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The mosquito antiviral response has been mainly studied in the case of arthropod-borne virus (arbovirus) infection in female mosquitoes. However, in nature, both female and male mosquitoes are abundantly infected with insect-specific viruses (ISVs). ISVs are capable of infecting the reproductive organs of both sexes and are maintained primarily by vertical transmission. Since the RNA interference (RNAi)-mediated antiviral response plays an important antiviral role in mosquitoes, ISVs constitute a relevant model to study sex-dependent antiviral responses. Using a naturally generated viral stock containing three distinct ISVs, Aedes flavivirus (AEFV), Menghai Rhabdovirus (MERV) and Shinobi tetra virus (SHTV), we infected adult Aedes albopictus females and males and generated small RNA libraries from ovaries, testes, and the remainder of the body. Overall, both female and male mosquitoes showed unique small RNA profiles to each co-infecting ISV regardless the sex or tissue tested. While all three ISVs generated virus-derived siRNAs, only MERV generated virus-derived piRNAs. We also studied the expression of PIWI genes in reproductive tissues and carcasses. Piwi1-4 were abundantly expressed in ovaries and testes in contrast to Piwi5-9, suggesting that Piwi 5-9 are involved in exogenous viral piRNA production. Together, our results show that ISV-infected Aedes albopictus produce viral small RNAs in a virus-specific manner and that male mosquitoes mount a similar small RNA-mediated antiviral response to that of females.

Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria., IMPORTANCE The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq-encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.

International audience; In bacteria-host interactions, competition for iron is critical for the outcome of the infection. As a result of its redox properties, this metal is essential for the growth and proliferation of most living organisms, including pathogenic bacteria. This metal is also potentially toxic, making the precise maintenance of iron homeostasis necessary for survival. Iron acquisition and storage control is mediated in most bacteria by the global ferric uptake regulator (Fur) and iron-responsive small regulatory non-coding RNAs (RyhB in the model organism Escherichia coli). While the role of these regulators in iron homeostasis is well documented in both pathogenic and non-pathogenic bacteria, many recent studies also demonstrate that these regulators are involved in the virulence of pathogenic bacteria. By sensing iron availability in the environment, Fur and RyhB are able to regulate, either directly or indirectly via other transcriptional regulators or modulation of intracellular iron concentration, many virulence determinants of pathogenic bacteria. Iron is thus both a nutritional and regulatory element, allowing bacteria to adapt to various host environments by adjusting expression of virulence factors. In this review, we present evidences that Fur and RyhB are the major regulators of this adaptation, as they are involved in diverse functions ranging from iron homeostasis to regulation of virulence by mediating key pathogen responses such as invasion of eukaryotic cells, toxin production, motility, quorum sensing, stress resistance or biofilm formation. Therefore, Fur and RyhB play a major role in regulating an adaptative response during bacterial infections, making them important targets in the fight against pathogenic bacteria.

In Escherichia coli , the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a Δ ryhB mutant and a Δ fur Δ ryhB double mutant showed significantly reduced bladder colonization. The Δ fur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the Δ ryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the Δ ryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli .

International audience; The RpoS/σS sigma subunit of RNA polymerase (RNAP) controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses. σS also contributes to biofilm formation and virulence of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). In this study, we used directional RNA-sequencing and complementary assays to explore the σS-dependent transcriptome of S. Typhimurium during late stationary phase in rich medium. This study confirms the large regulatory scope of σS and provides insights into the physiological functions of σS in Salmonella. Extensive regulation by σS of genes involved in metabolism and membrane composition, and down-regulation of the respiratory chain functions, were important features of the σS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions. As an example, we show that arginine catabolism confers a competitive fitness advantage in stationary phase. This study also provides a firm basis for future studies to address molecular mechanisms of indirect regulation of gene expression by σS. Importantly, the σS-controlled downstream network includes small RNAs that might endow σS with post-transcriptional regulatory functions. Of these, four (RyhB-1/RyhB-2, SdsR, SraL) were known to be controlled by σS and deletion of the sdsR locus had a competitive fitness cost in stationary phase. The σS-dependent control of seven additional sRNAs was confirmed in Northern experiments. These findings will inspire future studies to investigate molecular mechanisms and the physiological impact of post-transcriptional regulation by σS.

