scholarly journals Recognition of Yeast by Murine Macrophages Requires Mannan but Not Glucan

2010 ◽  
Vol 9 (11) ◽  
pp. 1776-1787 ◽  
Author(s):  
Sabine Keppler-Ross ◽  
Lois Douglas ◽  
James B. Konopka ◽  
Neta Dean
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Early Recognition ◽  
Yeast Cells ◽  
Phagocytic Cells ◽  
Fungal Cell ◽  
Content Type ◽  
Murine Macrophages ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACT The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.

scholarly journals Insight into the Antiadhesive Effect of Yeast Wall Protein 1 of Candida albicans

2012 ◽  
Vol 11 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Bruce L. Granger
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Surrounding Medium ◽  
Yeast Cells ◽  
Content Type ◽  
A Cell ◽  
Opportunistic Fungal Pathogen ◽  
Green Fluorescent

ABSTRACTYwp1 is a prominent glycosylphosphatidylinositol (GPI)-anchored glycoprotein of the cell wall ofCandida 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 expressYWP1lose adhesion in anin vitroassay. 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 inC. albicansand 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 theCandidaclade. The cleaved propeptide, which carries the sole N-glycan of Ywp1, must participate in the antiadhesive effect of Ywp1.

scholarly journals Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Lytic Bacteriophage ◽  
Cell Wall Synthesis ◽  
Content Type ◽  
Ftsz Ring ◽  
Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals FLO11Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

2013 ◽  
Vol 79 (19) ◽  
pp. 6023-6032 ◽  
Author(s):  
Marc Bou Zeidan ◽  
Lourdes Carmona ◽  
Severino Zara ◽  
Jose F. Marcos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Antimicrobial Peptides ◽  
Gene Knockout ◽  
Yeast Cells ◽  
Knockout Mutant ◽  
Fungal Cell ◽  
Content Type ◽  
Cell Penetrating ◽  
Flor Yeasts

ABSTRACTSaccharomyces cerevisiae“flor” yeasts have the ability to form a buoyant biofilm at the air-liquid interface of wine. The formation of biofilm, also called velum, depends onFLO11gene length and expression.FLO11encodes a cell wall mucin-like glycoprotein with a highly O-glycosylated central domain and an N-terminal domain that mediates homotypic adhesion between cells. In the present study, we tested previously known antimicrobial peptides with different mechanisms of antimicrobial action for their effect on the viability and ability to form biofilm ofS. cerevisiaeflor strains. We found that PAF26, a synthetic tryptophan-rich cationic hexapeptide that belongs to the class of antimicrobial peptides with cell-penetrating properties, but not other antimicrobial peptides, enhanced biofilm formation without affecting cell viability in ethanol-rich medium. The PAF26 biofilm enhancement required a functionalFLO11but was not accompanied by increasedFLO11expression. Moreover, fluorescence microscopy and flow cytometry analyses showed that the PAF26 peptide binds flor yeast cells and that aflo11gene knockout mutant lost the ability to bind PAF26 but not P113, a different cell-penetrating antifungal peptide, demonstrating that theFLO11gene is selectively involved in the interaction of PAF26 with cells. Taken together, our data suggest that the cationic and hydrophobic PAF26 hexapeptide interacts with the hydrophobic and negatively charged cell wall, favoring Flo11p-mediated cell-to-cell adhesion and thus increasing biofilm biomass formation. The results are consistent with previous data that point to glycosylated mucin-like proteins at the fungal cell wall as potential interacting partners for antifungal peptides.

scholarly journals The Interaction between Candida krusei and Murine Macrophages Results in Multiple Outcomes, Including Intracellular Survival and Escape from Killing

