Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

Abstract

Tuberculosis remains the most pervasive infectious disease and the recent emergence of multiple drugresistant strains emphasizes the need for more efficient drug treatments. The experimentally versatile Dictyostelium discoideum -Mycobacterium marinum infection model provides a powerful system to study mycobacteria pathogenicity and host response. In this study, a time-resolved transcriptomic analysis of the amoeba D. discoideum was performed to decipher the different host pathways impacted during infection. We investigated how D. discoideum fine-tunes its gene expression in response to M. marinum infection by assessing the transcriptomic profile covering the critical stages of entry, establishment of a permissive niche, proliferation and dissemination (1, 3, 6, 12, 24 and 48 hours post infection). Differential gene expression provided a fingerprint of the transcriptome of the host cell in the presence of mycobacteria, and helped identify specific markers and molecular signatures of infection. Enrichment pathway analysis showed that most of the Biological Processes (BP) of upregulated genes at early time point of infection hinted towards damage response and cellular defence, especially in specific pathways involved in membrane repair (ESCRT) and bacteria elimination (autophagy). Whereas at late time points of infection, BP related to starvation were upregulated. Some other signatures were more unexpected, such as cell cycle (downregulation of cytosolic large & small ribosomal subunits) and upregulation of metabolic adaptations (lipids transport).

Transcriptional Responses of Dictyostelium discoideum Exposed to Different Classes of Bacteria

“…For example, in the presence of M. marinum, D. discoideum induces the transcription of genes related to endosome to lysosome transport (including the ESCRT machinery), lipid transport and proteolysis (including ubiquitin-mediated degradation) ( Figure 5 and Box 2 in Supplementary Figure S3). This data suggests that D. discoideum senses M. marinum as a stress or that M. marinum induces a starvation-like condition (Hanna et al, 2019). In contrast to M. marinum, the lipid transport group was down-regulated in the presence of B. subtilis (Figure 5).…”

“…The cDNA preparation and the RNA sequencing were performed as described previously (Hanna et al, 2019). Briefly, 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, CA, United States).…”

“…The bioinformatic analysis was performed as described previously (Hanna et al, 2019). Briefly, RNA-seq libraries of D. discoideum exposed to bacteria or folate were compared to D. discoideum incubated in HL5c.…”

“…For a Gene Ontology (GO) term analysis, topGO was used (version 2.36.0), as previously described (Hanna et al, 2019). In particular, a less stringent set of thresholds values were used to filter input of differentially expressed genes for the GO analysis: a p-value ≤ 0.05 and an absolute log2 fold change ratio ≥0.585 (corresponding to a fold change of at least 1.5).…”

“…For example, in some studies D. discoideum amoebae were fed exclusively with non-pathogenic bacteria for several generations (16 h) (Nasser et al, 2013) or for a shorter time (2 h) (Sillo et al, 2008). In other studies, D. discoideum amoebae were exposed to pathogenic Legionella pneumophila (Farbrother et al, 2006), Pseudomonas aeruginosa (Carilla-Latorre et al, 2008), or Mycobacterium marinum (Hanna et al, 2019). These experimental variations, as well as the use of widely different techniques presumably account for the fact that very diverse results were obtained.…”