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INTRODUCTION
Microbial contamination of cells, including Mycoplasma infection, is a frequent problem in the study of cultured cells (estimated frequency varying from 5% to 35%) (1). The contamination of cells influences cell-growth and causes unexpected cell-reactions. It also changes a wide array of immunological, biochemical and biological properties of the cells without apparent change in morphology of cell. In addition Mycoplasma is highly contagious and can rapidly spread through the cell stocks. The possible consequences of Mycoplasma infection for the host-cells are many and varied, ranging from no apparent effect to extensive changes which include inhibition of cell proliferation, induction of apoptosis, induction of cytokines and oxidative radicals, and malignant transformation (2-4). There is also a possibility that Mycoplasma biological activities may be interpreted erroneously as being of host origin (5).
Microbial contamination, however, is often difficult to detect as the contaminated culture grows well and appears normal by ordinary light microscopy. In human, the Mycoplasma may also lead to genitourinary and neonatal infections (6). In addition, Mycoplasma have been implicated in the pathogenesis of AIDS (7) and rheumatoid arthritis (8), although their precise contribution is still under debate. Understanding the molecular basis of a host's response to microbial infection is essential for preventing disease and tissue damage as a result of the inflammatory response. A better understanding of this process should allow for the design of drugs that can more specifically and effectively target infected cells with reduced side-effects. The host pathogen interaction can result in changes to the host cell which includes modulation of RNA expression, target receptor induction, actin cytoskeletal rearrangements, signal transduction pathway activation, and vacuolar trafficking (3, 9).
DNA microarray technology has enabled us to describe a unique biological phenomenon in terms of genome-wide gene expression analysis (10). It can provide a detailed insight into observed phenomenon as well as complete list of the genes involved. Gene expression profiling using DNA microarray offers the potential to define patterns of gene expression during normal biological or aberrant disease processes. Moreover, many of differentially expressed genes that may play an integral role in these processes can be identified.
In this paper, we have utilized spotted oligonucleotide microarray methodology to examine the expression of 10,416 known regulatory genes following microbial infection. We compared the distribution of patterns of gene expression in keratinocytes and chondrocytes. We also compared gene expression patterns at day 1, day 3, and day 10 post-infection. We selected 30 genes that were expressed differentially in whole experimental samples, as biomarkers for microbial infection. The model for the prediction of cell infection is also discussed.
MATERIALS AND METHODS
Cell culture and experimental design
Human keratinocytes and chondrocytes were cultured and infected with 8 types of Mycoplasma (M. hyorhinis, M. orale, M. arthritidis, M. bovis, M. pirum, M. pulminis, M. salivarium, M. neurolyticumv), fungus (Candida albicans), bacteria (Staphylococcus aureus) or Adenovirus. Cells were collected after 1 day, 3 days and 10 days depending on the source of infection. For C. albicans cells were collected after 1 day, for S. aureus and adenovirus cells were collected after 3 days, and for Mycoplasma keratinocytes were collected after 3 days or 10 days. In total 62 samples were analyzed; 37 samples of keratinocytes and 25 samples of chondrocytes. RNA from these infected cells was marked with Cy5 and the RNA from uninfected cells was marked with Cy3. Fig. 1 shows experimental design of our study in 3 dimensional structures. The x axis means source of infection (12 different types of source of infection), y axis means cell types (keratinocyte or chondrocyte) and z axis means day of culture (1 day, 3 days or 10 days). The color of cube means a biological repetition. The dark blue means the triplicate sample. The light blue means that there is no biological repetition.
