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Staphylococcus aureus sRNA targetomes and regulatory networks involved in fast adaptive responses: structure, mechanisms and dynamics

Periodic Reporting for period 1 - SaRNAReg (Staphylococcus aureus sRNA targetomes and regulatory networks involved in fast adaptive responses: structure, mechanisms and dynamics)

Reporting period: 2018-06-04 to 2020-06-03

Although Staphylococcus aureus is a commensal bacterium of the human nose and skin, this opportunistic pathogen is responsible for a wide range of nosocomial and community-acquired infections. Due to the worldwide spread of hypervirulent multidrug-resistant strains, S. aureus infections are now considered as a major health issue.

S. aureus has developed many strategies to regulate the synthesis of its various and redundant virulence factors in response to host and perceived stresses. These networks are coordinated by complex interactions between numerous two-component systems, transcription factors, and small regulatory RNAs (sRNAs). Since their discovery in the early 1980’s, accumulated results depict sRNAs as key actors in all aspect of bacterial physiology. Described as 50 to 500 nt-long RNA fragments, sRNAs generally form short and imperfect pairings with their target mRNAs, thus regulating their translation and/or stability. This limited complementarity enables them to modulate the expression of multiple mRNAs, forming their interactome. By directly targeting mRNAs, sRNA-dependent regulation provides a rapid and specific reprogramming of gene expression according to changing physiological conditions. Remarkably, several staphylococcal sRNAs are described as key virulence regulators. For instance, RNAIII is part of a regulatory network controlling the expression of numerous virulence factors such as cell surface proteins, exoproteins and toxins required for host immune evasion and survival.

Hundreds of S. aureus sRNAs have been identified but little to no targets are known. Indeed, the identification of mRNA targets remains one of the biggest challenges in the sRNA field. The main goal of SaRNAReg project is to draw a comprehensive regulatory network of small RNAs involved in virulence and stress responses in S. aureus. This should notably bring the missing information to better understand the mechanisms of S. aureus survival and the existing relationships between virulence, metabolism adaptation, and stress responses.
MAPS (MS2-affinity purification coupled with RNA sequencing) has been developed to unravel the whole interactome of a specific sRNA in vivo. Basically, a sRNA of interest is tagged with the bacteriophage MS2 RNA stem-loop, which is recognized with high specificity by the MS2 coat protein. Hence, this highly specific interaction allows in vivo copurification of bound RNAs. By performing MAPS technology, we notably revealed that S. aureus defenses against manganese (Mn) starvation and oxidative stress, both generated by host immune cells, are interconnected and balanced by RsaC sRNA.
RsaC is an atypical and large sRNA. Its length varies between different isolates due to the presence of repeated sequences at the 5’ end while its 3’ part is structurally independent and highly conserved. Among the potential mRNA targets revealed by MAPS, sodA mRNA was identified as the main target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase, which is crucial to response to oxidative stress generated by aerobic respiration or by host defense cells (e.g. macrophages, neutrophils). In absence of Mn, RsaC is co-transcribed with the major manganese ABC transporter MntABC. After its release, RsaC efficiently inhibits the translation of SodA, involved in reactive oxygen species (ROS) detoxification. By negatively regulating non-essential Mn-utilizing enzymes, RsaC spares Mn for essential Mn-containing proteins (e.g. involved in DNA synthesis and repair). In parallel, RsaC favors the oxidative stress response mediated by SodM, an alternative SOD enzyme able to use iron as cofactor, and restore the ROS detoxification pathway. Interestingly, both rsaC and sodM genes have been acquired exclusively by S. aureus and closely related strains (S. argenteus and S. schweitzeri), a clear advantage compared to other Staphylococcus spp. when facing host-imposed manganese starvation.
Remarkably, RsaC may have a broader role in oxidative stress response (ROS and NOS), metal homeostasis (Fe and Zn) but also virulence.

Exploitation and dissemination:
- Publications (5)
1. Lalaouna D and Romby P. Manganese: The Battle of the Two Armies. The Project Repository Journal 6:100-101
2. Desgranges E, Caldelari I, Marzi S and Lalaouna D (2020). Navigation Through the Twists and Turns of RNA Sequencing Technologies: Application to Bacterial Regulatory RNAs. BBA - Gene Regulatory Mechanisms 1863(3):194506
3. Georg J†, Lalaouna D†, Hou S, Lott SC, Caldelari I, Marzi S, Hess WR, Romby P (2020). The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs. Molecular Microbiology 113(3):603-612
†co-first author
4. Lalaouna D, Baude J, Wu Z, Tomasini A, Chicher J, Marzi S, Vandenesch F, Romby P, Caldelari I and Moreau K (2019). RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation. Nucleic Acids Research 47(18):9871-9887
5. Lalaouna D, Desgranges E, Caldelari I and Marzi S (2018). MS2-affinity purification coupled with RNA sequencing approach in the human pathogen Staphylococcus aureus. Methods in Enzymology 612:394-407

- Invited presentations (7)
1. 14th Conference on Plant-Bacteria interactions (Aussois, France, 2020)
2. 2019 Junior scientists microbiology meeting of Marseille (Marseille, France, 2019)
3. “A day with the speaker” - Institut de Biologie Intégrative de la Cellule (Gif-sur-Yvette, France, 2019)
4. Laboratoire de Microbiologie, Adaptation et Pathogénie - Claude Bernard University Lyon 1 (Lyon, France, 2018)
5. Institut de Biologie Moléculaire et Cellulaire (IBMC) - University of Strasbourg (Strasbourg, France, 2018)
6. University of Strasbourg (Strasbourg, France, 2018)
7. Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements extrêmes (LEMIRE) - CEA Cadarache (St-Paul-lès-Durance, France, 2018)

- Oral presentation (3)
1. Horizon 2020 MSCA cluster event - Antimicrobial Resistance (Barcelona, Spain, 2019)
2. Séminaire de microbiologie de Strasbourg 2019 (Strasbourg, France, 2019)
3. Académie des Sciences, rencontre Strasbourg-Weizmann Institute (Strasbourg, France, 2018)

- Poster presentation (4)
1. 2019 Staphylococcal Diseases Conference GRC (Barcelona, Spain, 2019)
2. sRNA 2019 (Freiburg, Germany, 2019)
3. New Approaches and Concepts in Microbiology (Heidelberg, Germany, 2019)
4. 11th SifrARN (Nancy, France, 2018)

- Science popularization (3)
1. Esperluette website
2. INSB - CNRS (in French)
3. Twitter
As main breakthrough, the discovery of RsaC reconciles a decade of research, providing the missing link between two crucial stress responses. Indeed, RsaC sRNAs is a connection node between oxidative stress responses and metal homeostasis.
This strongly emphasizes the importance of exploring and understanding regulatory networks in pathogens such as S. aureus to improve human health. This might suggest new potential drug targets for antibiotic therapy (e.g. identification of virulence-related targets or sRNAs involved in antibiotic resistance/virulence) and open new therapeutic avenues (e.g. RNA-based drugs).