Staphylococcus aureus is a major pathogen responsible for a variety of infections. It expresses a wide range of factors to precisely coordinate gene expression in response to the ever-changing conditions. Among them, regulatory RNAs appear as key players of post-transcriptional and translational regulations. Here, we investigated the role of Srn_9342, a sRNA candidate previously identified in a cluster of five srna genes in Newman strain. We showed that Srn_9342 is expressed under two isoforms of different lengths (Srn_9342S and Srn_9342L) whose transcript levels are divergent as a function of growth phase with Srn_9342S being expressed at low cell-density, then being substituted by Srn_9342L at high cell-density. Using MS2-Affinity Purification Coupled with RNA Sequencing, we searched for RNA molecular partners of both Srn_9342S and Srn_9342L. Interestingly, we found that Srn_9342S was mainly bound to sRNAs whereas the expression of Srn_9342L led to the enrichment of mRNAs often linked with transport and metabolism. Among the sRNAs identified, the master regulator of virulence RNAIII appeared as an attractive partner. Using various constructs, we showed that the 5’ end of Srn_9342 specifically binds the 3’ end of RNAIII with high affinity in vitro, and that Srn_9342 mitigate RNAIII transcript level and stability. Finally, we report that the deletion of srn_9342 modulates the expression of the RNAIII encoded toxin δ-hemolysin and hemolytic activity, suggesting that the binding of Srn_9342 onto RNAIII may induce structural changes of RNAIII, and hence expression of the toxin. Overall, we showed that Srn_9342 has an unusual pattern of expression and that uncovering its targetome suggests a potential role in S. aureus virulence.

The development of in vitro models that recapitulate critical liver functions is essential for accurate assessments of drug toxicity. Although liver organoids can be used for drug discovery and toxicology, they are limited by (i) the lack of expression and activity of xenobiotic-metabolizing enzymes, and (ii) the difficulty of mimicking non-alcoholic fatty liver disease (NAFLD, which influences the expression of these enzymes) in vitro. Here, we generated three-dimensional multi-cell-type liver organoids (hereafter "HML organoids") from HepaRG cells, primary human macrophages, and hepatic-stellate-cell-derived LX-2 cells. We also developed an NAFLD model by culturing HML organoids for 9 days with a mixture of stearic and oleic acids. The exposed organoids showed typical features of steatosis and expressed fibrosis markers. We subsequently used HML and NAFLD-HML organoids to model drug-induced liver injury. By estimating the IC50 and benchmark doses, we were able to improve the in vitro detection of drugs likely to be toxic in fatty livers. Thus, HML and NAFLD-HML organoids exhibited most of the liver's functions and are relevant in vitro models of drug metabolism, drug toxicity, and adverse drug event in NAFLD.

Staphylococcus aureus RNAIII is a dual-function regulatory RNA that controls the expression of multiple virulence genes and especially the transition from adhesion to the production of exotoxins. However, its contribution to S. aureus central metabolism remains unclear. Using MS2-affinity purification coupled with RNA sequencing, we uncovered more than 50 novel RNAIII-mRNA interactions. Among them, we demonstrate that RNAIII is a major activator of the rpiRc gene, encoding a regulator of the pentose phosphate pathway (PPP). RNAIII binds the 5' UTR of rpiRc mRNA to favor ribosome loading, leading to an increased expression of RpiRc and, subsequently, of two PPP enzymes. Finally, we show that RNAIII and RpiRc are involved in S. aureus fitness in media supplemented with various carbohydrate sources related to PPP and glycolysis. Collectively, our data depict an unprecedented phenotype associated with the RNAIII regulon, especially the direct implication of RNAIII in central metabolic activity modulation. These findings show that the contribution of RNAIII in Staphylococcus aureus adaptation goes far beyond what was initially reported. IMPORTANCE:Staphylococcus aureus is a major human pathogen involved in acute and chronic infections. Highly recalcitrant to antibiotic treatment, persistent infections are mostly associated with the loss of RNAIII expression, a master RNA regulator responsible for the switch from colonization to infection. Here, we used the MS2 affinity purification coupled with RNA sequencing approach to identify novel mRNA targets of RNAIII and uncover novel functions. We demonstrate that RNAIII is an activator of the expression of genes involved in the pentose phosphate pathway and is implicated in the adjustment of bacterial fitness as a function of carbohydrate sources. Taken together, our results demonstrate an unprecedented role of RNAIII that goes beyond the knowledge gained so far and contributes to a better understanding of the role of RNAIII in bacterial adaptation expression and the coordination of a complex regulatory network.

