scholarly journals Small extracellular vesicles secreted by Candida albicans hyphae have highly diverse protein cargoes that include virulence factors and stimulate macrophages

2020 ◽  
Author(s):  
Raquel Martínez-López ◽  
Maria Luisa Hernáez ◽  
Esther Redondo ◽  
Guillermo Calvo ◽  
Sonja Radau ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Extracellular Vesicles ◽  
Virulence Factors ◽  
Ribosomal Proteins ◽  
Protein Transport ◽  
Surface Proteins ◽  
Immune Reactivity ◽  
Exosome Biogenesis ◽  
Virulence Trait ◽  
Human Sera

ABSTRACTExtracellular vesicles (EVs) have been described as mediators of microorganism survival and interaction with the host. In Candida albicans, a relevant commensal fungal pathogen, the dimorphic transition is an important virulence trait in candidiasis. We have analyzed EVs secreted by yeast (YEVs) or hyphal cells (HEVs) from C. albicans, finding interesting differences in both size distribution and protein loading. In general, HEVs were smaller and carried a much more diverse protein cargo than YEVs, including most of the proteins identified in YEVs, which were mainly cell surface proteins. Virulence factors such as phospholipases, aspartic proteases (Saps), adhesins and invasins, and the precursor protein of candidalysin toxin Ece1p were also detected. HEVs were also enriched in proteasomal and ribosomal proteins, and in enzymes from amino acid biosynthetic pathways, all involved in protein metabolism, as well as proteins related to intracellular protein transport and components of the ESCRT pathway related to exosome biogenesis. Both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis. In our conditions, only HEVs could elicit the release of TNFα by activated macrophages. This first analysis of C. albicans HEVs shows their relevance to pathogenesis and possible new diagnostics or treatments.

scholarly journals Tell me what type of extracellular vesicles you secrete, and I will tell you who you are: yeast or hypha

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Raquel Martínez-López ◽  
Catarina Vaz ◽  
Esther Redondo ◽  
Guillermo Calvo ◽  
María Luisa Hernáez ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ribosomal Proteins ◽  
Culture Medium ◽  
Surface Proteins ◽  
Cellular Transport ◽  
Tem Analysis ◽  
Host Interaction ◽  
Human Sera ◽  
Great Relevance ◽  
Shed Light

The transition between yeast and hyphal morphologies plays a crucial role in the pathogenicity of Candida albicans. Recent studies have pointed out the great relevance of extracellular vesicles (EVs) secreted by microorganisms in a wide variety of biological processes including interaction with the host. Therefore, the main objective of this work was to compare the EVs secreted by yeast and hyphal forms to shed light on C. albicans-host interaction. EVs were obtained by ultracentrifugation of the culture medium supernatant and analysed by mass spectrometry. They were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS). DLS and TEM analysis showed that yeast EVs were significantly bigger than hyphal EVs, being most of them in the range between 400 to 500nm while hyphal EVs were ranged mostly around 100-200nm. Proteomic analysis showed greater protein diversity in hyphal EVs when compared to yeast EVs (up to 1700 different proteins identified versus 300), although less amount of total protein was obtained. Gene Ontology (GO) analysis showed that yeast EVs were enriched in surface proteins while hyphal EVs, although containing also most of these surface proteins, were also significantly and exclusively enriched in proteins involved in protein metabolism (ribosomal proteins, many aminoacid-pathway enzymes and proteasome) and cellular transport. The differences between YEVs and HEVs also prompted a different immune host response, as tested with macrophage cell cultures and human sera from patients with invasive candidiasis. All these differences point out a possible different biogenesis and roles of EVs secreted by both morphologies.

scholarly journals Gene Overexpression/Suppression Analysis of Candidate Virulence Factors of Candida albicans

2008 ◽  
Vol 7 (3) ◽  
pp. 483-492 ◽  
Author(s):  
Yue Fu ◽  
Guanpingsheng Luo ◽  
Brad J. Spellberg ◽  
John E. Edwards ◽  
Ashraf S. Ibrahim
Keyword(s):  
Candida Albicans ◽  
Virulence Factors ◽  
Surface Proteins ◽  
Screening Tests ◽  
Vaginal Candidiasis ◽  
Loss Of Function ◽  
Genes Encoding ◽  
Gpi Proteins ◽  
Protein Cell

ABSTRACT We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.

scholarly journals A202 ROLE OF EXTRACELLULAR VESICLES IN GIARDIA-MICROBIOTA INTERACTIONS.

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 226-228
Author(s):  
A Siddiq ◽  
T Allain ◽  
G Dong ◽  
M Olivier ◽  
A Buret
Keyword(s):  
Epithelial Cells ◽  
Extracellular Vesicles ◽  
Virulence Factors ◽  
Proteomic Analysis ◽  
Diarrheal Disease ◽  
Cysteine Proteases ◽  
Surface Proteins ◽  
Commensal Bacteria ◽  
Swimming Motility ◽  
And Behavior

