Computational protocol: Foreign peptide triggers boost in pneumococcal metabolism and growth

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Protocol publication

[…] RNA-Seq was used to identify differentially expressed genes between wild type and ΔORF 2 mutant as well as wild type compared to wild type exposed to the peptide ligand and the ΔORF 2 mutant compared to ORF 2 treated with the ligand.110.58 and ΔORF 2 were plated out on CSBA plates and incubated at 37 °C, 5% CO2 overnight, colonies picked and cultured overnight in 5 ml BHI + FCS as described above until OD600nm = 0.4 then centrifuged at 2000 g for 5 min and the pellet resuspended in CDM. The centrifugation was repeated and the pellet resuspended in 5 ml CDM. This 5 ml bacterial suspension was added to 10 ml CDM. For each strain, at exactly OD600nm = 0.2 the culture was split into two tubes, each containing 5 ml. To one tube for each strain the peptide FPPQSV was added to give a final concentration of 0.07 mg/ml, mixed and all tubes incubated at 37 °C for exactly 15 min. Transcription was stopped by adding RNAprotect (Qiagen) and RNA extracted as described previously []. RNA was isolated from three separate experiments performed on different days for RNA-Seq and from a further three separate experiments performed on different days for real-time RT-PCR.For RNA-Seq, ribosomal RNA was depleted using Ribominus (Invitrogen) and then the RNA purified using RNA Clean & Concentration™-5 kit (Zymo Research) according to the manufacturers’ instructions, eluting in 6 μl. Libraries were prepared using TruSeq® Stranded mRNA (Illumina). 13 μl Fragment, Prime, Finish Mix was added to 5 μl of the mRNA. Fragmentation was performed for 40 s at 94 °C. Raw reads were obtained from an Illumina 3000 Hi-seq paired end sequencing platform. Reads were mapped to Streptococcus pneumoniae strain NT_110_58 assembly gca 000817005 ASM81700v1). Preprocessing and adapter triming was done using the Trimommatic tool (0.35) []. Alignments were performed with Bowtie (bowtie2–2.2.8) []. Cufflinks (cufflinks-2.2.1) and Cuffdiff were used to count reads and calculate differential gene expression []. Quality control and coverage were examined using FastQC (0.11.5) and Qualimap 2.2.1 []. The sequencing and coverage was on average 3557 times the genome size (ranging between 543 and 5220). The mean length of sequenced fragments was 141 bp. Mapping to the reference genome was above 99.09% for all samples.Expression of genes of interest identified by RNA-Seq was quantified by real-time RT-PCR as described previously [] and normalized against 16S using the following primers and probes: 16S forward primer 5’-GACGATACATAGCCGACCTGAGA-3′; reverse primer 5’-GTAGGAGTCTGGGCCGTGTCT-3′; probe 6-carboxyfluorescein (6-FAM)-CCAGTGTGGCCGATC-minor groove binder (MGB); afr forward primer 5′- TCTTCATCACCGAAATGTTCACCTT-3′; reverse primer 5′- ATGCCTGAAACTGTAACCATGACA-3′; probe, (6FAM)- ATGGGCCACATTTCCA-(MGB); adhE forward primer 5’-GAAGGAAGTTTCATCCATTGCATGT-3′; reverse primer 5’-ACGTTAGTGCCATTAACCTCTTGAA-3′; probe (6-FAM)-CCGTCTTCCGACTTTT-(MGB); nanA_3 forward primer 5’-CACCACTTCACCAGCAGATGTATAA-3′; reverse primer 5’-GAGACTAAAGTTCCAATAACGACTGGTT-3′; probe (6-FAM)-CACGCACCATTTTCTT--(MGB); nanB forward primer 5’-GTTAACCCAACTTTAGCAATGGCAAT-3′; reverse primer 5’-GGAGCAATCATGTCGAGACTACT-3′; probe (6-FAM)-TTCCCACCAATTTTG-(MGB); yesO_2 forward primer 5’-ACTTGGTTAGGAAGAGCTGTACTGA-3′; reverse primer 5’-GAATGACTTCTATACTAAATGGACTACAGGTT-3′; probe (6-FAM)-ATCTGGCACATTTCC-(MGB); ycjO forward primer 5’-GAAGTGGCGTACTCTGTGAAGA-3′; reverse primer 5’-TGGACCGTTTTCTCATTAGTTGGT-3′; probe (6-FAM)-AAGCCAATACAAACCC-(MGB). [...] Sample processing, LC-MS/MS and data interpretation was essentially done as described previously [] with the following minor changes. LC-MS/MS analysis was carried out on an Ultimate3000 nanoLC coupled to an Orbitrap Fusion Lumos instrument (ThermoFisher Scientific) acquiring full MS scans in the m/z range 400–1400 in the orbitrap at resolution 120′000 with AGC set to 4e5 and maximal ion injection time of 50 ms. Peptide precursors with charge 2–8 were fragmented once in the iontrap then excluded for 30s. The iontrap setting were data-dependent MS2 cycle time of 3 s, isolation width of 1.6 m/z, fragmentation HCD mode with 30% normalized collision energy, AGC of 1e4 with maximal ion injection time of 35 ms. The LC-MS/MS data was processed with MaxQuant (version using default settings for peak detection, strict trypsin cleavage rule allowing for up to three missed cleavages, variable oxidation on methionine and acetylation of protein N-termini with strict carbamidomethylation of cysteines. Match between runs was activated with a retention time window of 0.7 min. The fragment spectra were interpreted with the Streptococcus pneumoniae ensemble database (version gca_000817005_ASM81700v1). For data analysis, peptide feature intensities reported in the evidence file were median normalized and missing values were imputed from the low end of the LOG2 transformed intensity distribution of each LC-MS/MS run using Perseus (version as suggested by Lazar et al. []. The three most intense peptide feature intensities were summed for the individual protein group intensity. […]

Pipeline specifications

Software tools MaxQuant, Perseus
Application MS-based untargeted proteomics
Organisms Streptococcus pneumoniae, Escherichia coli, Homo sapiens, Rattus norvegicus, Bacteria
Chemicals Carbohydrates