scholarly journals Overexpression of Candida albicans Secreted Aspartyl Proteinase 2 or 5 Is Not Sufficient for Exacerbation of Immunopathology in a Murine Model of Vaginitis

2017 ◽  
Vol 85 (10) ◽  
Author(s):  
Hubertine M. E. Willems ◽  
Winter S. Bruner ◽  
Katherine S. Barker ◽  
Junyan Liu ◽  
Glen E. Palmer ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Genetic Interaction ◽  
Constitutive Expression ◽  
Hyphal Growth ◽  
Expression Vectors ◽  
Symptomatic Infection ◽  
Content Type ◽  
Lactate Dehydrogenase Release ◽  
Morphogenetic Factors ◽  
Secreted Aspartyl Proteinase

ABSTRACT The secreted aspartyl proteinases of Candida albicans have long been implicated in virulence at the mucosal surface, including contributions to colonization and immunopathogenesis during vulvovaginal candidiasis. In an effort to disentangle hypha-associated virulence factor regulation from morphological transition, the purpose of this study was to determine if overexpression of SAP2 or SAP5 in an efg1Δ/Δ cph1Δ/Δ mutant could restore the capacity to cause immunopathology during murine vaginitis to this avirulent hypofilamentous strain. Two similar yet distinct genetic approaches were used to construct expression vectors to achieve SAP overexpression, and both genetic and functional assays confirmed elevated SAP activity in transformed strains. Similar to previous findings, intravaginal challenge of C57BL/6 mice with hypha-defective strains attained high levels of mucosal colonization but failed to induce robust vaginal immunopathology (neutrophil recruitment, interleukin-1β [IL-1β] secretion, and lactate dehydrogenase release) compared to that with the hypha-competent control. Moreover, constitutive expression of SAP2 or SAP5 in two distinct sets of such strains did not elicit immunopathological markers at levels above those observed during challenge with isogenic empty vector controls. Therefore, these results suggest that the physiological contributions of SAPs to vaginal immunopathology require hypha formation, other hypha-associated factors, or genetic interaction with EFG1 and/or CPH1 to cause symptomatic infection. Additionally, the outlined expression strategy and strain sets will be useful for decoupling other downstream morphogenetic factors from hyphal growth.

scholarly journals Candida albicans Ume6, a Filament-Specific Transcriptional Regulator, Directs Hyphal Growth via a Pathway Involving Hgc1 Cyclin-Related Protein

2010 ◽  
Vol 9 (9) ◽  
pp. 1320-1328 ◽  
Author(s):  
Patricia L. Carlisle ◽  
David Kadosh
Keyword(s):  
Candida Albicans ◽  
Molecular Mechanisms ◽  
Constitutive Expression ◽  
Transcriptional Regulator ◽  
Hyphal Growth ◽  
Related Protein ◽  
Content Type ◽  
Epistasis Analysis ◽  
Dependent Inactivation ◽  
Hyphal Formation

ABSTRACT The ability of Candida albicans, the most common human fungal pathogen, to transition from yeast to hyphae is essential for pathogenicity. While a variety of transcription factors important for filamentation have been identified and characterized, links between transcriptional regulators of C. albicans morphogenesis and molecular mechanisms that drive hyphal growth are not well defined. We have previously observed that constitutive expression of UME6, which encodes a filament-specific transcriptional regulator, is sufficient to direct hyphal growth in the absence of filament-inducing conditions. Here we show that HGC1, encoding a cyclin-related protein necessary for hyphal growth under filament-inducing conditions, is specifically important for agar invasion, hyphal extension, and formation of true septa in response to constitutive UME6 expression under non-filament-inducing conditions. HGC1-dependent inactivation of Rga2, a Cdc42 GTPase activating protein (GAP), also appears to be important for these processes. In response to filament-inducing conditions, HGC1 is induced prior to UME6 although UME6 controls the level and duration of HGC1 expression, which are likely to be important for hyphal extension. Interestingly, an epistasis analysis suggests that UME6 and HGC1 play distinct roles during early filament formation. These findings establish a link between a key regulator of filamentation and a downstream mechanism important for hyphal formation. In addition, this study demonstrates that a strain expressing constitutive high levels of UME6 provides a powerful strategy to specifically dissect downstream mechanisms important for hyphal development in the absence of complex filament-inducing conditions.

