobility of ConA receptors in the membrane plane. The extent of ConA-mediated cell association is not influenced significantly by colchicine, cytochalasin B (CB) or hydrocortisone. A mild treatment of macrophages with glutaraldehyde reduces, however, the association of yeast cells, further indicating a need for lateral mobility of ConA receptors. ConA-mediated yeast cell attachment could be totally reversed by α-methyl mannoside in the case of MConA-Y and only partially in the case of M-YConA. Yeast cell ingestion is highly temperature-dependent; in MConA-Y a 50% interiorization of the associated yeast cells is reached at 32 °C and detectable interiorization starts only above 19 °C, while in M-YConA a 50% value of interiorization is reached at 18 °C and about 15% of yeast cells are interiorized already at 5 °C. Interiorization of attached yeast cells is not affected by colchicine. Cytochalasin B (CB) (10 μg/ml) inhibits 82% of yeast interiorization in MConA-Y and only 12% in M-YConA. Hydrocortisone has a similar differential effect of inhibition of ingestion; at 25 °C inhibition in MConA-Y amounts to 78% and in M-YConA to 22%. Sodium azide inhibits 90% of interiorization of yeast cells in both MConA-Y and M-YConA. The following working hypothesis was proposed to explain both the characteristics of attachment and the remarkable difference in ingestion pattern of yeast cells in MConA-Y and M-YConA. ConA-mediated yeast cell attachment to macrophages involves multipoint interaction between the two cells achieved by a certain clustering of ConA receptors in the membrane plane. To achieve interiorization a higher extent of bridge formation between the cells is required, and a higher number of ConA-membrane receptors have to be recruited to the area of apposition of the two membranes. This requires lateral mobility of either ConA receptors conjugates (in the case of MConA) or of mobile non-crosslinked ConA receptors in macrophages interacting with YConA). Mobility of ConA receptor conjugates is more sensitive to membrane fluidity than that of non-crosslinked receptors and hence the differential temperature-dependence of ingestion. The effect of CB suggests an involvement of the cytoskeleton in the reorganization of ConA receptors at the membrane level.