Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata

Namiki, Yuichi; Ueno, Keigo; Mitani, Hiroki; Virtudazo, Eric V.; Ohkusu, Misako; Shimizu, Kiminori; Kawamoto, Susumu; Chibana, Hiroji; Yamaguchi, Masashi

doi:10.1093/jmicro/dfq082

Cited by 14 publications

(9 citation statements)

References 12 publications

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“…The Lyticase buffer was applied to the cells before applying the cell fixation medium. The morphology of yeast cells should change significantly when converting them to protoplasts following the removal of the cell wall, exhibiting smooth cell surface with characteristic invaginations [48]. However, our results indicate no significant changes on the surface of Lyticase treated non-budding yeast cells after 15 min.…”

Section: Resultsmentioning

confidence: 52%

High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

et al. 2014

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Ultrastructural analysis of cells can reveal valuable information about their morphological, physiological, and biochemical characteristics. Scanning electron microscopy (SEM) has been widely used to provide high-resolution images from the surface of biological samples. However, samples need to be dehydrated and coated with conductive materials for SEM imaging. Besides, immobilizing non-adherent cells during processing and analysis is challenging and requires complex fixation protocols. In this work, we developed a novel dielectrophoresis based microfluidic platform for interfacing non-adherent cells with high-resolution SEM at low vacuum mode. The system enables rapid immobilization and dehydration of samples without deposition of chemical residues over the cell surface. Moreover, it enables the on-chip chemical stimulation and fixation of immobilized cells with minimum dislodgement. These advantages were demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment.

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Section: Resultsmentioning

confidence: 52%

High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

et al. 2014

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“…Also, KNH1 [28], which is involved in 1,6-glucan synthesis, was upregulated, while a 1,4-alpha-glucosidase (CAGL0G02717g) was downregulated. In addition to the glucan signature, CHS3B, a Class IV chitin synthase that has a calcineurin pathway- and Tor1p-dependent role in cell wall integrity [49, 50] was upregulated. Nevertheless, major quantitative or qualitative differences for content of chitin, β1,3-, and β1,6-glucans could not be established by employed biochemical methods.…”

Section: Resultsmentioning

confidence: 99%

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host

et al. 2017

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Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

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“…The measurement of plasma membrane fluidity was performed on yeast protoplasts containing 0.6 mM diphenylhexatriene (DPH), as described previously (Namiki et al 2011;Alexandre et al 1994). Octanoic acid was added to the protoplast suspension before…”

Section: Membrane Fluidity Measurementmentioning

confidence: 99%

Membrane stress caused by octanoic acid in Saccharomyces cerevisiae

Liu

Chernyshov

Najdi

et al. 2013

Appl Microbiol Biotechnol

112

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In order to compete with petroleum-based fuel and chemicals, engineering a robust biocatalyst that can convert renewable feedstocks into biorenewable chemicals, such as carboxylic acids, is increasingly important. However, product toxicity is often problematic. In this study, the toxicity of the carboxylic acids hexanoic, octanoic, and decanoic acid on Saccharomyces cerevisiae was investigated, with a focus on octanoic acid. These compounds are completely inhibitory at concentrations of magnitude 1 mM, and the toxicity increases as chain length increases and as media pH decreases. Transciptome analysis, reconstruction of gene regulatory network, and network component analysis suggested decreased membrane integrity during challenge with octanoic acid. This was confirmed by quantification of dose-dependent and chain length-dependent induction of membrane leakage, though membrane fluidity was not affected. This induction of membrane leakage could be significantly decreased by a period of pre-adaptation, and this preadaptation was accompanied by increased oleic acid content in the membrane, significantly increased production of saturated lipids relative to unsaturated lipids, and a significant increase in the average lipid chain length in the membrane. However, during adaptation cell surface hydrophobicity was not altered. The supplementation of oleic acid to the medium not only elevated the tolerance of yeast cells to octanoic acid but also attenuated the membrane leakiness. However, while attempts to mimic the oleic acid supplementation effects through expression of the Trichoplusia ni acyl-CoA Δ9 desaturase OLE1(TniNPVE desaturase) were able to increase the oleic acid content, the magnitude of the increase was not sufficient to reproduce the supplementation effect and increase octanoic acid tolerance. Similarly, introduction of cyclopropanated fatty acids through expression of the Escherichia coli cfa gene was not helpful for tolerance. Thus, we have provided quantitative evidence that carboxylic acids damage the yeast membrane and that manipulation of the lipid content of the membrane can increase tolerance, and possibly production, of these valuable products.

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Scanning and negative-staining electron microscopy of protoplast regeneration of a wild-type and two chitin synthase mutants in the pathogenic yeast Candida glabrata

Cited by 14 publications

References 12 publications

High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host

Membrane stress caused by octanoic acid in Saccharomyces cerevisiae

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