1: Construction of DNA microarrays for gene expression profiling concerning the pathogenesis of C. albicans
a. We have completed the construction of a set of DNA microarray of Candida albicans as well as the tuning of noise and normalization of the background of the data obtained from the array.
b. We have established a standard experimental procedure and operation platform for employment of the DNA microarray.
In order to study the response of C. albicans to its host environments and the functions of virulence factors of Candida albicans, we have set out to generate DNA microarrays representative of the whole genome of this pathogen. In the first year of this grant period, we have focused on the construction of DNA microarrays, optimization of sample preparation and microarray processing and facilitating practice for data analysis (an overview see Fig. 1). We have reached several goals that would provide important foundation for the continuing experiments in the next stages of the study.
(1) Construction C. albicans DNA microarrays. We have constructed the spotter arrays for C. albicans. The QIAGEN Operon C. albicans Array-Ready Oligo Set (AROS) (v. 1.2) containing 6,266 probes and C. albicans AROS upgrade set (v. 1.1) containing 1,659 probes has been used. These 70mer oligo sets contain a total of 7,925 optimized probes that represent the entire genome of C. albicans and more than 10 different controls. The oligo sets have been also successfully used in the studies of different aspects of C. albicans biology and pathogenesis.
The C. albicans oligos were spotted onto the UltraGAPS coated slides (Corning, New York, NY) with the use of the OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor,MI),according to themanufacturer’sinstructions.To ensurethequality of the microarray printing, one or two microarrays of each batch are randomly chosen and are labed with the Cy5 fluorescent dye. The quality of the test spotted microarrays is determined by visualization. At this stage, we have generated less than one hundred such C. albicans spotted arrays.
(2) Optimization of experimental protocols for RNA isolation, sample labeling and microarray hybridization. In addition to the quality of microarray per se, another important issues in microarray studies is the source and quality of the RNA used. To this end, we have examined various protocols for RNA isolation from either the yeast (balstspore) or filamentous cells of C. albicans. For the yeast form of C.
albicans, a single colony of the clinically isolated wild-type strain SC5314 was inoculated into 10 ml of YPD medium and grown overnight at 30°C. The overnight cultures were harvested by centrifugation and the cell pellets were washed twice with sterile water. The same amount of cells (OD600= 0.05) were inoculated into YPD liquid medium and incubated at 30°C. To obtain hyphael cells, equivalent overnight cultures (OD600= 0.1) were inoculated into Lee's and YPD + 10% bovine serum and incubated at 37°C. Both yeast and filament forms of C. albicans were grown until the cells reached early to mid-exponential phase. At each sampling, each culture was immediately collected by centrifugation at 4°C, and the cell pellets were snap-frozen in an ethanol-dry ice bath and stored at −80°C. Total RNA was then extracted as previously described (Lan et al., 2004). Briefly, the cell pellets were resuspended in 3 ml of extraction buffer (0.1M LiCl, 0.02M dithiothreitol, 0.1M Tris-HCl, pH 7.5) and vortexed. Then, the cell suspension was added to an ice-cold tube containing 6g of acid-washed glass beads, 0.5 ml of 10% SDS, 5ml of phenol-holoroform. The mixture was vortexed for five minutes to disrupt the cells. After centrifugation, the upper aqueous phase of the mixture was transferred to a fresh tube. Finally, the samples were extracted by phenol/cholorform twice, ethanol precipitation, treated with RQ1 DNase (Promega, Madison, WI) for 1 h and cleaned by using an RNeasy Column (QIAGEN, Valencia, CA). For the hyphal cells, the protocol for cell disruption were slightly modified: the lysed cells were disrupted by five cycles or vortexing (3 min each);
between cycles the tubes were incubated alternatively for 3 min at 42oC and then at 0oC.
The quality and quantity of total RNA are determined using Bioanalyzer 2100 (Agilent Technologies) and/or ND-1000 spectrophotometer (NanoDrop Inc).
