Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Physiological characterization of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from agro-industrial and environmental origins with possible probiotic function

Probiotics are defined as live microorganisms, which when administered in adequate amount confer a health benefit to the host. Most of studied or commercialized probiotics contain bacteria and very few of them present yeast in its composition. In this last case, the microorganisms almost always belong to Saccharomyces genus. In the present study, it was of interest to screen among 103 non-Saccharomyces yeasts a candidate for probiotic by using in vitro and in vivo criteria. In vitro assays included growth at 37°C and production of antagonistic compounds against enteropathogenic indicators, and the in vivo assays evaluated the colonization ability of mouse gastrointestinal tract without pathologic consequences and the protective ability in mice experimentally challenged with Clostridium difficile. In conclusion, Pichia kluyveri strain 898 showed to be a potential candidate for probiotic use, based on the criteria cited above, particularly as demonstrated by its protective effect against experimental infection in mice. Interestingly, an in vivo inhibition against C. difficile observed in the animal models did not correlate with the results obtained with the in vitro assays.     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

The ultrastructure of Candida albicans infections

Scrapings of Candida albicans plaques from the tongue and buccal mucosa of patients with oral candidiasis were examined by electron microscopy. In addition, urine sediment from patients with infection of their catheterized urinary tracts was similarly examined. Three types of C. albicans-oral epithelial cell interactions were noted: a loose adherence apparently mediated by a ruthenium red positive matrix, a "tight" adherence where no space could be seen between the host and yeast cell. and invasion of host cells by yeast hyphal elements. Adhesion of Candida blastospores to hyphal elements and adhesion of bacteria to Candida cells was also frequently observed. Urine sediments from patients with mixed bacteria-yeast infections demonstrated adhesion of the bacteria to the yeast cells. This phenomenon was also demonstrated in in vitro experiments and fibrous ruthenium red material invariably occupied the zone of adhesion. Phagocytosis of yeast by polymorphonuclear leukocytes was found in urinary, but not in oral. candidiasis. Our in vivo and in vitro observations indicate that a ruthenium red positive matrix covers the surfaces involved in the yeast to yeast, yeast to host, and yeast to bacteria adhesion.     

Adenosine 3'',5''-phosphate phosphodiesterase and pheromone response in the yeast Saccharomyces cerevisiae

Theophylline, aminophylline, and isobutylmethylxanthine, compounds reported to be inhibitors of adenosine 3',5'-phosphate (cAMP) phosphodiesterase, prevented the alpha-factor-induced cell cycle arrest of Saccharomyces cerevisiae a cells. To determine whether the in vivo effect of these methylxanthines on yeast pheromone response was related to their known biochemical mode of action, two assays for cAMP phosphodiesterase based on affinity of the product of the reaction (5'-AMP) for boronate groups were developed and were used to monitor the activity of the low Km cAMP phosphodiesterase present in yeast extracts. It was found that the relative efficacy of the methylxanthines as inhibitors of this enzyme in vitro was correlated with the degree to which they antagonized alpha-factor action in vivo. These results were consistent with our previous proposal that pheromone action involves a lowering of cAMP level in the target cell.     

Surface-active properties of Candida albicans

Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces.     

RNA/nucleotide enhances antibody production in vitro and is moderately mitogenic to murine spleen lymphocytes.

Suppression of immune functions was demonstrated in both humans and animals when exogenous RNA was eliminated from the diet. However, direct actions of RNA/nucleotide on the immune system are virtually unknown. Thus, in this study, we explored effects of RNA and nucleotide on lymphocyte functions in vitro. Yeast whole RNA, which is free of endotoxin, was supplemented to culture media, and changes in mitogen responses, thymocyte proliferation, or in vitro antibody production by murine spleen lymphocytes were analyzed. Yeast whole RNA potentiated the proliferation of spleen lymphocytes and it also strikingly enhanced in vitro antibody production in response to sheep red blood cells at least 10-fold. However, it did not potentiate the proliferation of thymocytes (immature lymphocytes). These enhancing activities of yeast RNA were significantly reduced by RNAse treatment, but not by treatments with DNAse or polymyxin B. Certain mononucleotides exhibited less, but similar, action on murine spleen lymphocytes. The whole yeast RNA employed was already degraded to small nucleotide (less than 1 kb). Therefore, it may be suggested that certain components of RNA degraders can function as powerful immunomodulators, indicating that exogenous RNA or nucleotide may be important in facilitating immune responses under certain circumstances.     