scholarly journals Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

2013 ◽  
Vol 13 (1) ◽  
pp. 2-9 ◽  
Author(s):  
Frans M. Klis ◽  
Chris G. de Koster ◽  
Stanley Brul
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Cell Size ◽  
Pathogenic Fungus ◽  
Turgor Pressure ◽  
Hyphal Growth ◽  
Biological Research ◽  
Content Type ◽  
Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

scholarly journals Overlapping Functions betweenSWR1Deletion and H3K56 Acetylation in Candida albicans

2015 ◽  
Vol 14 (6) ◽  
pp. 578-587 ◽  
Author(s):  
Zhiyun Guan ◽  
Haoping Liu
Keyword(s):  
Candida Albicans ◽  
Genetic Interaction ◽  
Regulation Of Gene Expression ◽  
Histone Variants ◽  
Content Type ◽  
Repressive Chromatin ◽  
Highly Sensitive ◽  
H3k56 Acetylation ◽  
A Subunit ◽  
Nucleosome Mapping

ABSTRACTNucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion ofSWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching inCandida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter ofWOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at theWOR1promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion ofYNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that ofswr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction betweenswr1and elevated H3K56ac with the discovery that theswr1deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects.

scholarly journals Adaptations of the Secretome of Candida albicans in Response to Host-Related Environmental Conditions

2015 ◽  
Vol 14 (12) ◽  
pp. 1165-1172 ◽  
Author(s):  
Frans M. Klis ◽  
Stanley Brul
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Human Body ◽  
Environmental Conditions ◽  
Surface Stress ◽  
Hyphal Growth ◽  
Content Type ◽  
General Response ◽  
Culture Supernatants

ABSTRACTThe wall proteome and the secretome of the fungal pathogenCandida albicanshelp it to thrive in multiple niches of the human body. Mass spectrometry has allowed researchers to study the dynamics of both subproteomes. Here, we discuss some major responses of the secretome to host-related environmental conditions. Three β-1,3-glucan-modifying enzymes, Mp65, Sun41, and Tos1, are consistently found in large amounts in culture supernatants, suggesting that they are needed for construction and expansion of the cell wall β-1,3-glucan layer and thus correlate with growth and might serve as diagnostic biomarkers. The genesENG1,CHT3, andSCW11, which encode an endoglucanase, the major chitinase, and a β-1,3-glucan-modifying enzyme, respectively, are periodically expressed and peak in M/G1. The corresponding protein abundances in the medium correlate with the degree of cell separation during single-yeast-cell, pseudohyphal, and hyphal growth. We also discuss the observation that cells treated with fluconazole, or other agents causing cell surface stress, form pseudohyphal aggregates. Fluconazole-treated cells secrete abundant amounts of the transglucosylase Phr1, which is involved in the accumulation of β-1,3-glucan in biofilms, raising the question whether this is a general response to cell surface stress. Other abundant secretome proteins also contribute to biofilm formation, emphasizing the important role of secretome proteins in this mode of growth. Finally, we discuss the relevance of these observations to therapeutic intervention. Together, these data illustrate thatC. albicansactively adapts its secretome to environmental conditions, thus promoting its survival in widely divergent niches of the human body.

scholarly journals Conjugated Linoleic Acid Inhibits Hyphal Growth in Candida albicans by Modulating Ras1p Cellular Levels and Downregulating TEC1 Expression

