Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

In Saccharomyces cerevisiae, withdrawal of the carbon source results in detachment of glycolytic enzymes from the cytoskeleton and in actin reorganization.

Metabolons are dynamic associations of enzymes catalyzing consecutive reactions within a given pathway. Association results in enzyme stabilization and increased metabolic efficiency. Metabolons may use cytoskeletal elements, membranes and membrane proteins as scaffolds. The effects of glucose withdrawal on a putative glycolytic metabolon/F-actin system were evaluated in three Saccharomyces cerevisiae strains: a WT and two different obligate fermentative (OxPhos-deficient) strains, which obtained most ATP from glycolysis. Carbon source withdrawal led to inhibition of fermentation, decrease in ATP concentration and dissociation of glycolytic enzymes from F-actin. Depending on the strain, inactivation/reactivation transitions of fermentation took place in seconds. In addition, when ATP was very low, green fluorescent protein-labeled F-actin reorganized from highly dynamic patches to large, non-motile actin bodies containing proteins and enzymes. Glucose addition restored fermentation and cytoskeleton dynamics, suggesting that in addition to ATP concentration, at least in one of the tested strains, metabolon assembly/disassembly is a factor in the control of the rate of fermentation.

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments.

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes.

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.

Effectiveness of UV-C light assisted by mild heat on Saccharomyces cerevisiae KE 162 inactivation in carrot-orange juice blend studied by flow cytometry and transmission electron microscopy.

The aim of this study was to analyze the effectiveness of UV-C light (0-10.6 kJ/m2) assisted by mild heat treatment (50 °C) on the inactivation of Saccharomyces cerevisiae KE 162 in peptone water and fresh carrot-orange juice blend (pH: 3.8; 9.8°Brix; 707 NTU; absorption coefficient: 0.17 cm-1). Yeast induced damage by single UV-C and mild heat (H) and the combined treatment UV-C/H, was investigated by flow cytometry (FC) and transmission electron microscopy (TEM). When studying induced damage by FC, cells were labeled with fluorescein diacetate (FDA) and propidium iodide (PI) to monitor membrane integrity and esterase activity. UV-C/H provoked up to 4.7 log-reductions of S. cerevisiae; whereas, only 2.6-3.3 log-reductions were achieved by single UV-C and H treatments. FC revealed a shift with treatment time from cells with esterase activity and intact membrane to cells with permeabilized membrane. This shift was more noticeable in peptone water and UV-C/H treated juice. In the UV-C treated juice, double stained cells were detected, suggesting the possibility of being sub-lethally damaged, with compromised membrane but still metabolically active. TEM images of treated cells revealed severe damage, encompassing coagulated inner content, disorganized lumen and cell debris. FC and TEM provided additional information regarding degree and type of damage, complementing information revealed by the traditional plate count technique.

Impact of a combined processing technology involving ultrasound and pulsed light on structural and physiological changes of Saccharomyces cerevisiae KE 162 in apple juice.

This study analyzed the effect of single ultrasound (US) (600 W, 20 kHz and 95.2 µm wave amplitude, 10 or 30 min at 20 or 44 ± 1 °C), or combined with pulsed light technology (PL) with controlled heat build-up (Xenon lamp, 3 pulses/s, 71.6 J/cm2, temperature ranges: 2-20 ± 1 °C and 44-56 ± 1 °C) on the inactivation of Saccharomyces cerevisiae KE 162 cells in commercial (pH: 3.5 ± 0.1; 12.5 ± 0.1 °Brix) and freshly pressed (pH: 3.4 ± 0.1; 12.6 ± 0.1 °Brix) apple juices. Structural damages were analyzed by transmission electronic microscopy (TEM) and induced damage by flow cytometry (FC). Cells were labeled with fluorescein diacetate (FDA) and propidium iodide (PI) for monitoring membrane integrity and esterase activity. US+PL treatment at the highest heat build-up led up to 6.4 and 5.8 log-cycles of yeast reduction in commercial and freshly apple juices, respectively. TEM images of treated cells revealed severe damage, encompassing loss and coagulated inner content and cell debris. In addition, FC revealed a shift of yeasts cells with esterase activity and intact membrane to cells with permeabilized membrane. This effect was more notorious after single 30-min US and all combined US+PL treatments, as 91.6-99.0% of treated cells showed compromised membrane. Additionally, heat build-up enhanced this shift when applying 10 min US (20 °C) in both juices.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

BACKGROUND: Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction. METHODS: ApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8µM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. GENERAL SIGNIFICANCE: We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.