Surface-active properties of Candida albicans.

Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces.     

Anti-angiogenesis: making the tumor vulnerable to the immune system

Ongoing angiogenesis has been shown to possess immune suppressive activity through several mechanisms. One of these mechanisms is the suppression of adhesion receptors, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin-adhesion molecules involved in leukocyte interactions-on the vascular endothelium. This phenomenon, when happening to the tumor endothelium, supports tumor growth due to escape from immunity. Since angiogenesis has this immune suppressive effect, it has been hypothesized that inhibition of angiogenesis may circumvent this problem. In vitro and in vivo data now show that several angiogenesis inhibitors are able to normalize endothelial adhesion molecule expression in tumor blood vessels, restore leukocyte vessel wall interactions, and enhance the inflammatory infiltrate in tumors. It is suggested that such angiogenesis inhibitors can make tumors more vulnerable for the immune system and may therefore be applied to facilitate immunotherapy approaches for the treatment of cancer.     

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1–8 of mature glucoamylase greatly enhance excretion of both glucoamylase and β-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro . Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro .     

Screening of yeasts as probiotic based on capacities to colonize the gastrointestinal tract and to protect against enteropathogen challenge in mice

Probiotics are defined as viable microorganisms that exhibit a beneficial effect on the host's health when they are ingested. Two important criteria are used for selection of probiotic microorganisms: they must be able to survive in the gastrointestinal environment and to present at least one beneficial function (colonization resistance, immunomodulation or nutritional contribution). Generally, in vitro assays demonstrating these properties were used to select probiotics but it is unclear if the data can be extrapolated to in vivo conditions. In the present work, twelve Saccharomyces cerevisiae strains isolated from different environments (insect association, tropical fruit, cheese and "aguardente" production) and pre-selected for in vitro resistance to simulated gastrointestinal conditions were inoculated in germ-free mice to evaluate their real capacity to colonize the mammal digestive tract. Using these data, one of the yeasts (S. cerevisiae 905) was selected and tested in gnotobiotic (GN) and conventional (CV) mice for its capacity to protect against oral challenge with two enteropathogenic bacteria (Salmonella Typhimurium and Clostridium difficile). The yeast reached populational levels potentially functional in the gastrointestinal portions where the enteropathogens tested act. No antagonism against either pathogenic bacterium by the yeast was observed in the digestive tract of GN mice but, after challenge with S. Typhimurium, mortality was lower and liver tissue was better preserved in CV animals treated with the yeast when compared with a control group (p<0.05). Histopathological results of intestines showed that the yeast also presented a good protective effect against oral challenge with C. difficile in GN mice (p<0.05). In conclusion, among the 12 S. cerevisiae tested, strain 905 showed the best characteristics to be used as a probiotic as demonstrated by survival capacity in the gastrointestinal tract and protective effect of animals during experimental infections.     

Eap1p, an adhesin that mediates Candida albicans biofilm formation in vitro and in vivo

Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.     

T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria

In invertebrates, cellular and humoral components are evolved to maintain their body immunity and integrity. Both these factors respond to different antigens such as microorganisms, vertebrate erythrocytes and foreign proteins. In this article, we report a study of a lectin (HSL) involved in immune response in the echinoderm, sea cucumber (Holothuria scabra). Correlative studies indicate that the expression of this defensive lectin is induced by bacterial challenge, wherein cell wall glycoconjugates of bacteria are involved in lectin induction. HSL showed strong broad spectrum antibacterial activity against both gram-positive and gram-negative bacteria. Under in vitro conditions, purified HSL mediate agglutination of the test bacteria, there by indicating a possible mode of action in physiological situation.     

