Dictyostelium discoideum amoebae feed by ingesting bacteria, then killing them in phagosomes. Ingestion and killing of different bacteria have been shown to rely on largely different molecular mechanisms. One would thus expect that D. discoideum adapts its ingestion and killing machinery when encountering different bacteria. In this study, we investigated by RNA sequencing if and how D. discoideum amoebae respond to the presence of different bacteria by modifying their gene expression patterns. Each bacterial species analyzed induced a specific modification of the transcriptome. Bacteria such as Bacillus subtilis, Klebsiella pneumoniae, or Mycobacterium marinum induced a specific and different transcriptional response, while Micrococcus luteus did not trigger a significant gene regulation. Although folate has been proposed to be one of the key molecules secreted by bacteria and recognized by hunting amoebae, it elicited a very specific and restricted transcriptional signature, distinct from that triggered by any bacteria analyzed here. Our results indicate that D. discoideum amoebae respond in a highly specific, almost non-overlapping manner to different species of bacteria. We additionally identify specific sets of genes that can be used as reporters of the response of D. discoideum to different bacteria.
Overall design: After co-incubation for 4h between bacteria and wild-type D. discoideum cells, the uningested bacteria were removed by centrifugation at 1000 g for 2 min, RNA was extracted from cells using the directzol RNA extraction kit (Zymo research) following the manufacturer’s instructions for total RNA isolation. To remove contaminating genomic DNA, samples were treated with 0.25U of DNase I (Zymo) per 1 μg of RNA for 15 min at 25°C. RNA was quantified using Qubit 4.0 (Invitrogen) and its quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). The total RNA preparation was used as a template for cDNA synthesis and NGS library construction using the Ovation Universal System (NuGEN Technologies, San Carlos, California, USA). 100 ng of total DNAse I-treated RNA was used for first- and second-strand cDNA synthesis following the manufacturer’s protocol. In order to obtain a comparable library size, a double bead cut strategy was applied using the 10X genomics protocol. cDNA was recovered using magnetic beads with two ethanol washing steps, followed by enzymatic end repair of the fragments. Barcoded adapters were ligated to each sample before strand selection. Ribosomal RNAs were targeted for depletion by the addition of custom-designed oligonucleotides specific for D. discoideum (5S, 18S and 28S). To amplify the libraries, 18 cycles of PCR were performed. The quality of the libraries was monitored by TapeStation (Agilent, High Sensitivity D1000 ScreenTape, # 5067–5584). Samples were pooled in approximately equimolar amounts and analyzed in 50 bp single read flow cells (Illumina, # 15022187; Hiseq 4000). The numbers of replicates for each conditions are indicated below.
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