2011 ◽  
Vol 10 (4) ◽  
pp. 565-577 ◽  
Author(s):  
Julie Shareck ◽  
André Nantel ◽  
Pierre Belhumeur
Keyword(s):  
Candida Albicans ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Cellular Localization ◽  
Therapeutic Potential ◽  
Transcriptional Profiling ◽  
Hyphal Growth ◽  
Cellular Growth ◽  
Content Type ◽  
Protein Levels

ABSTRACTThe polymorphic yeastCandida albicansexists in yeast and filamentous forms. Given that the morphogenetic switch coincides with the expression of many virulence factors, the yeast-to-hypha transition constitutes an attractive target for the development of new antifungal agents. Since an untapped therapeutic potential resides in small molecules that hinderC. albicansfilamentation, we characterized the inhibitory effect of conjugated linoleic acid (CLA) on hyphal growth and addressed its mechanism of action. CLA inhibited hyphal growth in a dose-dependent fashion in both liquid and solid hypha-inducing media. The fatty acid blocked germ tube formation without affecting cellular growth rates. Global transcriptional profiling revealed that CLA downregulated the expression of hypha-specific genes and abrogated the induction of several regulators of hyphal growth, includingTEC1,UME6,RFG1, andRAS1. However, neitherUME6norRFG1was necessary for CLA-mediated hyphal growth inhibition. Expression analysis showed that the downregulation ofTEC1expression levels by CLA depended onRAS1. In addition, whileRAS1transcript levels remained constant in CLA-treated cells, its protein levels declined with time. With the use of a strain expressing GFP-Ras1p, CLA treatment was also shown to affect Ras1p localization to the plasma membrane. These findings suggest that CLA inhibits hyphal growth by affecting the cellular localization of Ras1p and blocking the increase inRAS1mRNA and protein levels. Combined, these effects should prevent the induction of the Ras1p signaling pathway. This study provides the biological and molecular explanations that underlie CLA's ability to inhibit hyphal growth inC. albicans.

scholarly journals Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

2014 ◽  
Vol 14 (2) ◽  
pp. 182-193 ◽  
Author(s):  
Areti Gkourtsa ◽  
Janny van den Burg ◽  
Karin Strijbis ◽  
Teja Avula ◽  
Sietske Bijvoets ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Associated Functions ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
And Function

ABSTRACT Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro . A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3 Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167 Δ/Δ and rvs161 Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans : the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

scholarly journals Linking Sfl1 Regulation of Hyphal Development to Stress Response Kinases in Candida albicans

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ohimai Unoje ◽  
Mengli Yang ◽  
Yang Lu ◽  
Chang Su ◽  
Haoping Liu
Keyword(s):  
Candida Albicans ◽  
Stress Response ◽  
Map Kinase ◽  
Hyphal Growth ◽  
Acidic Ph ◽  
Transcriptional Repressors ◽  
Growth Forms ◽  
Content Type ◽  
Hyphal Development ◽  
Study Results

ABSTRACT Candida albicans is an important human pathogen responsible for causing both superficial and systemic infections. Its ability to switch from the yeast form to the hyphal growth form is required for its pathogenicity. Acidic pH inhibits hyphal initiation, but the nature of the mechanism for this inhibition is not completely clear. We show that acidic pH represses hyphal initiation independently of the temperature- and farnesol-mediated Nrg1 downregulation. Using a collection of transcription factor deletion mutants, we observed that the sfl1 mutant induced hyphae in acidic pH but not in farnesol at 37°C. Furthermore, transcription of hyphal regulators BRG1 and UME6 was not induced in wild-type (WT) cells but was induced in the sfl1 mutant during hyphal induction in acidic pH. Using the same screening conditions with the collection of kinase mutants, we found that deletions of the core stress response mitogen-activated protein (MAP) kinase HOG1 and its kinase PBS2, the cell wall stress MAP kinase MKC1, and the calcium/calmodulin-dependent kinase CMK1 allowed hyphal initiation in acidic pH. Furthermore, Hog1 phosphorylation induced by high osmotic stress also retarded hyphal initiation, and the effect was abolished in the sfl1 and three kinase mutants but was enhanced in the phosphatase mutant ptp2 ptp3. We also found functional associations among Cmk1, Hog1, and Sfl1 for cation stress. Our study results suggest that robust hyphal initiation requires downregulation of both Nrg1 and Sfl1 transcriptional repressors as well as timely BRG1 expression. Acidic pH and cationic stress retard hyphal initiation via the stress-responsive kinases and Sfl1. IMPORTANCE Candida albicans is a commensal as well as a pathogen of humans. C. albicans is able to mount a cellular response to a diverse range of external stimuli in the host and switch reversibly between the yeast and hyphal growth forms. Hyphal development is a key virulence determinant. Here, we studied how C. albicans senses different environmental signals to control its growth forms. Our study results suggest that robust hyphal development requires downregulation of two transcriptional repressors, Nrg1 and Sfl1. Acidic pH or cationic stress inhibits hyphal formation via stress-responsive kinases and Sfl1.