Abstract Background Extracellular vesicles (EVs) have emerged as important mediators of host-parasite interactions. Studies in various parasites have shown that EVs have important roles in pathogenesis. The protozoan parasite Giardia duodenalis, which causes diarrheal disease, produces EVs in response to changes in the environment. However, their exact role in the pathogenesis of giardiasis remains unclear. In this study, we investigate the effects of Giardia EVs on the gut commensal bacteria. EVs mediated disruption of the microbiota represents a novel mechanism that has not been investigated before. Aims We hypothesize that Giardia EVs contain virulence factors that will modify the growth and behavior of commensal bacteria. Based on our hypothesis, the study has three aims: 1. Quantify and characterize the EVs produced by Giardia under varying conditions. 2. Examine the effects of Giardia EVs on commensal bacteria. 3. Identify proteins and virulence factors in Giardia EVs using proteomics. Methods G. duodenalis (isolate NF) was exposed to bile or left untreated and the EVs were isolated using Exo-Easy Maxi Kit (Qiagen). The isolated EVs were characterized using Nanosight track analysis (NTA) and transmission electron microscopy. To examine the effects of EVs on bacteria, we used a lab strain E.coli HB101 and human isolate E. cloacae. The bacteria were incubated with Giardia EVs and their growth rate was monitored using a bacterial kinetics assay. The swimming motility of bacteria exposed to Giardia EVs was assessed on a 0.3% agar. Additionally, the ability of the EVs treated bacteria to adhere to epithelial cells was also examined. Proteomic analysis of EVs was conducted using liquid chromatography with tandem mass spectrometry. Results Results indicate that bile treated Giardia trophozoites produce more EVs as compared to untreated trophozoites. Giardia EVs exerted bacteriostatic effects on E.coli HB101 and E. cloacae. Additionally, Giardia EVs significantly increased the swimming motility of both E.coli HB101 and E. cloacae as well as their adhesion to the intestinal epithelial cells. Finally, proteomic analysis of EVs revealed the presence of well-characterized Giardia virulence factors such as cysteine proteases, tenascins, variant surface proteins as well as metabolic enzymes such as arginine deaminase and ornithine carbamoyltransferase. Conclusions Our research highlights a novel mechanism of Giardia’s interaction with commensal bacteria. Our results indicate that Giardia can release EVs in response to changes in the environment. These EVs also contain virulence factors that can modify the growth and behavior of commensal bacteria. These effects may have significant implications in disease pathophysiology. The results of the study can also be applied to other parasitic diseases where the production of EVs has been shown to play a role in the pathogenesis process. Funding Agencies NSERC

scholarly journals Isolation of Small Extracellular Vesicles from Human Sera

2021 ◽  
Vol 22 (9) ◽  
pp. 4653
Author(s):  
Małgorzata S. Małys ◽  
Christof Aigner ◽  
Stefan M. Schulz ◽  
Helga Schachner ◽  
Andrew J. Rees ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Human Serum ◽  
Extracellular Vesicles ◽  
Plasma Lipids ◽  
Surface Proteins ◽  
Nanoparticle Tracking Analysis ◽  
Disease Biomarkers ◽  
Exclusion Chromatography ◽  
Human Sera ◽  
Specific Proteins

Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.

A comparative analysis of protein virulence factors released via extracellular vesicles in two Candida albicans strains cultivated in a nutrient-limited medium

2019 ◽  
Vol 136 ◽  
pp. 103666 ◽  
Author(s):  
Klára Konečná ◽  
Jana Klimentová ◽  
Oldřich Benada ◽  
Ivana Němečková ◽  
Ondřej Janďourek ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Comparative Analysis ◽  
Extracellular Vesicles ◽  
Virulence Factors

scholarly journals Comparative Molecular and Immunoregulatory Analysis of Extracellular Vesicles from Candida albicans and Candida auris

mSystems ◽  
2021 ◽  
Vol 6 (4) ◽  
Author(s):  
Daniel Zamith-Miranda ◽  
Heino M. Heyman ◽  
Sneha P. Couvillion ◽  
Radames J. B. Cordero ◽  
Marcio L. Rodrigues ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Extracellular Vesicles ◽  
Virulence Factors ◽  
Nosocomial Infections ◽  
Pathogenic Fungus ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type

Candida auris is a recently described multidrug-resistant pathogenic fungus that is responsible for outbreaks across the globe, particularly in the context of nosocomial infections. Its virulence factors and pathogenesis are poorly understood.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Candida Albicans Virulence Factors and Its Pathogenicity

2021 ◽  
Vol 9 (4) ◽  
pp. 704
Author(s):  
Mariana Henriques ◽  
Sónia Silva
Keyword(s):  
Candida Albicans ◽  
Virulence Factors ◽  
Healthy Individuals ◽  
Mucosal Surfaces

Candida albicans lives as commensal on the skin and mucosal surfaces of the genital, intestinal, vaginal, urinary, and oral tracts of 80% of healthy individuals [...]

scholarly journals TUP1, CPH1 and EFG1 Make Independent Contributions to Filamentation in Candida albicans

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Burkhard R Braun ◽  
Alexander D Johnson
Keyword(s):  
Candida Albicans ◽  
Environmental Conditions ◽  
Target Genes ◽  
Single Cells ◽  
Filamentous Growth ◽  
Surface Proteins ◽  
Pathway Expression ◽  
Separate Pathway ◽  
The Common ◽  
Growth Pathway

Abstract The common fungal pathogen, Candida albicans, can grow either as single cells or as filaments (hyphae), depending on environmental conditions. Several transcriptional regulators have been identified as having key roles in controlling filamentous growth, including the products of the TUP1, CPH1, and EFG1 genes. We show, through a set of single, double, and triple mutants, that these genes act in an additive fashion to control filamentous growth, suggesting that each gene represents a separate pathway of control. We also show that environmentally induced filamentous growth can occur even in the absence of all three of these genes, providing evidence for a fourth regulatory pathway. Expression of a collection of structural genes associated with filamentous growth, including HYR1, ECE1, HWP1, ALS1, and CHS2, was monitored in strains lacking each combination of TUP1, EFG1, and CPH1. Different patterns of expression were observed among these target genes, supporting the hypothesis that these three regulatory proteins engage in a network of individual connections to downstream genes and arguing against a model whereby the target genes are regulated through a central filamentous growth pathway. The results suggest the existence of several distinct types of filamentous forms of C. albicans, each dependent on a particular set of environmental conditions and each expressing a unique set of surface proteins.

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