Project name: Streptococcus pneumoniae D39

Description: To investigate the response of S. pneumoniae to three distinct antimicrobial peptides (AMPs), i.e. bacitracin, nisin and LL-37, transcriptome analysis of challenged bacteria was performed. Only a limited number of genes was found to be up- or down-regulated in all cases. Several of these common highly induced genes were chosen for further analysis, i.e. SP0385-0387, SP0912-0913, SP0785-0787, SP1714-1715 and the blp cluster. Deletion of these genes in combination with MIC determinations showed that several putative transporters, i.e. SP0785-0787 and SP0912-0913, were indeed involved in resistance to lincomycin, LL-37 and to bacitracin, nisin, lincomycin, respectively. Mutation of blp-bacteriocin production and its immunity genes resulted in an increased of sensitivity to LL-37. Interestingly, a putative ABC transporter (SP1715) protected against bacitracin and Hoechst 33342, but confered sensitivity to LL-37. A GntR-like regulator, SP1714, was identified as a negative regulator of itself and two of the putative transporters. In conclusion, we show that resistance to three different AMPs in S. pneumoniae is mediated by several putative ABC transporters, some of which have not been associated with antimicrobial resistance in this organism before. In addition, a GntR-like regulator was identified, which regulates two of these transporters. Our findings extend the undertstanding of defense mechanisms of this important human pathogen against antimicrobial compounds and points toward novel proteins, i.e. putative ABC transporters, which can be used as targets for the development of new antimicrobials.Overall design: Experimental design. 1 ml aliquots of S. pneumoniae D39 were used to 100 ml THY medium, and were grown at 37°C until early logarithmic phase to an OD600 of ~0.25. Subsequently, cultures were split in two and exposed to either 0.7 μg/ml bacitracin, 0.1 μg/ml nisin or 4.5 μg/ml LL-37 (end concentrations) for 15 (early response) and 30 min (late response), respectively. These concentrations of AMPs were chosen, as they are able to just inhibit growth of S. pneumoniae D39 for approximately 10%. For each AMP, three replicates were performed.RNA isolation, cDNA preparation and hybridization. RNA was isolated from 50 ml of three independent cultures exposed to either no AMPs or to each AMP. After centrifugation, pellets were frozen in liquid nitrogen and stored at -80°C. Subsequently, pellets were suspended in 500 μl of 10 mM Tris-HCl, 1 mM EDTA pH 8.0, after which 50 μl of 10% SDS, 500 μl of phenol:chloroform:isoamyloalcohol (24:24:1), 500 mg of glass beads (Sigma,75-150 μm), 175 μl of Macaloid solution (Bentone) were added. RNA was isolated with a High Pure RNA Isolation Kit (Roche). Subsequently, cDNA was obtained from 15-20 μg of total RNA and the labeling Cy3/Cy5-dCTPs of cDNA was performed with the CyScribe Post labeling kit (Amersham Biosciences). Hybridization was carried out at 45°C for 16 h in Ambion Slide hybridyzation buffer (Ambion Europe) on superamine glass slides (Array-It; SMMBC). Slides contained replicates of amplicons of 2.087 open reading frames (ORFs) of S. pneumoniae Tigr4 and 184 unique ORFs of S. pneumoniae R6. Amplicon sequences are available on the World Wide Web at molgen.biol.rug.nl. Slides were scanned using a GeneTac LSIV confocal laser scanner (Genomics Solutions).Data analysis. ArrayPro 4.5 (Media Cybernetics Inc., Silver Spring, MD) was used to analyze the data. For the processing and normalization of the data the MicroPrep software was used as described previously (74,75). Genes with p < 0.0001 and with a differential expression greather than 1.5 or 2 – fold were considered significantly differentially expressed.

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: Medical

Release date: 2010-01-08

Last updated: 2009-06-08