Streptococcus pneumoniae is opportunistic bacteria cause’s acute otitis media (AOM) in children. It colonizes the nasopharynx in the form of biofilms, and these biofilms act as reservoir, and are vital for pneumococcal infections. The pneumococcal biofilms are regulated by LuxS/AI-2 media quorum sensing. In this study, we confirmed the role of LuxS/AI-2 for in vitro formation of biofilms, assessed the effects of the absence of LuxS/AI-2 signaling, for pneumococcal middle ear infection and identified global genes regulated by LuxS/AI-2 during formation of pneumococcal biofilms. In the cDNA-microarray analysis, 117 genes were differentially expressed in D39 luxS mutant when compared with D39 wild type. Among the 66 genes encoding putative proteins and previously characterized proteins, 60 were significantly down-regulated and 6 were significantly up-regulated. The functional annotation revealed that genes involve in DNA replication and repair, ATP synthesis, capsule biosynthesis, cell division and cell cycle, signal transduction, transcription regulation, competence, virulence, and carbohydrate metabolism were down-regulated in the absence of LuxS/AI-2.
Overall design: The in vitro biofilm formation ability of S. pneumoniae D39 wild type and an isogenic luxS strain were evaluated using a static microtiter plate assay as described previously. Briefly, the pneumococcal strains were grown on BAP overnight, and a single colony from the plate was transferred to BHI broth and grown to mid-exponential phase. These log-phase cells were diluted 1:200, and 1 mL of this cell suspension was seeded into 24-well polystyrene flat-bottom microtiter plates (BD Falcon, Sparks, MD, USA) and incubated at 37°C for various times. After incubation, the planktonic cells and medium were removed, and the biofilms remaining in the wells were washed twice with PBS. Global gene expression of S. pneumoniae D39 wild type and D39luxS biofilms was analyzed by cDNA-microarray. For the experiment, S. pneumoniae D39 wild type and D39luxS biofilms were grown in a 24-well plate for 18 h as described above. The biofilms were washed twice with PBS, scraped, and suspended in PBS. Then, the biofilm cells were pelleted by centrifugation and treated with 100 µL of lysozyme (3 mg/mL in TE; Sigma-Aldrich, St. Louis, MO, USA) for 4 min to lyse the cells. Total RNA was extracted using the RNeasy Total RNA Isolation System Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Contaminating DNA was removed by on-column RNase-free DNase (Qiagen) treatment for 10 min at 20–25°C. The quantity and quality of total RNA was detected using a Nano-drop, and the integrity of RNA was assessed with a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). RNA probe synthesis and hybridization were performed using the Agilent Low Input Quick Amp WT Labeling Kit according to the manufacturer’s protocol. Briefly, 200 ng of total biofilm RNA were mixed with WT primer mix, and the samples were incubated at 65°C for 10 min. Then, cDNA master mix was prepared with 5× first strand buffer, 0.1M DTT, 10mM dNTP mix, and RNase Block Mix (AffinityScript) and added to the RNA + WT primer reaction. The samples were incubated at 40°C for 2 h, and then RT and dsDNA synthesis were terminated by incubation at 70°C for 15 min. The transcription master mix was prepared according to the manufacturer’s protocol (5× Transcription buffer, 0.1M DTT, NTP mix, T7-RNA polymerase Blend, and Cyanine 5-CTP in nuclease-free water). Transcription of dsDNA was performed by adding transcription master mix to the dsDNA reaction samples and incubating the mix at 40°C for 2 h. Amplified and labeled cRNA was purified on an RNase mini column (Qiagen) according to the manufacturer’s protocol. The labeled cRNA target was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). After checking the labeling efficiency of the cyanine 5-labeled cRNA target, the cRNA was fragmented by adding 10× blocking agent and 25× fragmentation buffer and incubating at 60°C for 30 min. The fragmented cRNA was resuspended in 2× hybridization buffer and directly pipetted onto an assembled S.pneumoniae_6x7k V2 Microarray (Mycroarray.com). The arrays were hybridized at 57°C for 17 h in an Agilent Hybridization oven. The hybridized microarrays were washed according to the manufacturer’s washing protocol (Agilent Technology). After an overnight incubation at 42°C, the slide was washed twice with washing solution 1 (containing 2× SSC, 0.1% SDS) for 5 min at 42°C, washed once with washing solution 2 (containing 0.1× SSC and 0.1% SDS) for 10 min at room temperature, and finally washed four times with 0.1× SSC for 1 min at room temperature. The slide was dried by centrifugation at 650 rpm for 5 min. The hybridization image on the slide was scanned with a 4000B apparatus (Axon Instruments, Union City, CA, USA). The hybridization image was analyzed with GenePix Pro 3.0 software (Axon Instruments, Union City, CA, USA) to obtain the gene expression ratios in D39 wild type and D39luxS biofilms. The microarray data were analyzed with Genowiz 4.0TM (Ocimum Biosolutions, Hyderabad, India), and normalized with Global LOWESS. The cutoffs for up-regulated and down-regulated genes were +2-fold and -2-fold, respectively. The microarray experiment was performed with three biological replicates. Statistical significance was calculated by Student’s t-test, and P value less than 0.05 was considered significant. STRING version 10.5 (https://string-db.org) was used for the functional annotation, and the UniProtKB database(http://www.uniprot.org/uniprot/P0A4M0) was used to search for clusters of biological processes in the gene ontology database within the two sets of differentially expressed genes (D39luxS mutant and D39 wild type). Microarray data has been deposited in NCBI's Gene Expression Omnibus (GEO) database (/geo/info/linking.html) and are accessible through GEO Series accession number.
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