scholarly journals Fungal Morphogenetic Pathways Are Required for the Hallmark Inflammatory Response during Candida albicans Vaginitis

2013 ◽  
Vol 82 (2) ◽  
pp. 532-543 ◽  
Author(s):  
Brian M. Peters ◽  
Glen E. Palmer ◽  
Andrea K. Nash ◽  
Elizabeth A. Lilly ◽  
Paul L. Fidel ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Inflammatory Responses ◽  
Lavage Fluid ◽  
Health Issues ◽  
Innate Immune Responses ◽  
Childbearing Age ◽  
Symptomatic Infection ◽  
Lactate Dehydrogenase Activity ◽  
Content Type

ABSTRACTVulvovaginal candidiasis, caused primarily byCandida albicans, presents significant health issues for women of childbearing age. As a polymorphic fungus, the ability ofC. albicansto switch between yeast and hyphal morphologies is considered its central virulence attribute. Armed with new criteria for defining vaginitis immunopathology, the purpose of this study was to determine whether the yeast-to-hypha transition is required for the hallmark inflammatory responses previously characterized during murine vaginitis. Kinetic analyses of vaginal infection withC. albicansin C57BL/6 mice demonstrated that fungal burdens remained constant throughout the observation period, while polymorphonuclear leukocyte (PMN), S100A8, and interleukin-1β levels obtained from vaginal lavage fluid increased by day 3 onward. Lactate dehydrogenase activity was also positively correlated with increased effectors of innate immunity. Additionally, immunodepletion of neutrophils in infected mice confirmed a nonprotective role for PMNs during vaginitis. Determination of the importance of fungal morphogenesis during vaginitis was addressed with a two-pronged approach. Intravaginal inoculation of mice withC. albicansstrains deleted for key transcriptional regulators (bcr1Δ/Δ,efg1Δ/Δ,cph1Δ/Δ, andefg1Δ/Δcph1Δ/Δ) controlling the yeast-to-hypha switch revealed a crucial role for morphogenetic signaling through the Efg1 and, to a lesser extent, the Bcr1 pathways in contributing to vaginitis immunopathology. Furthermore, overexpression of transcription factorsNRG1andUME6, to maintain yeast and hyphal morphologies, respectively, confirmed the importance of morphogenesis in generating innate immune responsesin vivo. These results highlight the yeast-to-hypha switch and the associated morphogenetic response as important virulence components for the immunopathogenesis ofCandidavaginitis, with implications for transition from benign colonization to symptomatic infection.

scholarly journals In Vitro Antibiofilm Activity of Eucarobustol E against Candida albicans

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Rui-Huan Liu ◽  
Zhi-Chun Shang ◽  
Tian-Xiao Li ◽  
Ming-Hua Yang ◽  
Ling-Yi Kong
Keyword(s):  
Candida Albicans ◽  
Antifungal Susceptibility ◽  
Hyphal Growth ◽  
Negative Regulator ◽  
Ergosterol Biosynthesis ◽  
Pcr Analysis ◽  
Antibiofilm Activity ◽  
Biofilm Inhibition ◽  
Content Type

