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TRANSCRIPT PROFILING OF CANDIDA ALBICANS IN BLOOD AND BLOOD FRACTIONS

TRANSCRIPT PROFILING OF CANDIDA ALBICANS IN BLOOD DEPLETED OR NOT OF CD15⁺ CELLS

Introduction

Despite the emergence of other Candida species as causes of invasive infection, C. albicans remains the leading cause of life threatening disseminated candidosis. Although several mechanisms and attributes by which C. albicans can colonize and penetrate host tissue have been described, it is not known how this fungus can survive in human blood and escape from blood vessels as an essential step in dissemination of infection. This fungus is polymorphic, with cells developing as yeast, pseudohyphal or true hyphal forms. There are two major pathways by which C. albicans may enter the bloodstream: penetration from mucosal surfaces into blood vessels or direct transmission via intravascular catheters.

Blood is a complex and — to microorganisms — a hostile milieu composed of several types of immunoactive cells and molecules to which C. albicans has to adapt and respond. Furthermore, to cause disseminated infections, the fungal cells need mechanisms to escape from the bloodstream by invasion of endothelial cells and their surrounding tissues. In a previous report we began to elucidate how C. albicans responds to the challenge of the blood environment (Fradin et al., 2003) . The expression patterns provided evidence that the fungus is able to adapt very quickly to a new environment. In addition, we were able to show that blood cells significantly influence the expression of certain fungal genes. 

To elucidate the influence of blood components on fungal growth and transcript profile during bloodstream infections, we exposed C. albicans to blood and fractions enriched in erythrocytes, polymorphonuclear or mononuclear leukocytes and plasma. C. albicans cells were also exposed to blood depleted of CD15⁺ cells, which are mainly neutrophils. 

The data provided on this page belong to the study "Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood" performed by Chantal Fradin, Piet de Groot, Donna Mac Callum, Martin Schaller, Frans Klis, Frank C. Odds and Bernhard Hube and published in Mol. Microbiol. 56:397-415 (2005).

This work was supported by the European Commission (QLK2-2000-00795).

Download the 2 tables with normalized data for differentially expressed genes:

Table corresponding to transcript profiling of Candida albicans in blood and blood fractions

Table corresponding to transcript profiling of Candida albicans in blood depleted or not of CD15+ cells

Go to all microarray data

Experiment design

(i) Comparison of Candida albicans transcript profiles in blood and different blood fractions (RC (Red Cells), PMN (Polymorphonuclear cells), MNC (Mononuclear cells) and plasma). 

(ii) Comparison of C. albicans transcript profiles in blood depleted of CD15 positive cells (CD15^-) and untreated blood(CD15⁺).

 

Inoculation of C. albicans with human samples 30 minutes at 37¡C.

Three biological replicate RNA samples were used for hybridization. Thus a total of 12 hybridizations were performed for (i) and 3 hybridizations for (ii). The three replicates include a dye swap. For (i) an extra hybridization was performed to check the correlation for the co-hybridization of the same sample.

In order to show that the major observations are independent of the blood donor, Candida albicans transcript profiles in blood and plasma from single and pooled donors were compared. Go to scatter plot analysis.

C. albicans strain and preculture 

Strain SC5314 (Gillum et al., 1984) was grown overnight in YPD (10g/L Yeast Extract, 10g/L Peptone and 20g/L glucose) at 37¡C.

Human blood, human blood fractions and inoculation

Blood was taken as described in Fradin et al., 2003.

(i)Red blood cells, polymorphonuclear cells and mononuclear cells were isolated from human blood by gradient density centrifugation in Histopaque 1077 and 1119 (Sigma). The different blood cells were resuspended in plasma obtained from the same blood by reconstituting their original concentration in blood.

(ii)CD15⁺ cells were depleted from fresh human blood with Dynabeads M-450 CD15 (Dynal) according to the manufacturer's instructions, except that blood was not diluted. Fresh blood was treated under the same condition but without dynabeads, corresponding to untreated blood.

Candida cells were inoculated in blood, plasma, or blood depleted of CD15⁺ cells and in the 3 different blood fractions and incubated for 30 min at 37¡C. The cells were collected and shock frozen. 

RNA isolation and labeling

RNA were isolated as described in Fradin et al., 2005.

mRNA were isolated and Cy3/Cy5 cRNA were obtained by linear amplification as described here. Additional information on Agilent Fluorescent Linear Amplification kit can be found here.

Hybridization procedures

as described here.

Array design

            Microchip features: C. albicans glass microarrays with probes for 5907 genes as described here

            Microchip manufacturer: Eurogentec SA

Measurement data and specifications

            Imaging: GenePix 4.1

            Data capture: GenePix 4.1

            Normalisation method: Intensity-dependent method (Lowess) in GeneSpring v 6.0

            Analysis: one-way analysis of variance (ANOVA) test with a P-value cut-off of 0.05

All microarray data 

* see experiment design

** stripped slides were used

 

Scatter plot analysis

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Fungal Biology and Pathogenicity
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gf2@pasteur.fr

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