Npa3/ScGpn1 carboxy-terminal tail is dispensable for cell viability and RNA polymerase II nuclear targeting but critical for microtubule stability and function.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

Fisetin yeast-based bio-capsules via osmoporation: effects of process variables on the encapsulation efficiency and internalized fisetin content.

Osmoporation is an innovative method that can be used with food-grade yeast cells of Saccharomyces cerevisiae as natural encapsulating matrices. This technique overcomes barriers that difficult encapsulation and enables the internalization of fragile bioactive molecules such as fisetin into yeasts. In the present study, we assessed the effects of concentration, osmotic pressure, and temperature on the encapsulation efficiency (EE) and internalized fisetin content (IF). Two different quantification strategies were investigated: direct extraction (DE) without cell washing or freeze-drying steps and indirect extraction (IE) performed after washings with ethanol and freeze-drying. Our results showed that osmoporation improved EE (33 %) and IF (1.199 mg). The best experimental conditions were found by using DE. High-resolution images showed that the yeast cell envelope was preserved during osmoporation at 30 MPa and 84 % of yeast cells remained viable after treatment. Washing cells with organic solvent led to decreased EE (0.65 %) and IF (0.023 mg). This was probably due to either damages caused to yeast cell envelope or fisetin dragged out of cell. Overall, the results demonstrated the adequacy and relevant biotechnological potential of yeasts as encapsulating matrices for hydrophobic compounds. This fresh biotechnological approach has proven to be a promising tool for the production of bioactive-rich food products.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

Brewer's yeast and sugarcane yeast as protein sources for dogs.

Brewer's yeast (BY), autolysed sugarcane yeast (ASCY) and integral sugar cane yeast (ISCY) were studied in two experiments as ingredients for dog diets. In the first experiment, 28 dogs were randomly assigned to four diets; one reference diet and three test diets containing 15% of BY, ASCY or ISCY and 85% of the reference diet (as-fed basis). The digestibilities of the yeasts were calculated by the substitution method. In the second experiment, 35 dogs were randomized to five diets with similar chemical composition but different levels of sugarcane yeast inclusion (0%, 7.5% ASCY, 15% ASCY, 7.5% ISCY and 15% ISCY). In both experiments, the coefficient of total tract apparent digestibility (CTTAD) of nutrients was determined through total collection of faeces. During experiment, two additional analyses of food palatability, nitrogen balance and urea postprandial responses were performed. The data were submitted to analysis of variance, and the means were compared by orthogonal or polynomial contrasts or Tukey's test (p < 0.05). In experiment 1, CTTAD of protein was lower for both sugarcane yeasts than for BY (p = 0.012), as was metabolizable energy content (p = 0.025). In experiment 2, a linear reduction in energy digestibility with ASCY inclusion (p = 0.05) was verified. Furthermore, faecal score and DM content were reduced with ISCY inclusion (p < 0.003). No effect of yeast inclusion on nitrogen balance or postprandial urea response was found. Also, the inclusion of 7.5% of ASCY or ISCY increased diet palatability (p < 0.01). Yeasts present adequate digestibility by dogs, but its effect on faecal formation needs to be considered. No clear advantage for the use of ASCY over ISCY was found. In conclusion, we find that sugarcane yeast is suitable for inclusion in dog food and can enhance the overall palatability of the diet.

Short transmembrane domains with high-volume exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex.

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the Golgi SNARE protein Sft1 and the plasma membrane SNARE protein Sso1 from Saccharomyces cerevisiae as model proteins, we modified the length of their TMDs and the volume of their exoplasmic hemi-TMD, and determined their subcellular localization both in yeast and mammalian cells. We found that short TMDs with high-volume exoplasmic hemi-TMDs confer Golgi membrane residence, whereas TMDs with low-volume exoplasmic hemi-TMDs, either short or long, confer plasma membrane residence to these proteins. Results indicate that the shape of the exoplasmic hemi-TMD, in addition to the length of the entire TMD, determine retention in the Golgi or exit to the plasma membrane of Type II membrane proteins.

