Cédric Romilly

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

A Non-Coding RNA Promotes Bacterial Persistence and Decreases Virulence by Regulating a Regulator in Staphylococcus aureus

Staphylococcus aureus produces a high number of RNAs for which the functions are poorly understood. Several non-coding RNAs carry a C-rich sequence suggesting that they regulate mRNAs at the post-transcriptional level. We demonstrate that the Sigma B-dependent RsaA RNA represses the synthesis of the global transcriptional regulator MgrA by forming an imperfect duplex with the Shine and Dalgarno sequence and a loop-loop interaction within the coding region of the target mRNA. These two recognition sites are required for translation repression. Consequently, RsaA causes enhanced production of biofilm and a decreased synthesis of capsule formation in several strain backgrounds. These phenotypes led to a decreased protection of S. aureus against opsonophagocytic killing by polymorphonuclear leukocytes compared to the mutant strains lacking RsaA. Mice animal models showed that RsaA attenuates the severity of acute systemic infections and enhances chronic catheter infection. RsaA takes part in a regulatory network that contributes to the complex interactions of S. aureus with the host immune system to moderate invasiveness and favour chronic infections. It is the first example of a conserved small RNA in S. aureus functioning as a virulence suppressor of acute infections. Because S. aureus is essentially a human commensal, we propose that RsaA has been positively selected through evolution to support commensalism and saprophytic interactions with the host.

Current knowledge on regulatory RNAs and their machineries in Staphylococcus aureus

Staphylococcus aureus is one of the major human pathogens, which causes numerous community-associated and hospital-acquired infections. The regulation of the expression of numerous virulence factors is coordinated by complex interplays between two component systems, transcriptional regulatory proteins, and regulatory RNAs. Recent studies have identified numerous novel RNAs comprising cis-acting regulatory RNAs, antisense RNAs, small non coding RNAs and small mRNAs encoding peptides. We present here several examples of RNAs regulating S. aureus pathogenicity and describe various aspects of antisense regulation.

RNA-mediated regulation in bacteria: from natural to artificial systems

Bacteria use various means of RNA-mediated gene regulation. Regulatory RNAs include mRNA leaders that affect expression in cis or in trans, non-coding RNAs that trap regulatory proteins or interact with one or multiple target mRNAs, and RNAs that protect the bacteria against foreign and invasive DNA. The aim of this review is to outline the basic principles of bacterial RNA-mediated regulation, with a special focus on both cis-acting regulatory regions of mRNAs and antisense RNAs (asRNAs), and to give a brief overview of selected examples of RNA-based technology that have paved the way for biotechnological applications.

A nitric oxide regulated small RNA controls expression of genes involved in redox homeostasis in Bacillus subtilis.

RsaE is the only known trans-acting small regulatory RNA (sRNA) besides the ubiquitous 6S RNA that is conserved between the human pathogen Staphylococcus aureus and the soil-dwelling Firmicute Bacillus subtilis. Although a number of RsaE targets are known in S. aureus, neither the environmental signals that lead to its expression nor its physiological role are known. Here we show that expression of the B. subtilis homolog of RsaE is regulated by the presence of nitric oxide (NO) in the cellular milieu. Control of expression by NO is dependent on the ResDE two-component system in B. subtilis and we determined that the same is true in S. aureus. Transcriptome and proteome analyses revealed that many genes with functions related to oxidative stress and oxidation-reduction reactions were up-regulated in a B. subtilis strain lacking this sRNA. We have thus renamed it RoxS. The prediction of RoxS-dependent mRNA targets also suggested a significant enrichment for mRNAs related to respiration and electron transfer. Among the potential direct mRNA targets, we have validated the ppnKB mRNA, encoding an NAD+/NADH kinase, both in vivo and in vitro. RoxS controls both translation initiation and the stability of this transcript, in the latter case via two independent pathways implicating RNase Y and RNase III. Furthermore, RNase Y intervenes at an additional level by processing the 5′ end of the RoxS sRNA removing about 20 nucleotides. Processing of RoxS allows it to interact more efficiently with a second target, the sucCD mRNA, encoding succinyl-CoA synthase, thus expanding the repertoire of targets recognized by this sRNA.

