Final Report Summary - SARHYB (Large scale elucidation of the biological functions of the Staphylococcal RNome in genetic hybrid systems) Every year in the European Union, an estimated 25000 patients die because of a serious resistant bacterial infection acquired in hospital. The bacterium Staphylococcus aureus is a main opportunistic pathogen responsible for food poisoning, skin infections and nosocomial infections. In the recent years, small ribonucleic acids (sRNAs) have attracted great interests as ubiquitous regulators. Some of them have been directly related to commitment of virulence. However, a serious bottleneck in unraveling their biological roles is due to the difficulty in identifying targets of the sRNome (any nonconventional RNA with regulatory functions). SarHyb intends to identify the primary molecular targets (RNAs and proteins) of staphylococcal RNome with a multidisciplinary approach. Therefore, SarHyb combines the use of novel bacterial genetic hybrid systems, molecular biology and biochemistry. The principal novelty consists in development bacterial three-hybrid and RNA-hybrid systems in S. aureus, and if unsuccessful to enhance the yeasts systems already available for better expression of A/U rich RNAs. At beginning of the project, it appeared that many of the sRNAs decribed in S. aureus were questionable. The lack of consensus in the community and the presence of a large number of repeated sequences appeared deleterious to define a clear list of sRNA candidates to work with. Therefore, during the first period of the project, the researcher added a new task. RNAseq experiments were performed as a collaborative effort between several members of the laboratory prior starting the project. Together, we defined a new set of sRNAs that should be used for Sarhyb. RNAseq data analysis revealed significant differences with previous work published in the community and strongly suggested that several sequences published as sRNAs were UTRs (untranslated regions of mRNAs) or repeated DNA sequences. Thus, in association with a postdoctoral fellow specialized in bioinformatics, we have cured all the sRNAs data published so far and created a sRNA database for the Staphylococci exempted of redundancies. This work was recently published in RNA (2015 21(5):1005-17. doi: 10.1261). In the meantime, the researcher focused on improving the transformation efficiency of S. aureus and tried to directly transform strain RN1_HG003 or Newman directly from E. coli. The goal here was to assess whether such strains could be used for the S. aureus genetic hybrid system. Although strain RN1 HG003 is transformable directly from E. coli, the efficiency remains modest (1.103). Therefore, strain RN4220, an RN1 derivative, is the only strain suitable for implementing an hybrid system. The researcher combined the use of sucrose, glycerol, DMSO or Ethanol to increase the transformation efficiency of strains RN4220 that reached the 8.105 transformants per microgram of DNA which was close to the initial goal. Then, the researcher started to delete the SprA family toxin-antitoxin systems. A thorough investigation of the family revealed the presence of SprA/SpAs1, SprA2/SprAs2, SprA3 and SprAs2’in RN4220. Although the researcher activities on the SprA2/SprAS2 module, with the help of an Assistant Engineer from the lab, did not evidenced for a cross-regulation in vitro, the researcher decided to delete all the modules. Using a markless deletion strategy, he was successful in deleting the first three modules although it took more time than expected. However, the researcher was unsuccessful when he tried to delete the SpAs2’ locus, which is very small. While deletions were performed in RN4220, the toxicity of SprA1 and SprA2 toxins were investigated by using synthesized peptides (with the help of chemical lab in University of Rennes 1) and also by overexpressing peptides directly in S. aureus. Both approach led to the conclusion that SprA2 is more toxic than SprA1. Those results shall be published soon as they describe the SprA2/SprAs2 system. The next step will be to test the decoupling of both toxins and the efficacy of the antitoxin for a library screening. This task was not achieved because of the time necessary to generated of recombination in S. aureus. All of them will be pursued after the project since the researcher is appointed at University of Rennes 1 and receiving the help of a postdoc for the next 6 months.Additionally, we assessed the robustness of the yeast system. It appeared that the system is sensitive to uridines tract within the S. aureus sRNAs to be cloned that lead to premature arrest of transcription in yeast. Several genetic modifications were prepared to stabilize the expression and restriction sites suitable for cloning the S. aureus libraries have been generated. The constructs generated allow the stable expression of the hybrid RNA constructs. However, this stabilized expression led to the loss of any interactions, including the positive controls SprA1/S4 and IRE/IRP which were working previously. This was due to the fact that changing of the use of RNA polymerase led to target the sRNA outside the nucleus. While the researcher focused of the deletions of toxin-antitoxins system in S. aureus, further investigation in yeast were postponed. As genetic manipulation were difficult in S. aureus because of the region to delete the researcher only came back on yeast at the end of the project to investigated whether it would be possible to destabilize the targeting of sRNA outside the nucleus by generating site directed mutation in yeast for genes involved in this mechanisms. This has been documented in the literature and therefore strategy has been employed during the last months of the project and is still under development. Major Scientific findings expected during the course of the project include the development of the first bacterial hybrid systems devoted to RNA-target identification, an exhaustive list of mRNA and proteins directly regulated by sRNAs, and an in-depth understanding of the molecular signals allowing S. aureus to adapt to its environment. If successful, this ambitious research project will impact both basic and translational research at the European Union level.