The essential gene YMR134W from Saccharomyces cerevisiae is important for appropriate mitochondrial iron utilization and the ergosterol biosynthetic pathway.

A thermosensitive strain (YMR134W(ts)) of the essential gene YMR134W presented up to 40% less ergosterol, threefold lower oxygen consumption and impaired growth on respiratory conditions. The iron content in the mitochondrial fraction of YMR134W(ts) cells was considerably low, despite these cells uptake and accumulate more iron from the culture media than wild-type cells. YMR134W(ts) cells were also more susceptible to oxidative stress. The results suggest that Ymr134wp is essential to aerobic growth due to its function in ergosterol biosynthesis, playing a role in maintaining mitochondrial and plasma membrane integrity and consequently impacting the iron homeostasis, respiratory metabolism and antioxidant response.

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness.

AIMS: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B1 binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. METHODS AND RESULTS: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. CONCLUSIONS: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin-adsorbent interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.

Capsicum annuum L. trypsin inhibitor as a template scaffold for new drug development against pathogenic yeast.

A 6,000 Da peptide, named CaTI, was isolated from Capsicum annuum L. seeds and showed potent inhibitory activity against trypsin and chymotrypsin. The aim of this study was to determine the effect of CaTI on Saccharomyces cerevisiae, Candida albicans, Candida tropicalis and Kluyveromyces marxiannus cells. We observed that CaTI inhibited the growth of S. cerevisiae, K. marxiannus as well as C. albicans and induced cellular agglomeration and the release of cytoplasmic content. No effect on growth was observed in C. tropicalis but morphological changes were noted. In the spot assay, different degrees of sensitivity were shown among the strains and concentrations tested. Scanning electron microscopy showed that S. cerevisiae, K. marxiannus and C. albicans, in the presence of CaTI, exhibited morphological alterations, such as the formation of pseudohyphae, cellular aggregates and elongated forms. We also show that CaTI induces the generation of nitric oxide and interferes in a dose-dependent manner with glucose-stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells.

Using the residue of spirit production and bio-ethanol for protein production by yeasts.

The residue (vinasse) formed during the distillation of bio-ethanol and cachaça, a traditional rum-type spirit produced from sugar-cane in Brazil, is highly harmful if discharged into the environment due to high values of COD and BOD. One possibility for minimizing the impact of vinasse in soils and waters is to use the residue in the production of microbial biomass for use as an animal feed supplement that will provide high levels on nitrogen (>9% d.m.) and low content of nucleic (≤ 10% d.m.) This paper reports the production and quality of biomass produced from fermentation of Saccharomyces cerevisiae and Candida parapsilosis in culture media under 12 different culture conditions and the respective effects of each variable (glucose, yeast extract, peptone, potassium phosphate, vinasse, pH and temperature). Of the S. cerevisiae isolates tested, two (VR1 and PE2) originating from fuel alcohol-producing plants were identified as offering the best potential for the industrial production of single cell protein from vinasse due to highest biomass productivity. Our results showed a potential viable and economic use of vinasse.

Scanning electron and confocal scanning laser microscopy imaging of the ultrastructure and viability of vaginal Candida albicans and non- albicans species adhered to an intrauterine contraceptive device.

Although bacterial biofilms have been studied in detail, adhesion of Candida albicans and non-albicans species to an intrauterine contraceptive device (IUD) is not clear. The objective of this study was to evaluate aspects of imaging of the ultrastructure and viability of vaginal yeasts adhered to different parts of an IUD, through scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). We studied yeasts isolated from different patients with vulvovaginal candidiasis: C. albicans, C. glabrata, C. guillermondii, C. parapsilosis, C. tropicalis, and Saccharomyces cerevisiae. A suspension of the each yeast was prepared and incubated with IUD parts (tail, without copper, and copper-covered). SEM and CSLM showed that all the vaginal yeasts adhered to all the parts of the IUD and demonstrated viability, including 30 days after contact for C. albicans. Possibly irregularities of IUD surface contribute to the adherence process. Although all of the IUD parts contribute to retention of yeasts in the genital tract, high concentration of yeast cells on the tail may indicate the importance of this segment in maintaining the colonization by yeast cells because the tail forms a bridge between the external environment, the vagina that is colonized by yeast cells, and the upper genital tract where there is no colonization.

Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

BACKGROUND: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.