Staphylococcus aureus is a major human and animal pathogen, colonizing diverse ecological niches within its hosts. Predicting whether an isolate will infect a specific host and its subsequent clinical fate remains unknown. In this study, we investigated the S. aureus pangenome using a curated set of 356 strains, spanning a wide range of hosts, origins, and clinical display and antibiotic resistance profiles. We used genome-wide association study (GWAS) and random forest (RF) algorithms to discriminate strains based on their origins and clinical sources. Here, we show that the presence of sak and scn can discriminate strains based on their host specificity, while other genes such as mecA are often associated with virulent outcomes. Both GWAS and RF indicated the importance of intergenic regions (IGRs) and coding DNA sequence (CDS) but not sRNAs in forecasting an outcome. Additional transcriptomic analyses performed on the most prevalent clonal complex 8 (CC8) clonal types, in media mimicking nasal colonization or bacteremia, indicated three RNAs as potential RNA markers to forecast infection, followed by 30 others that could serve as infection severity predictors. Our report shows that genetic association and transcriptomics are complementary approaches that will be combined in a single analytical framework to improve our understanding of bacterial pathogenesis and ultimately identify potential predictive molecular markers. IMPORTANCE Predicting the outcome of bacterial colonization and infections, based on extensive genomic and transcriptomic data from a given pathogen, would be of substantial help for clinicians in treating and curing patients. In this report, genome-wide association studies and random forest algorithms have defined gene combinations that differentiate human from animal strains, colonization from diseases, and nonsevere from severe diseases, while it revealed the importance of IGRs and CDS, but not small RNAs (sRNAs), in anticipating an outcome. In addition, transcriptomic analyses performed on the most prevalent clonal types, in media mimicking either nasal colonization or bacteremia, revealed significant differences and therefore potent RNA markers. Overall, the use of both genomic and transcriptomic data in a single analytical framework can enhance our understanding of bacterial pathogenesis.

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient’s plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

Staphylococcus aureus est un agent pathogène humain, qui peut être à l’origine d’un large spectre d’infections communautaires et nosocomiales. Les évènements facilitant le passage d’un état commensal à pathogène chez cette bactérie restent mal connus. Néanmoins, ils sont à corréler à la régulation fine de l’expression de nombreux gènes (dont des facteurs de virulence) permettant à la bactérie de s’adapter efficacement à son environnement. Outre les facteurs globaux de transcription, les ARN régulateurs (ARNreg) ont été montrés comme jouant un rôle clé dans la régulation de l’expression de ces gènes. Le développement de l'utilisation du séquençage ARN à haut débit a montré que le répertoire en ARNreg de S. aureus ne serait pas entièrement décrit. Ainsi, le premier objectif de ce travail était de rechercher de nouveaux ARNreg chez la souche Newman de S. aureus, une souche jamais utilisée pour ce type d’étude. Cette recherche a permis la découverte de 24 nouveaux ARNreg. Parmi ces 24 ARNreg, 7 appartiennent au core génome de S. aureus, confirmant que le répertoire en ARNreg de cette espèce n’est pas encore entièrement décrit. La caractérisation de ces ARN a mis en évidence la présence d’un locus original regroupant 5 ARNreg nommés Srn_9342 à Srn_9346. L’étude du premier ARN de ce locus Srn_9342, a révélé qu’il était transcrit sous deux formes (Srn_9342court et Srn_9342long) ayant des profils d’expression opposés et dépendants de la phase de croissance. L’identification des cibles de ces deux formes par MAPS, indique qu’elles auraient des rôles distincts. Ainsi, Srn_9342court interagirait préférentiellement avec des ARNreg ayant un rôle dans la virulence (ARNIII, SprD et RsaC). Une hypothèse que nous suggérons serait que la forme courte aurait un rôle d’ARN éponge. Concernant la forme longue, d’après les cibles enrichies par MAPS, cette ARNreg contribuerait à l’adaptation de la bactérie face aux différents stress rencontrés au cours de l’infection. La validation et l’étude de l’ensemble de ces partenaires potentiels permettra de mieux comprendre le rôle de cet ARNreg au sein de la bactérie.