International audience; The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen.

International audience; RydC pseudoknot aided by Hfq is a dynamic regulatory module. We report that RydC reduces expression of curli-specific gene D transcription factor required for adhesion and biofilm production in enterobacteria. During curli formation, csgD messenger RNA (mRNA) synthesis increases when endogenous levels of RydC are lacking. In Escherichia coli and Salmonella enterica, stimulation of RydC expression also reduces biofilm formation by impairing curli synthesis. Inducing RydC early on in growth lowers CsgA, -B and -D protein and mRNA levels. RydC's 5'-domain interacts with csgD mRNA translation initiation signals to prevent initiation. Translation inhibition occurs by an antisense mechanism, blocking the translation initiation signals through pairing, and that mechanism is facilitated by Hfq. Although Hfq represses csgD mRNA translation without a small RNA (sRNA), it forms a ternary complex with RydC and facilitates pseudoknot unfolding to interact with the csgD mRNA translation initiation signals. RydC action implies Hfq-assisted unfolding and mRNA rearrangements, but once the pseudoknot is disrupted, Hfq is unnecessary for regulation. RydC is the sixth sRNA that negatively controls CsgD synthesis. Hfq induces structural changes in the mRNA domains targeted by these six sRNAs. What we describe is an ingenious process whereby pseudoknot opening is orchestrated by a chaperone to allow RNA control of gene expression.

RNA–mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2−/− mice and that the Sox9 paramutation was also not established in Dnmt2−/− embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA–mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA–mediated epigenetic heredity., Author Summary The possibility of a mode of inheritance distinct from the Mendelian model has been considered since the early days of genetics. Only recently, however, suitable experimental models were created. We now see the development of new experimental systems detecting non-Mendelian inheritance in a variety of organisms, from worms to mice. We have previously shown that RNA molecules act as transgenerational inducers of epigenetic variations in mice. We are currently using Mendelian genetics to dissect the factors involved in RNA–mediated transgenerational signalling. By showing an absolute requirement for Dnmt2 in this process, our study extends our knowledge of this still somewhat enigmatic protein. We confirmed that RNA rather than DNA methylation by the protein is involved in epigenetic heredity, and our genetic results indicate a requirement during an early step in the reproductive process, between parental gametogenesis and the preimplantation stage.

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen., Author Summary The emergent human pathogen Clostridium difficile is a major cause of nosocomial diarrhea associated with antibiotic therapy. During the last few years, severe forms of C. difficile infections became more frequent due to the emergence of hypervirulent isolates. Despite intensive studies, many questions regarding the mechanisms controlling C. difficile virulence remain unanswered. We hypothesized that C. difficile, a member of an ancient group of bacteria, might widely use ancestral RNA–based mechanisms to control its gene expression for better adaptation to host conditions. Indeed, using next-generation sequencing technology, we identified a great number and a large diversity of potential RNA regulators in this pathogen. We obtained experimental evidence for regulatory roles of a particular class of regulatory RNAs responding to c-di-GMP, a universal bacterial signaling molecule regulating motility, biofilm formation, and virulence. We also detected abundant small RNA products of recently discovered adaptive prokaryotic immunity CRISPR-Cas systems that might be important for C. difficile survival in gut communities. Our findings suggest that small RNA molecules may play a major role in regulatory processes during C. difficile infection cycle and as such are promising targets of new therapeutic strategies.

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes. ISSN:1362-4962 ISSN:0301-5610

International audience; Non-coding RNAs (ncRNAs) play critical roles in all physiological processes. Many ncRNAs have also been implicated in a variety of pathological disorders, including cancer. This review provides an overview of the roles of small ncRNAs in infectious diseases and genetic disorders, including diseases of the CNS and various cancers. There is a focus on the consequences of microRNA (miRNA) de-regulation in human diseases, and recent progress in understanding other disease-related classes of ncRNAs, such as small nucleolar RNAs (snoRNAs), is presented. Finally, this review provides a brief perspective on new and powerful diagnostic strategies for human diseases that are based on ncRNA analysis, and presents novel potential approaches using ncRNAs as tools or targets for the treatment of such diseases.