RNA extraction and oligonucleotide microarray
Total RNA from control or microbial-infected cells was used for experiments done in triplicate. Experiments were performed using the microarray system (Oligo-Human 10K, Macrogen Inc., Seoul, Korea) according to the manufacturer's protocol. Briefly, 100 µg of total RNA was labelled by incubation with Cyanine-3-dUTP (6.0 mM) or Cyanine-5-dUTP (4.0 mM) (Perkin Elmer Life Sciences, Waltham, MA, USA), dNTP mixture, 0.1 M DTT, RnaseOUT, inorganic pyrophosphatase and reverse transcriptase at 40℃ for 3 hr. Before hybridization 8 µg of Cyanine-3-CTP labelled cRNA and 8 µg of Cyanine-5-CTP labelled cRNA were mixed with 2.5 µL of Mouse Cot-1 DNA (Invitrogen, Carlsbad, CA, USA), 2.5 µL of Deposition control target (Operon Technologies, Alameda, CA, USA) and 12.5 µL of 2 × hybridization buffer (Agilent Technologies, Santa Clara, CA, USA). Cy3 and Cy5 fluorescent intensity was determined using the GenePix scanner (Axon Instruments, Union City, CA, USA) and images were analyzed using the built-in software to calculate relative ratios and to determine confidence intervals.
Microarray data analysis and marker genes identification
Fluorescence intensity was processed and measured using GenePix Pro software (Axon Instruments) and intensity data were imported to the Xperanto in-house microarray database (11). Variance stabilizing normalization (12) was applied using the 'vsn' package in Bioconductor with the R statistical package. After performing intensity-dependent global LOWESS regression, spatial and intensity dependent effects were managed by pin-group LOWESS normalization using the method of Yang et al. (13).
Each sample represents a specific condition of infection (see Fig. 1) taking into account of infected cell type, source of infection and collection time after infection. We can regard the differentially expressed genes for a given sample as a response to the specific conditions of infection. We used the log-odds value for each gene (14) to identify differentially expressed genes for each sample (single chip). In order to select a gene list for combined samples, we use an average of log-odds values in each sample and the number 1.0 as a threshold value to define differentially expressed genes. Contrary to simple and widely-used 2-fold method, Newton's method relies on the average intensity of the varying critical zone which takes into consideration the uncertainty of small-intensity areas. From the definition of logodds value (14), almost all genes have negative log-odds values and genes with high values have high probabilities of being differentially expressed. An infection score function L(g) for a gene g, is defined as the average of log-odds values under a given set of samples. This generates the equation
where log-odds(g, s) is the log-odds value for a gene g under the sample s, and s1, ..., sK represent all samples within our experimental design or a subset of samples which are of particular interest. The score L(g) of a gene g represents the degree to which the gene g is associated with the general process of infection with respect to the samples s1, ..., sK.
The top ranking genes g1, ..., gn were selected using the Score L(g) that defined them as possible marker genes for microbial infection. Using the marker genes g1, ..., gn, we defined the microbial contamination index (MCI) for a given sample (details are in supplement pages). To determine optimal number of marker genes (n) we performed a leave-one-out cross-validation method and obtained cross-validation score, named cross-validation (CV). The high score of CV means that the marker genes are consistent to predict microbial infection. Based on the CV score, we select the number of genes which has highest CV score. Statistical analyses were done using R/Bioconductor package. Using this MCI and CV score, we also select group of marker genes to predict source of infection, cell type and infection time.
RESULTS
Overall distribution of differentially expressed genes with regard to the conditions of cell-infection
We examined the gene expression profiles of microbial-infected cells using Oligo-Human 10K chips. In order to select differentially expressed genes, we use the log-odds values of each sample. Table 1 shows an overall distribution of differentially expressed genes following infection by Mycoplasma, Fungus, Bacteria and Virus. Up-regulated and down-regulated genes were numbered separately and samples were divided according to cell type.
Fig. 2 shows a dendrogram of samples clustered using the Hierarchical clustering algorithm with centered-correlation and the average linkage method. From over 10,000 candidate genes, the 2,465 genes were selected for clustering which had no constant expression pattern with respect to the samples. Statistics V(g), were calculated for each gene g, defined by the variance of expression of gene g over a median of all variance of gene expressions. A total of 2,465 genes satisfying a V(g) chi-square value of P < 0.001 were selected.
