Identification of Pseudomonas aeruginosa-induced genes in human mast cells using suppression subtractive hybridization: up-regulation of IL-8 and CCL4 production

Abstract

Mast cells have recently been found to be a major player in the host defence against bacterial infection through secretion of potent mediators. Identification of bacteria-induced mast cell mediators and intracellular signalling molecules involved during bacterial infection remains a major area of investigation. Recently we found an active interaction between mast cells and Pseudomonas aeruginosa bacteria. To further characterize specific genes in mast cells modulated by P. aeruginosa, we used a new approach for the study of mast cell-bacteria interaction; the suppression subtractive hybridization (SSH). SSH approach does not require a prerequisite knowledge of target genes and does not rely on the availability of the assay reagents for the specific genes. Using SSH, 94 clones were randomly selected from the subtracted cDNA library for differential screening leading to the identification of 14 P. aeruginosa-up-regulated transcripts. Sequence analysis revealed that expression of IL-1, IL-8 and CCL4 was increased by human mast cells after P. aeruginosa infection. Increased production of IL-1, IL-8 and CCL4 was confirmed at the protein levels. In addition, sequence analysis of the clones also suggests that ribosomal protein S3 and cytochrome b as well as additional 4 uncharacterized genes may potentially be involved in P. aeruginosa pathogenesis. Thus, SSH is an effective approach by identifying potential molecular targets for the study of mechanisms involved in P. aeruginosa and mast cell interaction.

Fig. 1

Evaluation of the subtraction efficiency by amplifying the cDNA of GAPDH in both the subtracted and nonsubtracted cDNA pools by PCR. (a). The products amplified for different cycles were electrophoresized on the 1·5% agarose gel. The numerical number indicated the number of PCR cycles. Lane M: 100 bp molecular mass markers. Note that the GAPDH band appeared at the 13rd cycle in the nonsubtracted pool, while it appeared at the 18th cycle in the subtracted pool. (b). The cDNA of GAPDH in both the subtracted and nonsubtracted cDNA pools were amplified for 18–24 cycles by PCR. The numerical number indicated the number of PCR cycles. Lane M1: 100 bp molecular markers, lane M2: 1kb molecular markers. The difference in GAPDH band density between the subtracted and nonsubtracted pool was detected in the PCR less than 20 cycles.

Fig. 2

PCR assays to verify the insert of each colony. The PCR amplified subtracted cNDA was ligated into the PCR 2·1 TA cloning vector, and transformed into DH5α competent cells. The transformed DH5α competent cells were grown overnight at 37 °C on LB agar plates containing ampicillin, X-gal, and isopropyl-β-D-thiogalactopyranoside for white/blue colony selection. White colonies were isolated and grown individually overnight at 37 °C in 200 µl of LB broth containing ampicillin. The insert of each colony was PCR amplified using a 1-µl aliquot of the bacteria growth. The size of inserts showing in this example varied from about 350 base pair (bp) to about 1100 bp. Lanes 7 and 10 showed that bacteria colonies had more than one plasmid. Lanes 2 and 14 showed no insert in these colonies. Lane 20 is 100 bp DNA markers.

Fig. 3

Virtual Northern assay of gene expression. The SMART double strand cDNAs from P. aeruginosa stimulated mast cells (lane Psa), and untreated mast cells (lane NT) were hybridized with the probes prepared from the positive clone psa1D, psa8C, psa10C, psa8B and GAPDH (control).

Fig. 4

CCL4 and IL-8 production by human cord blood-derived mast cells following P. aeruginosa stimulation. Human cord blood-derived mast cells (5 × 10⁵ cells/ml) from two individual donors were treated with P. aeruginosa (1/50 cell-bacteria ratio) for 24 h. Cell free supernatants were collected for the determination by ELISA. Treatment of mast cells with P. aeruginosa induced increased CCL4 and IL-8 production. Error bars represent replicate.