BioProject

Purpose of the study The encounter between Candida albicans and phagocytes (macrophages) is considered the initial step in the development of host immune defenses. More...

Purpose of the study The encounter between Candida albicans and phagocytes (macrophages) is considered the initial step in the development of host immune defenses. The single utility of proteomic and genomic approaches to study host - pathogen interaction has been previously described in the literature (1, 2, 3); but up to now few studies have employed both techniques as complementary approaches. Because of the importance of the macrophages in the innate immune response against fungal infectious, we have investigated the transcriptional profiling and the differential protein expression (using a proteomic approach) of C. albicans after the interaction with these phagocytes. Description We have developed an in vitro system by employing the murine macrophage cell line RAW264.7 and the wild-type Candida albicans yeast strain SC5314. The study of the differential C. albicans protein expression in these conditions (3h. of interaction) has been performed by 2D-PAGE. The comparative analyse of the bidimensional silver stained gels obtained, was performed using the bioinformatic software Melanie 3. A complementary approach for this proteomic work is the study of the yeast transcriptional profiling after two time points of interaction (1.5 and 3h). We have analyzed differential C. albicans gene expression using Microarrays from Eurogentec. Results and conclusions A total of 132 protein species differentially expressed have been detected; 79 with increased expression and 53 with their expression decreased. The most interesting proteins up and down regulated, have been identified by MALDI-TOF TOF (59 proteins) and classified according to their biological function.The majority of these proteins belong to different metabolic pathways. Up-regulated proteins belong to oxidation of fatty acids and detoxification, meanwhile, other metabolic proteins belonging to glycolysis, fermentation and gluconeogenesis are clearly down-regulated. Proteins belonging to cell rescue, defence and virulence, proteosome and oxidative stress response are also up-regulated. A total of 240 C. albicans differentially expressed genes have been detected after 1,5 and 3h of macrophage interaction (122 over-expressed and 118 under-expressed). The yeast transcriptional response pointed out an increase in genes belonging to oxidative stress, metal homeostasis, DNA damage repair, lipid metabolism and pathogenesis and a down-regulation profile in genes belonging to cytoskeleton, morphogenesis, metabolism (carbohydrate, amino acid and nucleotide) and vesicular-vacuolar transport. The results obtained after this proteomic/genomic analyses indicate that a great percent of these proteins/genes belong to different cellular pathways showing the attempt of C. albicans to avoid being engulfed by macrophages, and once inside, points out the hostile environment that surrounds the yeast. References 1-Lorenz MC, Bender JA, Fink GR. Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell. 2004 Oct;3(5):1076-87. 2-Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B. Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol. 2005 Apr;56(2):397-415. 3- Martínez-Solano L, Nombela C, Molero G, and Gil C. Differential protein expression of murine macrophages upon interaction with Candida albicans. Proteomics, in press. Keywords: stress response Overall design: Hybridization protocol cDNA was synthesized from 20-30 microgrammes of total RNA by reverse transcription using CyScribe Post-Labeling kit, incorporating Cy3-cUTP or Cy5-dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. The amount of cDNA as well as the incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified on an Ultrospec 3300 Pro UV/ Visible spectrophotometer (Amersham Biosciences). Both labeled cDNA populations were combined, dried in a vacuum trap, and used as a hybridization probe after resuspension in hybridization solution and 100 micrograms Salmon Sperm. Slides were hybridized overnight with labeled probe at42 degrees in a water bath. Before scanning, the chips were washed and dried following the manufactures instructions Scan protocol Microarrays were scanned with a GenePix 4000B scanner (Axon Instruments. Union City, CA) at a resolution of 5um (PMT values ranging from 550 to 700 and laser power 100%) Description 18-24 hrs prior to initiation of the co-culture experiment, macrophages were collected and counted with a Neubauer chamber (hemocytometer). A total of 3,5 x 107 cells were plated in complete culture media in 750-ml cell culture flask and grown overnight at 37ºC in a humidified atmosphere with 5% CO2. C. albicans strain SC5314 was grown overnight at 30ºC on solid YED medium (to maintain cells in the yeast form). On day 1, these yeast cells were harvested, washed twice with phosphate buffered saline (PBS), counted and diluted to the desired density in 50 ml of complete culture media. 5 x 107 yeast cells were added per flask to obtain a fungus-macrophage ratio 1:1, (since repeated cell counts of the overnight macrophage cultures indicated a growth rate of 1,4) and incubated for 1.5 h. (for microarray experiments) and 3h. at 37°C and 5% CO2 . Non ingested and unbound Candida cells were removed by washing 3 times with ice-cold PBS. The macrophages and bounded/ingested yeast cells were dislodged by scraping the flask with rubber scrapers in ice-cold water, and pooled by centrifugation for 10 min at 4000 rpm.The resulting cell pellet was resuspended in a 0.25 % Triton X114 solution (50 mM Tris-HCl, 2mM EDTA, pH 7.5), vortexed 3 times and left/chilled on ice in order to eliminate most of the lysated macrophages. After 30 min, the sample was washed 4-6 times with ice-cold MilliQ water, in order to eliminate any trace of the detergent employed. Data processing GenePix Pro 4.0 analysis software (Axon Instruments) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to manufacturer instructions. Flagged spots and spots with an average intensity minus background below the media of the intensity minus background for all the non flagged spots in any channel (Cy3 or Cy5) were not retained for further analysis. Within this group those spots which showed in at least one channel a value of intensity minus background higher than 5 times the media of the background for all spots in the microarray for this channel were recovered. Two biological samples. Dye swap. Four microarrays per condition Less...