Loop-loop interactions involved in antisense regulation are processed by the endoribonuclease III in Staphylococcus aureus

The endoribonuclease III (RNase III) belongs to the enzyme family known to process double-stranded RNAs. Staphylococcus aureus RNase III was shown to regulate, in concert with the quorum sensing induced RNAIII, the degradation of several mRNAs encoding virulence factors and the transcriptional repressor of toxins Rot. Two of the mRNA-RNAIII complexes involve fully base paired loop-loop interactions with similar sequences that are cleaved by RNase III at a unique position. We show here that the sequence of the base pairs within the loop-loop interaction is not critical for RNase III cleavage, but that the co-axial stacking of three consecutive helices provides an ideal topology for RNase III recognition. In contrast, RNase III induces several strong cleavages in a regular helix, which carries a sequence similar to the loop-loop interaction. The introduction of a bulged loop that interrupts the regular helix restrains the number of cleavages. This work shows that S. aureus RNase III is able to bind and cleave a variety of RNA-mRNA substrates, and that specific structure elements direct the action of RNase III.

In vivo mapping of RNA-RNA interactions in Staphylococcus aureus using the endoribonuclease III.

Ribonucleases play key roles in gene regulation and in the expression of virulence factors in Staphylococcus aureus. Among these enzymes, the double-strand specific endoribonuclease III (RNase III) is a key mediator of mRNA processing and degradation. Recently, we have defined, direct target sites for RNase III processing on a genome-wide scale in S. aureus. Our approach is based on deep sequencing of cDNA libraries obtained from RNAs isolated by in vivo co-immunoprecipitation with wild-type RNase III and two cleavage-defective mutants. The use of such catalytically inactivated enzymes, which still retain their RNA binding capacity, allows the identification of novel RNA substrates of RNase III. In this report, we will summarize the diversity of RNase III functions, discuss the advantages and the limitations of the approach, and how this strategy identifies novel mRNA targets of small non-coding RNAs in S. aureus.

A Current Overview of Regulatory RNAs in Staphylococcus Aureus

Staphylococcus aureus is a common commensal bacterial species that is usually found in the nose and on the skin of 30 % of healthy adults. However, the bacterium is a remarkably versatile pathogen that is one of the main causes of community as well as hospital-acquired infections (Cheung et al., 2004; Novick, 2003). S. aureus is responsible for systemic infections such as sepsis and endocarditis, which can be difficult to treat due to the acquisition of resistance towards numerous antibiotics in clinical use. S. aureus causes a wide spectrum of human diseases in part due to its ability to produce an array of virulence factors, which are mostly encoded by laterally acquired genomic regions, the so-called pathogenicity islands. These factors include surface proteins responsible for the adhesion and invasion of the host, exoproteins required for host immune evasion, and toxins involved in dissemination in host tissues and acquisition of nutrients (Novick, 2003). Redundancies exist to ensure that a productive infection still occurs even though one factor may be lost. In recent decades, many studies have been carried out to understand how S. aureus is able to coordinate the expression of a large panel of virulence factors at the appropriate time in order to facilitate successful infections (Novick and Geisinger, 2008). These works offer the possibility for developing anti-virulence therapies as alternative strategies for affecting the bacteria viability, i. e. by inhibiting the expression of the virulence factors that cause host damage or the interaction between the pathogen and the host (Clatworthy et al., 2007). Inhibiting virulence instead of viability may have little impact on human flora and result in weaker selective pressure for the development of antibiotic resistance. Hence, determining the regulatory networks and the dynamics involved in virulence and in fast adaptive responses are of prime importance to combating S. aureus infections.