[
International Worm Meeting,
2017]
The host-microbe interaction depends on molecular patterns and damage signals that reciprocally modulate bacterial colonization and the host immune response, triggering changes in gene expression in both players. Bacterial small RNAs (sRNAs) are emerging actors of this inter-kingdom crosstalk, but many questions about the mechanisms by which they affect host transcriptomic profiles remain unanswered. Our group observed that C. elegans enters diapause, after two generations of feeding on P. aeruginosa PAO1 and S. Typhimurium MST1, as an RNAi-dependent defensive mechanism against infection. We hypothesize that bacterial sRNAs trigger this behavior by targeting host RNAs. Our aim is to identify which sRNAs from P. aeruginosa PAO1 sensed by C. elegans, are able to trigger an RNAi mediated transcriptomic change by targeting host RNAs. We compared the transcriptomic profiles of naive bacteria (never exposed to worms) with those residing in C. elegans intestine for one, and two generations, using simultaneous worm-bacteria RNAseq. Sequencing enriched for small RNAs was done using Illumina Truseq, yielding an average of 2,5 M reads from naive bacteria and 1,2 M reads from intestinal bacteria (bacteria-worm samples) trimmed and quality filtered (Q = 30; lenght = 17). After mapping (Bowtie, Bowtie2, Segemehl/Lack, Star) reads from bacteria-worm samples were separated in silico (Picard tools), counted, and annotated using NRDR database and SRF prediction (Structure RNA Finder). Differentially expressed genes between naive and experienced bacteria using Deseq2 identified 23 small RNAs from P. aeruginosa PAO1 overexpressed in contact with host (15 in F1, 19 in F2), far less than the 88 sRNAs from E. coli OP50 (61 in F1, 63 in F2). Targets were predicted by IntaRNA, RNA duplex, RNAplex, RNAup and PITA softwares, with 12956 RNA-RNA interactions consistently predicted by all five. Pre-miRNAs constituted the most frequent target class in the host (1214). Prediction of structure and target; analyzes of functional RNA motifs, sites and their homology (RegRNA 2.0), will clarify functional roles and specificity of sRNAs expressed in pathogens. In summary, there is a set of sRNAs uniquely overexpressed in pathogens, candidates of triggering a behavior response in host. We propose that there is a bidirectional dynamic interaction between bacteria and C. elegans that involves the activation of signaling pathways in the host, in which the RNAi machinery may play a central role. This work is the base for future validation studies.