The optimal conditions for sample labeling and microarray hybridization are currently working in-progress. Briefly, cDNA containing a T7 RNA polymerase recognition sequence are prepared from total RNA amplified with T7 RNA polymerase. After processing, blunt-ended cDNAs are used as templates to produce antisense RNA using a T7 Ampliscribe kit. A second round of double-stranded cDNA synthesis is performed in the presence of random hexamer primers and reverse transcriptase; aminoallyl-dUTP. The Cy3-/Cy5-dyes are incorporated into single-stranded cDNAs with monofunctional NHS esters that bind to free amino groups. Un-incorporated dyes are removed by ultrafiltration using a Centricon 30 unit
(Amicon). DNA microarrays will be analyzed using Axon 4000B scanner (Axon Technologies). A representative of these tested microarrays is shown in Fig 2. In this figure, the overall good distributions, fine shapes of each oligo spots and fine hybridization are demonstrated.
(3) Practice toward statistical analysis for expression profiles. Microarray data could be noisy and no single statistical method has been chosen as a standard approach for the data normalization. We have collected some of the data sets in the public domain and used these available data to practice for the statistical analysis. We have practiced for the three major steps of data analysis: (1) image processing (2) statistical analysis for differential expressed genes, and (3) clustering genes with similar time profiles. For image processing, we were focused on the most widely adopted software, GenePix Pro. With these practice, we try to extract reliable signals even though our budget can only support for the spotted microarrays, which may not be the best platform comparing to the commercially available ones. For statistical analysis of significant genes, we have used classical linear model approach in the JMP®Genomics software. Several results of our practices are shown in Fig 3.
In conclusion, our DNA microarray is ready for our study in the second year.
Figure 1. An overview for working on the C. albicans oligo microarrays and data analysis.
Microarray scan & data analysis
Total RNA
Synthesis of ds-cDNA
CyDye coupling to aa-aRNA Purify Labeled aa-aRNA
UV Cross-linking
Baking for 6 hrs
Pre-Hybridization 42C, 1hr
Hybridization 65C, 16hr
Wash II
Pre-warm prehybridization buffer, 42C, 1hr
Wash I
Prepare pre-hyb wash buffer I
DNase I treatment
Synthesis of
purification
purification
Prepare wash buffer II pre-warm at 65C
Sample preparation
DNA microarrays
(A) (B)
(A) (B)
Figure 2. A representative of the C. albicans spotted oligo microarrays. (A) The whole view of a test microarray with sample hybridized. (B) A portion of the microarray is enlarged and shown.
(A) (B)
Figure 3. A parctice for the DNA microarray data analysis. (A) Scatter plot for two pairs of arrays. The upper panel shows a scatter plot for two arrays that belong to different treatments and the lower panel shows a scatter plot for two arrays that belong to biological replicates of the same treatment. (B) Principal component analysis for time course data. In the upper panel, the first two principal components clearly separate the time profiles of a few hundreds of genes. The lower panel shows the clustering results with different groups indicated by different colors.
2: Unveiling the molecular involvement of Efg1 pathway in the pathogenesis of C. albicans
a. We have constructed Caeno1/Caeno1 homozygous mutant for the purpose of understand the effects of the null mutation on C. albicans.
b. We have performed deletion analysis on CaENO1 for the purpose of determination of the signals on CaEno1 responsible for its various localities.
CaEno1 is a multi-functional glycolytic protein regulated by Efg1 and it is also a major component of cell wall as well as a secreted antigen in Candida infection. Its location in the cell is related to its function and subjected to the regulation of Efg1.
Hence, understand the signal and mechanism will help to reveal the role of the Efg1 pathway and the controlling mechanism of pathogenesis. The approach we used is the mutagenesis analysis to unveil its functions.
In the first year of this research, our effort is to construct CaENO1 mutants for functional study. We started by constructing the knockout mutation of CaENO1 to understand the role of CaEno1p in morphogenesis/virulence. At the same time, we also fused various sequence fragments of CaENO1 to reporter GFP to monitor the location of the GFP to determine the sequences necessary for the various locations of CaEno1p, which is in connection to the virulence and pathogenesis of C. albicans.