All of the replicated samples had a strong correlation with infection and hence the reliability of experiment could be considered sufficiently high. We also discovered that differential gene expression patterns exist depending on the cell types assayed and that the tree could be divided into two sub-trees for keratinocytes and chondrocytes. With regard to the time point of the type of the assay post infection, we can conclude that the gene expression pattern was similar regardless of the type of infection.
Selection of marker genes for microbial infection
In this section we have elucidated the genes associated with a general microbial infection process and suggest a model to predict whether a sample is infected or not. According to our prediction model associated with whole samples, the marker genes for cell infection are NM_005298, NM_016408, NM_014588, S76389, and NM_001853 (See Tables 2 and 3). Also, the cross-validation score of CV(n*)=3.01>1.0 confirmed that the marker genes were correct. Fig. 3 shows the CV score according to the number of marker genes for detecting microbial infection (A) and Mycoplasma specific infection (B). We selected the number of genes which has highest CV score.
We also built a model to determine whether the origin of infection was a species of Mycoplasma. We let Lmyco(g) and Lnon-myco(g) be the infection scores associated with 48 Mycoplasma infection samples and 14 non-Mycoplsma infection samples respectively. With a newly defined infection score L(g)=Lmyco(g)-Lnon-myco(g), we found two marker genes S76389 and NM_001853 by applying the cross-validation model. Three marker genes (NM_005298, NM_014588, NM_001853) and four marker genes (NM_005298, NM_016408, NM_014588, S76389) were selected to build prediction model for detecting keratinocyte or chondrocyte cell type, respectively. We also build prediction model for an early infected cell using following five marker genes; NM_005298, NM_016408, NM_014588, S76389, and NM_001853. Table 2 shows the marker genes depending on each prediction model. Table 3 shows the gene name, symbol and overlap of cell type specific, time specific and mycoplasma specific marker genes.
Classifications for sample groups
If a sample s is contaminated by certain source infection, it can be classified using PAM (Prediction Analysis for Microarrays) which is a class prediction program for data mining that finds genes and classifies them with prediction error using a cross-validation method. Table 4 shows the results of the detection of 4 sources of infection with 20 genes designated as classifier with a classification accuracy of 93.55%. Since the data on fungus was poor, we tried to classify 3 sources infection excluding fungus. This gave a better result showing 19 genes with a classification accuracy of 98.33%. These 19 genes were included in the 20 genes detected using 4 sources of infection. For classification of Mycoplasma and non-Mycoplasma we detected 34 classifier genes with a classification accuracy of 91.94%. We easily conclude that the source of infection may cause different patterns of gene expressions.
For classifying the species of Mycoplasma we obtained poor classification results. Table 4 shows that the prediction accuracy was 43.75% when classifying the 8 species of Mycoplasma, and 77.78% when classifying two species, M. hyorinis and M. orale, which are a common source of infection. Therefore the similarities between infections by different species of Mycoplasma are more pronounced than the differences between them.
Finally, for classifying the infection time of Mycoplasma, four classification models were built. The classification result was shown in last 4 rows of Table 4. Classification of groups of early-collected samples using two time points (1 day or 3 day) and late-collected samples (3 day or 10 day) gave better results than when classifying groups using 3 time points (1, 3, and 10 day).
Implementation of prediction of the microbial infection
We implemented a web based tool, named Prediction of Microbial Infection (PMI) to predict microbial infection in microarray data using MCI which we defined in this study (http://www.snubi.org/software/PMI). Input to PMI is a common tab-delimited text file of log-odds value of gene-expression. The first row must contain column heading (i.e., Index, Reporter_ID, condition 1, condition 2...). The first column contains index of probe in array. The second column must contain either Entrez Gene ID, GenBank accession number, or an official gene symbol. The third to i-th columns contain log-odds value of gene expression levels across experimental conditions. PMI calculates Jaccard similarity coefficient as a prediction score of microbial infection between 5 marker genes (NM_005298, NM_016408, NM_014588, S76389, and NM_001853) and candidate marker genes calculated from user's input gene expression file by MCI.
