Complex Regulation of Gamma-Hemolysin Expression Impacts Staphylococcus aureus Virulence.

Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome-encoded pore-forming toxin that targets the C5a receptor, similar to the phage-encoded Panton-Valentine leucocidin (PVL). Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in the HlgC and HlgB yields between isolates. Moreover, although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. To decipher the molecular basis for the variation in hlgCB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Both the HlgCB level and the HlgC/HlgB ratio were found to depend on hlgC promoter activity and mRNA processing and translation. Strikingly, only one single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR) of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in the level of HlgC but not in HlgB. Finally, we found that high levels of HlgCB synthesis led to mortality in a rabbit model of pneumonia, correlated with the implication of the role of HlgCB in severe S. aureus CAP. Taken together, this work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect where subtle genomic variations have a major impact on phenotype and virulence. IMPORTANCE S. aureus virulence in pneumonia results in its ability to produce several virulence factors, including the leucocidin PVL. Here, we demonstrate that HlgCB, another leucocidin, which targets the same receptors as PVL, highly contributes to S. aureus virulence in pvl-negative strains. In addition, considerable variations in HlgCB quantities are observed among clinical isolates from patients with CAP. Biomolecular analyses have revealed that a few SNPs in the promoter sequences and only one SNP in the 5' UTR of hlgCB mRNA induce the differential expression of hlgCB, drastically impacting hlgC mRNA translation. This work illustrates the subtlety of regulatory mechanisms in bacteria, especially the sometimes major effects on phenotypes of single nucleotide variation in noncoding regions.

RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.

The 3'UTR-derived sRNA RsaG coordinates redox homeostasis and metabolism adaptation in response to glucose-6-phosphate uptake in Staphylococcus aureus.

Staphylococcus aureus RsaG is a 3'-untranslated region (3'UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5'- to 3'-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5'-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3'UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3'UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches.

Assembling the Current Pieces: The Puzzle of RNA-Mediated Regulation in Staphylococcus aureus.

The success of the major opportunistic human Staphylococcus aureus relies on the production of numerous virulence factors, which allow rapid colonization and dissemination in any tissues. Indeed, regulation of its virulence is multifactorial, and based on the production of transcriptional factors, two-component systems (TCS) and small regulatory RNAs (sRNAs). Advances in high-throughput sequencing technologies have unveiled the existence of hundreds of potential RNAs with regulatory functions, but only a fraction of which have been validated in vivo. These discoveries have modified our thinking and understanding of bacterial physiology and virulence fitness by placing sRNAs, alongside transcriptional regulators, at the center of complex and intertwined regulatory networks that allow S. aureus to rapidly adapt to the environmental cues present at infection sites. In this review, we describe the recently acquired knowledge of characterized regulatory RNAs in S. aureus that are associated with metal starvation, nutrient availability, stress responses and virulence. These findings highlight the importance of sRNAs for the comprehension of S. aureus infection processes while raising questions about the interplay between these key regulators and the pathways they control.

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria.

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures.

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5'UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.

Navigation through the twists and turns of RNA sequencing technologies: Application to bacterial regulatory RNAs.

Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.

Differential evolution in 3'UTRs leads to specific gene expression in Staphylococcus.

The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.

The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs.

Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable.

RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation.

The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. 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. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3'UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.

A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.

Complete Genome Sequence and Annotation of the Staphylococcus aureus Strain HG001.

Staphylococcus aureus is an opportunistic Gram-positive pathogen responsible for a wide range of infections from minor skin abscesses to life-threatening diseases. Here, we report the draft genome assembly and current annotation of the HG001 strain, a derivative of the RN1 (NCT8325) strain with restored rbsU (a positive activator of SigB).

The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.

The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non-coding RNAs (sRNAs). Many of the sRNAs regulate gene expression through base-pairings with mRNAs. However, characterization of the direct sRNA targets in Gram-positive bacteria remained a difficult challenge. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaA sRNA of S. aureus, known to repress the synthesis of the transcriptional regulator MgrA. Several mRNAs were enriched with RsaA expanding its regulatory network. Besides mgrA, several of these mRNAs encode a family of SsaA-like enzymes involved in peptidoglycan metabolism and the secreted anti-inflammatory FLIPr protein. Using a combination of in vivo and in vitro approaches, these mRNAs were validated as direct RsaA targets. Quantitative differential proteomics of wild-type and mutant strains corroborated the MAPS results. Additionally, it revealed that RsaA indirectly activated the synthesis of surface proteins supporting previous data that RsaA stimulated biofilm formation and favoured chronic infections. All together, this study shows that MAPS could also be easily applied in Gram-positive bacteria for identification of sRNA targetome.

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.

Various checkpoints prevent the synthesis of Staphylococcus aureus peptidoglycan hydrolase LytM in the stationary growth phase.

In Staphylococcus aureus, peptidoglycan metabolism plays a role in the host inflammatory response and pathogenesis. Transcription of the peptidoglycan hydrolases is activated by the essential 2-component system WalKR at low cell density. During stationary growth phase, WalKR is not active and transcription of the peptidoglycan hydrolase genes is repressed. In this work, we studied regulation of expression of the glycylglycine endopeptidase LytM. We show that, in addition to the transcriptional regulation mediated by WalKR, the synthesis of LytM is negatively controlled by a unique mechanism at the stationary growth phase. We have identified 2 different mRNAs encoding lytM, which vary in the length of their 5' untranslated (5'UTR) regions. LytM is predominantly produced from the WalKR-regulated mRNA transcript carrying a short 5'UTR. The lytM mRNA is also transcribed as part of a polycistronic operon with the upstream SA0264 gene and is constitutively expressed. Although SA0264 protein can be synthesized from the longer operon transcript, lytM cannot be translated because its ribosome-binding site is sequestered into a translationally inactive secondary structure. In addition, the effector of the agr system, RNAIII, can inhibit translation of lytM present on the operon without altering the transcript level but does not have an effect on the translation of the upstream gene. We propose that this dual regulation of lytM expression, at the transcriptional and post-transcriptional levels, contributes to prevent cell wall damage during the stationary phase of growth.

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.