2011 ◽  
Vol 79 (6) ◽  
pp. 2136-2144 ◽  
Author(s):  
Rocío García-Rodas ◽  
Fernando González-Camacho ◽  
Juan Luis Rodríguez-Tudela ◽  
Manuel Cuenca-Estrella ◽  
Oscar Zaragoza
Keyword(s):  
Cell Line ◽  
Actin Polymerization ◽  
Candida Krusei ◽  
Yeast Cells ◽  
Phagocytic Cells ◽  
Intrinsic Resistance ◽  
Specific Staining ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Murine Macrophages

ABSTRACTCandida kruseiis a fungal pathogen of interest for the scientific community for its intrinsic resistance to fluconazole. Little is known about the interaction of this yeast with host immune cells. In this work, we have characterized the outcome of the interaction betweenC. kruseiand murine macrophages. OnceC. kruseiwas internalized, we observed different phenomena. In a macrophage-like cell line,C. kruseisurvived in a significant number of macrophages and induced filamentation and macrophage explosion. Phagocytosis ofC. kruseiled to actin polymerization around the yeast cells at the site of entry. Fluorescent specific staining with anti-Lamp1 and LysoTracker indicated that after fungal internalization, there was a phagolysosome maturation defect, a phenomenon that was more efficient when the macrophages phagocytosed killed yeast cells. Using cell line macrophages, we also observed macrophage fusion after cell division. When we used primary resident peritoneal macrophages in addition to macrophage explosion, we also observed a strong chemotaxis of uninfected macrophages to regions whereC. krusei-infected macrophages were present. We also noticed yeast transfer phenomena between infected macrophages. Primary macrophages inhibited pseudohypha elongation more efficiently than the macrophage-like cell line, suggesting thatC. kruseiinfection was better controlled by the former macrophages. Primary macrophages induced more tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) than the macrophage-like cell line. Our results demonstrate thatC. kruseican exploit the macrophages for replication, although other different outcomes are also possible, indicating that the interaction of this pathogen with phagocytic cells is very complex and regulated by multiple factors.

scholarly journals Role of the ESCRT Pathway in Laccase Trafficking and Virulence of Cryptococcus neoformans

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Yoon-Dong Park ◽  
Shu Hui Chen ◽  
Emma Camacho ◽  
Arturo Casadevall ◽  
Peter R. Williamson
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Extracellular Vesicle ◽  
Multivesicular Bodies ◽  
Melanin Production ◽  
Content Type ◽  
Endosomal Sorting ◽  
Green Fluorescent

ABSTRACT The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans. Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect is not due to defects in actin-related localization. Extracellular vesicle maturation was also defective in the vps27Δ mutant, which had a larger vesicle size as measured by dynamic light scattering. Our data identify cryptococcal VPS27 as a required gene for laccase trafficking and attenuates virulence of C. neoformans in a mouse intravenous (i.v.) meningitis model.

scholarly journals Chitinases Are Essential for Cell Separation in Ustilago maydis

2015 ◽  
Vol 14 (9) ◽  
pp. 846-857 ◽  
Author(s):  
Thorsten Langner ◽  
Merve Öztürk ◽  
Sarah Hartmann ◽  
Stefan Cord-Landwehr ◽  
Bruno Moerschbacher ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Separation ◽  
Morphological Changes ◽  
Ustilago Maydis ◽  
Yeast Cells ◽  
In Planta ◽  
Fungal Cell ◽  
Content Type ◽  
Chitinolytic Enzymes ◽  
Complete Life Cycle

ABSTRACTChitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such asUstilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a myceliumin planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity ofU. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

scholarly journals Targeted Gene Disruption of OsCERK1 Reveals Its Indispensable Role in Chitin Perception and Involvement in the Peptidoglycan Response and Immunity in Rice

2014 ◽  
Vol 27 (9) ◽  
pp. 975-982 ◽  
Author(s):  
Yusuke Kouzai ◽  
Susumu Mochizuki ◽  
Keisuke Nakajima ◽  
Yoshitake Desaki ◽  
Masahiro Hayafune ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Leaf Sheath ◽  
Cell Wall Component ◽  
Fungal Cell ◽  
Receptor Like Kinase ◽  
Green Fluorescent ◽  
Molecular Patterns ◽  
Two Hybrid

OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.

scholarly journals Rapid Redistribution of Phosphatidylinositol-(4,5)-Bisphosphate and Septins during the Candida albicans Response to Caspofungin

2012 ◽  
Vol 56 (9) ◽  
pp. 4614-4624 ◽  
Author(s):  
Hassan Badrane ◽  
M. Hong Nguyen ◽  
Jill R. Blankenship ◽  
Shaoji Cheng ◽  
Binghua Hao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Regulatory Protein ◽  
Wall Material ◽  
Pleckstrin Homology Domain ◽  
Calcofluor White ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTWe previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintainingCandida albicanscell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response ofC. albicansto caspofungin. First, we studied caspofungin-hypersusceptibleC. albicans irs4andinp51mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5′-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes,irs4andinp51mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants.irs4andinp51were linked most closely to thegin4mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-typeC. albicansto caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those ofirs4,inp51, andgin4mutants. Taken together, our data suggest that theC. albicansIrs4-Inp51 5′-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

scholarly journals Highly Dynamic and Specific Phosphatidylinositol 4,5-Bisphosphate, Septin, and Cell Wall Integrity Pathway Responses Correlate with Caspofungin Activity against Candida albicans

2016 ◽  
Vol 60 (6) ◽  
pp. 3591-3600 ◽  
Author(s):  
Hassan Badrane ◽  
M. Hong Nguyen ◽  
Cornelius J. Clancy
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fluorescent Protein ◽  
Cell Wall Integrity ◽  
Ph Domain ◽  
Calcofluor White ◽  
Content Type ◽  
Pathway Activation ◽  
Cell Wall Integrity Pathway ◽  
Green Fluorescent

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] activates the yeast cell wall integrity pathway.Candida albicansexposure to caspofungin results in the rapid redistribution of PI(4,5)P2and septins to plasma membrane foci and subsequent fungicidal effects. We studiedC. albicansPI(4,5)P2and septin dynamics and protein kinase C (PKC)-Mkc1 cell wall integrity pathway activation following exposure to caspofungin and other drugs. PI(4,5)P2and septins were visualized by live imaging ofC. albicanscells coexpressing green fluorescent protein (GFP)-pleckstrin homology (PH) domain and red fluorescent protein-Cdc10p, respectively. PI(4,5)P2was also visualized in GFP-PH domain-expressingC. albicans mkc1mutants. Mkc1p phosphorylation was measured as a marker of PKC-Mkc1 pathway activation. Fungicidal activity was assessed using 20-h time-kill assays. Caspofungin immediately induced PI(4,5)P2and Cdc10p colocalization to aberrant foci, a process that was highly dynamic over 3 h. PI(4,5)P2levels increased in a dose-response manner at caspofungin concentrations of ≤4× MIC and progressively decreased at concentrations of ≥8× MIC. Caspofungin exposure resulted in broad-based mother-daughter bud necks and arrested septum-like structures, in which PI(4,5)P2and Cdc10 colocalized. PKC-Mkc1 pathway activation was maximal within 10 min, peaked in response to caspofungin at 4× MIC, and declined at higher concentrations. The caspofungin-induced PI(4,5)P2redistribution remained apparent inmkc1mutants. Caspofungin exerted dose-dependent killing and paradoxical effects at ≤4× and ≥8× MIC, respectively. Fluconazole, amphotericin B, calcofluor white, and H2O2did not impact the PI(4,5)P2or Cdc10p distribution like caspofungin did. Caspofungin exerts rapid PI(4,5)P2-septin and PKC-Mkc1 responses that correlate with the extent ofC. albicanskilling, and the responses are not induced by other antifungal agents. PI(4,5)P2-septin regulation is crucial in early caspofungin responses and PKC-Mkc1 activation.

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