ABSTRACT Formyl-phloroglucinol meroterpenoids (FPMs) are important types of natural products with various bioactivities. Our antifungal susceptibility assay showed that one of the Eucalyptus robusta-derived FPMs, eucarobustol E (EE), exerted a strong inhibitory effect against Candida albicans biofilms at a concentration of 16 μg/ml. EE was found to block the yeast-to-hypha transition and reduce the cellular surface hydrophobicity of the biofilm cells. RNA sequencing and real-time reverse transcription-PCR analysis showed that exposure to 16 μg/ml of EE resulted in marked reductions in the levels of expressions of genes involved in hyphal growth (EFG1, CPH1, TEC1, EED1, UME6, and HGC1) and cell surface protein genes (ALS3, HWP1, and SAP5). Interestingly, in response to EE, genes involved in ergosterol biosynthesis were downregulated, while the farnesol-encoding gene (DPP3) was upregulated, and these findings were in agreement with those from the quantification of ergosterol and farnesol. Combined with the obvious elevation of negative regulator genes (TUP1, NRG1), we speculated that EE's inhibition of carbon flow to ergosterol triggered the mechanisms of the negative regulation of hyphal growth and eventually led to biofilm inhibition.

scholarly journals Endosomal and AP-3-Dependent Vacuolar Trafficking Routes Make Additive Contributions to Candida albicans Hyphal Growth and Pathogenesis

2010 ◽  
Vol 9 (11) ◽  
pp. 1755-1765 ◽  
Author(s):  
Glen E. Palmer
Keyword(s):  
Candida Albicans ◽  
Double Mutant ◽  
Kidney Tissue ◽  
Hyphal Growth ◽  
Endosomal Trafficking ◽  
Transcriptional Responses ◽  
Transport Pathways ◽  
Content Type ◽  
Vacuolar Protein ◽  
Vacuolar Trafficking

ABSTRACT Candida albicans mutants deficient in vacuolar biogenesis are defective in polarized hyphal growth and virulence. However, the specific vacuolar trafficking routes required for hyphal growth and virulence are unknown. In Saccharomyces cerevisiae, two trafficking routes deliver material from the Golgi apparatus to the vacuole. One occurs via the late endosome and is dependent upon Vps21p, while the second bypasses the endosome and requires the AP-3 complex, including Aps3p. To determine the significance of these pathways in C. albicans hyphal growth and virulence, aps3Δ/Δ, vps21Δ/Δ, and aps3Δ/Δ vps21Δ/Δ mutant strains were constructed. Analysis of vacuolar morphology and localization of the vacuolar protein Mlt1p suggests that C. albicans Aps3p and Vps21p mediate two distinct transport pathways. The vps21Δ/Δ mutant has a minor reduction in hyphal elongation, while the aps3Δ/Δ mutant has no defect in hyphal growth. Interestingly, the aps3Δ/Δ vps21Δ/Δ double mutant has dramatically reduced hyphal growth. Overexpression of the Ume6p transcriptional activator resulted in constitutive hyphal growth of wild-type, aps3Δ/Δ, and vps21Δ/Δ strains and formation of highly vacuolated subapical compartments. Thus, Ume6p-dependent transcriptional responses are sufficient to induce subapical vacuolation. However, the aps3Δ/Δ vps21Δ/Δ mutant formed mainly pseudohyphae that lacked vacuolated compartments. The aps3Δ/Δ strain was virulent in a mouse model of disseminated infection; the vps21Δ/Δ mutant failed to kill mice but persisted within kidney tissue, while the double mutant was avirulent and cleared from the kidneys. These results suggest that while the AP-3 pathway alone has little impact on hyphal growth or virulence, it is much more significant when endosomal trafficking is disrupted.

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