DISCUSSION
In conclusion, we suggest a group of genes that are believed to be strongly implicated in the microbial infection of human keratinocytes and chondrocytes. We also suggest a model to predict whether a given sample is infected by the microbial contamination. This is a powerful model for the prediction of cell contamination, and is suitable for application to data consisting of gene expression profiles following infection under various experimental conditions. To obtain stronger prediction power for the non-contamination of a sample, structural experimental profiling data for host response to infection by a wide source of pathogens is required. To the best of our knowledge, this is first study to detect microbial contamination using gene expression profiles of host responses. This eliminates the need for additional microarray experiment to distinguish microbial contamination.
Previously, there are two basic testing methods for Mycoplasma contamination; direct culture in media, or indirect tests that measure specific characteristics of Mycoplasma. Direct culture is the most effective and a sensitive method for detecting Mycoplasma, but it is also the most difficult and time consuming (requiring up to 28 days). While DNA fluorochrome staining is an easy and relatively fast indirect procedures to perform (requiring up to 4 to 5 days), however it also has several limitations such as high cost, some equivocal staining results to interpret, and mandatory use of suitable positive and negative control slides (15).
In the present study, we examined the gene expression profiles of microbial infected cells to select marker genes that could identify microbial infection on sample cell. The five genes selected as marker genes by MCI could predict whether biological sample is infected or not by the microbial contamination. We also selected three and four marker genes to predict keratinocyte and chondrocyte cell type, respectively. But these three or four marker genes are included in the set of marker genes found in the model for whole samples, so we can conclude that our prediction model is robust for these types of cells, and also we can apply the general prediction model instead of cell type specific model without loss of prediction power.
The previous methods used to detect cell infection work poorly during the early stages of infection. Our microarray based prediction model can be used to detect infection in the early stages without the need for additional process or a loss of prediction power. Table 2 shows that the set of marker genes for early-collected samples coincide with the set of marker genes for whole samples thus substantiating our prediction model.
As previously mentioned, this is the first study to detect microbial contamination using gene expression profiles of host response. The five marker genes (NM_005298, NM_016408, NM_014588, S76389, and NM_001853), which were selected in this study, are novel genes to distinguish microbial contamination in microarray data. For understating biological mechanisms of these genes, we examined literature search. Among the five genes, three genes (NM_016408, NM_001853, and NM_014588) are related to the cell differentiation and cell cycle process (16-18). Especially NM_016408 is one of the marker genes of human cell cycle PCA array, which distinguishes between G2 phase and G2/M transition. These results indicated that the microbial infection affects cell cycle process of host cell.
Efforts in functional genomics related to cancer research have yielded major successes in the pursuit of gene expression signatures. Approaches to gene expression analysis such as time-series analysis, pattern discovery, clustering, and class prediction, have recently been reviewed (19). Expression-based criteria and class predictors have been defined by neighborhood joining analysis (20), a method based on a subset of genes whose expression strongly correlates with specific classes, as well as Bayesian regression models (21) and artificial neural networks (22). These predictors were successfully used to classify novel samples in a manner consistent with clinical assessments. Classifications based on gene expression alone or class discovery have also been demonstrated and suggest that gene expression profiling has the capacity to identify subtypes that have not been previously defined (20). Although these results are promising, it should be noted that many of the previously conducted cancer line gene expression profiles are one dimensional. In contrast, a host expression profile evoked by pathogen exposure would be expected to be temporal and may also exhibit dose dependence. Comprehensive sets of gene expression profiles that explore temporal and dose ranges for pathogen exposure must be produced to map the continuum of gene expression changes.
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