Vancomycin-resistant Enterococcus faecium is a leading cause of hospital-acquired infections and outbreaks. Regulatory RNAs (sRNAs) are major players in adaptive responses, including antibiotic resistance. They were extensively studied in gram-negative bacteria, but less information is available for gram-positive pathogens. No sRNAs are described in E . faecium . We sought to identify a set of sRNAs expressed in vancomycin-resistant E . faecium Aus0004 strain to assess their roles in daptomycin response and resistance. Genomic and transcriptomic analyses revealed a set of 61 sRNA candidates, including 10 that were further tested and validated by Northern and qPCR. RNA-seq was performed with and without subinhibitory concentrations (SICs) of daptomycin, an antibiotic used to treat enterococcal infections. After daptomycin SIC exposure, the expression of 260 coding and srna genes was altered, with 80 upregulated and 180 downregulated, including 51% involved in carbohydrate and transport metabolisms. Daptomycin SIC exposure significantly affected the expression of seven sRNAs, including one experimentally confirmed, sRNA_0160. We studied sRNA expression in isogenic mutants with increasing levels of daptomycin resistance and observed that expression of several sRNAs, including sRNA_0160, was modified in the stepwise mutants. This first genome-wide sRNA identification in E . faecium suggests that some sRNAs are linked to antibiotic stress response and resistance.

Periodontitis are bacterial inflammatory diseases, where the bacterial biofilms present on the tooth-supporting tissues switch from a healthy state towards a pathogenic state. Among bacterial species involved in the disease, Porphyromonas gingivalis has been shown to induce dysbiosis, and to induce virulence of otherwise healthy bacteria like Streptococcus gordonii. During biofilm development, primary colonizers such as S. gordonii first attach to the surface and allow the subsequent adhesion of periodontal pathogens such as P. gingivalis. Interactions between those two bacteria have been extensively studied during the adhesion step of the biofilm. The aim of the study was to understand interactions of both species during the growing phase of the biofilm, for which little knowledge is available, using a mathematical model. This two-species biofilm model was based on a substrate-dependent growth, implemented with damage parameters, and validated thanks to data obtained on experimental biofilms. Three different hypothesis of interactions were proposed and assayed using this model: independence, competition between both bacteria species, or induction of toxicity by one species for the other species. Adequacy between experimental and simulated biofilms were found with the last hypothetic mathematical model. This new mathematical model of two species bacteria biofilms, dependent on different substrates for growing, can be applied to any bacteria species, environmental conditions, or steps of biofilm development. It will be of great interest for exploring bacterial interactions in biofilm conditions.

The human pathogen Staphylococcus aureus expresses a set of transcriptional factors and small RNAs (sRNAs) to adapt to environmental variations. Recent harmonization of staphylococcal sRNA data allowed us to search for novel sRNAs using DETR'PROK, a computational pipeline for identifying sRNA in prokaryotes. We performed RNA-Seq on Newman strain and identified a set of 48 sRNA candidates. To avoid bioinformatic artefacts, we applied a series of cut-offs and tested experimentally each selected intergenic region. This narrowed the field to 24 expressed sRNAs, of which 21 were new and designated with Srn identifiers. Further examination of these loci revealed that one exhibited an unusual condensed sRNA cluster of about 650 nucleotides. We determined the transcriptional start sites within this region and demonstrated the presence of three contiguous sRNA genes (srn_9342, srn_9344 and srn_9345) expressed from the positive strand, and two others (srn_9343 and srn_9346) transcribed from the opposite one. Using comparative genomics, we showed that genetic organization of the srn_9342-9346 locus is specific to Newman and that its expression is growth-phase dependent and subjected to nutrient deprivation and oxidative stress. Finally, we demonstrated that srn_9343 encodes a secreted peptide that could belong to a novel S. aureus toxin-antitoxin system.