(1). Constructing Caeno1/Caeno1 homozygous mutant and to determine the effects on C. albicans.
Due to the diploid-only nature of C. albicans and the lack of known plasmid, the homozygous mutation on CaENO1 is in fact a null mutation constructed by knock-out procedure based on homologous replacement. An SAT1 expression cassette was flanked by the 5’and 3’CaENO1 sequences of CaENO1, introduced by restriction cloning (Fig. 4). This recombinant DNA fragment was then introduced into C.
albicans SC5314. Transformants were selected for nourseothricin resistance expressed by SAT1. In those cells, the SAT1 cassette replaces the center portion of CaENO1 ORF after homologous recombination. However, the SAT1 cassette contains two FRT sequences, which will then recombine to pup out the SAT1
sequence, leaving truncated CaENO1 with only the 5’and 3’sequences. After PCR and Southern blot confirmation (Fig. 5), this heterozygous mutant is subjected for second round knock-out to remove the second allele of CaENO1. The resultants were assessed again by PCR and Southern analysis (Fig. 5). Since CaENO1 is known to be the sole gene encoding enolase in C. albicans, its null mutant will not be able to grow on media containing glucose, which is what has been observed (Fig. 6).
In short, we have obtained the homozygous mutant strain and are in the process of
characterizing the effects on the phenotypes involved in morphogenesis and virulence.
Fig. 4. Genomic construction of CaENO1 null mutation. Acc1 is used to treat genomic DNA for Southern analysis.
Fig. 5. Southern Analysis of CaENO1 locus in various strains.
Fig. 6. CaENO1 null mutant can not grow on YPD. YCH78: wild-type CaENO1/CaENO1 strain; YCH61: eno1Δ::FRT/eno1Δ::FRT; YCH81: wild-type CaENO1/CaENO1 strain.
(2). Investigating the signals on CaEno1 responsible for its various cellular localizations.
The attempt is to perform deletion analysis to narrow the sequences responsible for the locations of CaENO1 till the minimal sequences are defined. However, due to the difficulty of molecular manipulation in C. albicans, our strategy is to perform the deletion analysis in the baker yeast first, and then assess the result in C. albicans.
Therefore, we have fused the sequence of CaENO1 to EFGP in a S. cerevisiae expression vector (Fig. 7) and then determined whether the CaEno1p expressed in S.
cerevisiae could still be retained in cytoplasm as well as secreted into media.
Plasmids carrying the secretion signal of-factor fused to the 5’of EGFP and plasmids carrying the EGFP gene alone serve as controls. The cells transformed with plasmids carrying the sequence of CaENO1-EGFP fusion can be seen to express EGFP (Fig. 8A), so does the plasmids carrying EGFP (Fig. 8B). Hence, the fusion of EGFP and CaENO1 can be expressed and the EGFP can serve as the reporter. In addition, cells transformed with plasmids carrying-factor secretion signal fused to the 5’of EGFP was also expressed but a lower intensity (Fig. 8C). One possible reason for the lower intensity is that the fusion proteins were indeed secreted. Hence, we decided to collect the media from the culture and analyzed with Western blot against EGFP. As it is shown in figure 9, Western analysis was able to reveal a band with the size of about 75 kD in the culture media of cells carrying the sequence of CaENO1-EGFP fusion. Hence, the CaEno1p-EGFP is secreted into media as the wild-type CaEno1p shall have. We are now completed the construction of various truncated CaENO1 fused with EGFP to determine whether different portions of the sequence can direct EGFP to different cellular location.
Fig 7. Schematic representation of fusion construct of CaENO1 on baker yeast expression vector YEP363.
Fig 8. Plasmid-based CaENO1-EGFP can express in S. cerevisiae. Cells carrying different plasmids were observed under fluorescence microscope. A, cells contain plasmid carrying CaENO1-EGFP; B, cells contain plasmid carrying EGFP alone; C, cells contain plasmid carrying secretion signal-EGFP.
A. B. C.
Fig. 9. Western analysis revealed that CaENO1-EGFP can be detected in media.
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