The viral killer system in yeast: from molecular biology to application

Since the initial discovery of the yeast killer system almost 40 years ago, intensive studies have substantially strengthened our knowledge in many areas of biology and provided deeper insights into basic aspects of eukaryotic cell biology as well as into virus-host cell interactions and general yeast virology. Analysis of killer toxin structure, synthesis and secretion has fostered understanding of essential cellular mechanisms such as post-translational prepro-protein processing in the secretory pathway. Furthermore, investigation of the receptor-mediated mode of toxin action proved to be an effective means for dissecting the molecular structure and in vivo assembly of yeast and fungal cell walls, providing important insights relevant to combating infections by human pathogenic yeasts. Besides their general importance in understanding eukaryotic cell biology, killer yeasts, killer toxins and killer viruses are also becoming increasingly interesting with respect to possible applications in biomedicine and gene technology. This review will try to address all these aspects.     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes,but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis

BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface. Here, deletion of the C-terminal EGF-like domain profoundly affected BAD1 function, leading to non-association with yeast, extracellular accumulation and impaired yeast adherence to macrophages. In equilibrium binding assays, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) compared with BAD1. Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF-like domain is critical for BAD1 interactions with chitin on yeast. DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains. However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis. Our results illustrate the previously undefined role of the EGF-like domain for BAD1 localization to yeast surfaces during cell wall biogenesis. They also demonstrate that the requirements for host cell binding and immune modulation by BAD1 can be dissociated from one another, and that the former is unexpectedly dispensable in the requisite role of BAD1 in pathogenesis.     

Influence of yeast quality on performance of gnotobiotically grown Artemia

Using axenically grown Artemia, a model system was developed to evaluate the effect of bacteria on the survival and development of this crustacean. Two strains of baker's yeast (Saccharomyces cerevisiae) were used in all experiments as feed for Artemia: a wild-type strain and its mnn9 mutant, defective in the synthesis of mannoproteins in the outer cell wall. The genetic background, yeast growth phase and growth medium appeared to be important parameters determining the quality of yeast cells as feed for Artemia. A strong positive correlation between Artemia performance and the yeast cell wall chitin and glucan content was obtained, while the mannoprotein content was negatively correlated. Mnn9 yeast cells grown till exponential phase in minimal medium proved to be excellent feed for Artemia, yielding an average 95% survival and 4-mm growth after 6 days at 28 °C, which is comparable to the best results obtained with algal feed. The standard growth test yields highly reproducible results and can become an excellent tool to study the mode of action of bacteria. Furthermore, yeast cell viability and the method used to kill/sterilize the cells are important parameters influencing nauplii performance.     

Insight into the antiadhesive effect of yeast wall protein 1 of Candida albicans

Ywp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall of Candida albicans; it is present in the yeast form of this opportunistic fungal pathogen but absent from filamentous forms and chlamydospores. Yeast cells that lack Ywp1 are more adhesive and form thicker biofilms, implying an antiadhesive activity for Ywp1, with a possible role in yeast dispersal. The antiadhesive effect of Ywp1 is transplantable from yeast to hyphae, as hyphae that are forced to express YWP1 lose adhesion in an in vitro assay. Deletion of the GPI anchor results in loss of Ywp1 to the surrounding medium and reduction of the antiadhesive effect, implying an importance of time-dependent residency in the cell wall. Anchor-negative versions of Ywp1 possessing or lacking a C-terminal green fluorescent protein (GFP) tag were created in C. albicans and harvested from culture supernatants; in addition to serving as quantifiable markers for Ywp1 secretion, they revealed that the cleaved 11-kDa propeptide of Ywp1 remains strongly but noncovalently associated with the Ywp1 core. This association is resistant to highly acidic and basic solutions, 8 M urea, and 1% SDS (below 45°C). Above 50°C, SDS dissociates the isolated complex, but even higher temperatures are required to dissociate the propeptide from native Ywp1 that is anchored in a cell wall. This property has permitted detection, for the first time, of orthologs of Ywp1 in other members of the Candida clade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.