Isabelle Caldelari

Sensing of 'danger signals' and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of Toll

In drosophila, fungal and Gram-positive bacterial molecular determinants are detected by circulating pattern recognition receptors (PRRs). Previous findings suggest that these PRRs activate yet unidentified serine protease cascades culminating in the cleavage of Spaetzle, the endogenous Toll receptor ligand, and triggering the immune response. We demonstrate here that the Grass protease defines a common activation cascade for PRR-mediated fungal and Gram-positive bacterial detection. The serine protease Persephone, previously shown to be specific for fungal detection in a cascade activated by secreted fungal proteases, was also required for sensing of proteases elicited by bacteria in the hemolymph. Hence, Persephone defines a parallel proteolytic cascade activated by danger signals such as abnormal proteolytic activities.

RNA-Mediated Regulation in Pathogenic Bacteria

Pathogenic bacteria possess intricate regulatory networks that temporally control the production of virulence factors, and enable the bacteria to survive and proliferate after host infection. Regulatory RNAs are now recognized as important components of these networks, and their study may not only identify new approaches to combat infectious diseases but also reveal new general control mechanisms involved in bacterial gene expression. In this review, we illustrate the diversity of regulatory RNAs in bacterial pathogens, their mechanism of action, and how they can be integrated into the regulatory circuits that govern virulence-factor production.

Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression.

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.

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.

Novel aspects of RNA regulation in Staphylococcus aureus

A plethora of RNAs with regulatory functions has been discovered in many non‐pathogenic and pathogenic bacteria. In Staphylococcus aureus, recent findings show that a large variety of RNAs control target gene expression by diverse mechanisms and many of them are expressed in response to specific internal or external signals. These RNAs comprise trans‐acting RNAs, which regulate gene expression through binding with mRNAs, and cis‐acting regulatory regions of mRNAs. Some of them possess multiple functions and encode small but functional peptides. In this review, we will present several examples of RNAs regulating pathogenesis, antibiotic resistance, and host‐pathogen interactions and will illustrate how regulatory proteins and RNAs form complex regulatory circuits to express the virulence factors in a dynamic manner.

Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression.

Staphylococcus aureus RNAIII is one of the main intracellular effectors of the quorum-sensing system. It is a multifunctional RNA that encodes a small peptide, and its noncoding parts act as antisense RNAs to regulate the translation and/or the stability of mRNAs encoding transcriptional regulators, major virulence factors, and cell wall metabolism enzymes. In this review, we explain how regulatory proteins and RNAIII are embedded in complex regulatory circuits to express virulence factors in a dynamic and timely manner in response to stress and environmental and metabolic changes.

The importance of regulatory RNAs in Staphylococcus aureus

RNA molecules with regulatory functions in pathogenic bacteria have benefited from a renewed interest these two last decades. In Staphylococcus aureus, recent genome-wide approaches have led to the discovery that almost 10-20% of genes code for RNAs with critical regulatory roles in adaptive processes. These RNAs include trans-acting RNAs, which mostly act through binding to target mRNAs, and cis-acting RNAs, which include regulatory regions of mRNAs responding to various metabolic signals. Besides recent analysis of S. aureus transcriptome has revealed an unprecedented existence of pervasive transcription generating a high number of weakly expressed antisense RNAs along the genome as well as numerous mRNAs with overlapped regions. Here, we will illustrate the diversity of trans-acting RNAs and illustrate how they are integrated into complex regulatory circuits, which link metabolism, stress response and virulence.

Multiple ways to regulate translation initiation in bacteria: Mechanisms, regulatory circuits, dynamics.

To adapt their metabolism rapidly and constantly in response to environmental variations, bacteria often target the translation initiation process, during which the ribosome assembles on the mRNA. Here, we review different mechanisms of regulation mediated by cis-acting elements, sRNAs and proteins, showing, when possible, their intimate connection with the translational apparatus. Indeed the ribosome itself could play a direct role in several regulatory mechanisms. Different features of the regulatory signals (sequences, structures and their positions on the mRNA) are contributing to the large variety of regulatory mechanisms. Ribosome heterogeneity, variation of individual cells responses and the spatial and temporal organization of the translation process add more layers of complexity. This hampers to define manageable set of rules for bacterial translation initiation control.

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.