Thirty Years of sRNA-Mediated Regulation in Staphylococcus aureus: From Initial Discoveries to In Vivo Biological Implications.

Staphylococcus aureus is a widespread livestock and human pathogen that colonizes diverse microenvironments within its host. Its adaptation to the environmental conditions encountered within humans relies on coordinated gene expression. This requires a sophisticated regulatory network, among which regulatory RNAs (usually called sRNAs) have emerged as key players over the last 30 years. In S. aureus, sRNAs regulate target genes at the post-transcriptional level through base-pair interactions. The functional characterization of a subset revealed that they participate in all biological processes, including virulence, metabolic adaptation, and antibiotic resistance. In this review, we report 30 years of S. aureus sRNA studies, from their discovery to the in-depth characterizations of some of them. We also discuss their actual in vivo contribution, which is still lagging behind, and their place within the complex regulatory network. These shall be key aspects to consider in order to clearly uncover their in vivo biological functions.

Analysis of the CodY RNome reveals RsaD as a stress-responsive riboregulator of overflow metabolism in Staphylococcus aureus.

In Staphylococcus aureus, the transcription factor CodY modulates the expression of hundreds of genes, including most virulence factors, in response to the availability of key nutrients like GTP and branched-chain amino acids. Despite numerous studies examining how CodY controls gene expression directly or indirectly, virtually nothing is known about the extent to which CodY exerts its effect through small regulatory RNAs (sRNAs). Herein, we report the first set of sRNAs under the control of CodY. We reveal that staphylococcal sRNA RsaD is overexpressed >20-fold in a CodY-deficient strain in three S. aureus clinical isolates and in S. epidermidis. We validated the CodY-dependent regulation of rsaD and demonstrated that CodY directly represses rsaD expression by binding the promoter. Using a combination of molecular techniques, we show that RsaD posttranscriptionally regulates alsS (acetolactate synthase) mRNA and enzyme levels. We further show that RsaD redirects carbon overflow metabolism, contributing to stationary phase cell death during exposure to weak acid stress. Taken together, our data delineate a role for CodY in controlling sRNA expression in a major human pathogen and indicate that RsaD may integrate nutrient depletion and other signals to mount a response to physiological stress experienced by S. aureus in diverse environments.

A novel Staphylococcus aureus cis-trans type I toxin-antitoxin module with dual effects on bacteria and host cells.

Bacterial type I toxin-antitoxin (TA) systems are widespread, and consist of a stable toxic peptide whose expression is monitored by a labile RNA antitoxin. We characterized Staphylococcus aureus SprA2/SprA2AS module, which shares nucleotide similarities with the SprA1/SprA1AS TA system. We demonstrated that SprA2/SprA2AS encodes a functional type I TA system, with the cis-encoded SprA2AS antitoxin acting in trans to prevent ribosomal loading onto SprA2 RNA. We proved that both TA systems are distinct, with no cross-regulation between the antitoxins in vitro or in vivo. SprA2 expresses PepA2, a toxic peptide which internally triggers bacterial death. Conversely, although PepA2 does not affect bacteria when it is present in the extracellular medium, it is highly toxic to other host cells such as polymorphonuclear neutrophils and erythrocytes. Finally, we showed that SprA2AS expression is lowered during osmotic shock and stringent response, which indicates that the system responds to specific triggers. Therefore, the SprA2/SprA2AS module is not redundant with SprA1/SprA1AS, and its PepA2 peptide exhibits an original dual mode of action against bacteria and host cells. This suggests an altruistic behavior for S. aureus in which clones producing PepA2 in vivo shall die as they induce cytotoxicity, thereby promoting the success of the community.

SRD: a Staphylococcus regulatory RNA database.

An overflow of regulatory RNAs (sRNAs) was identified in a wide range of bacteria. We designed and implemented a new resource for the hundreds of sRNAs identified in Staphylococci, with primary focus on the human pathogen Staphylococcus aureus. The "Staphylococcal Regulatory RNA Database" (SRD, http://srd.genouest.org/) compiled all published data in a single interface including genetic locations, sequences and other features. SRD proposes novel and simplified identifiers for Staphylococcal regulatory RNAs (srn) based on the sRNA's genetic location in S. aureus strain N315 which served as a reference. From a set of 894 sequences and after an in-depth cleaning, SRD provides a list of 575 srn exempt of redundant sequences. For each sRNA, their experimental support(s) is provided, allowing the user to individually assess their validity and significance. RNA-seq analysis performed on strains N315, NCTC8325, and Newman allowed us to provide further details, upgrade the initial annotation, and identified 159 RNA-seq independent transcribed sRNAs. The lists of 575 and 159 sRNAs sequences were used to predict the number and location of srns in 18 S. aureus strains and 10 other Staphylococci. A comparison of the srn contents within 32 Staphylococcal genomes revealed a poor conservation between species. In addition, sRNA structure predictions obtained with MFold are accessible. A BLAST server and the intaRNA program, which is dedicated to target prediction, were implemented. SRD is the first sRNA database centered on a genus; it is a user-friendly and scalable device with the possibility to submit new sequences that should spread in the literature.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission.

Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.

Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum.

Unique peptide-morpholino oligomer (PMO) conjugates have been designed to bind and promote the cleavage of specific mRNA as a tool to inhibit gene function and parasite growth. The new conjugates were validated using the P. falciparum gyrase mRNA as a target (PfGyrA). Assays in vitro demonstrated a selective degradation of the PfGyrA mRNA directed by the external guide sequences, which are morpholino oligomers in the conjugates. Fluorescence microscopy revealed that labeled conjugates are delivered into Plasmodium-infected erythrocytes during all intraerythrocytic stages of parasite development. Consistent with the expression of PfGyrA in all stages of parasite development, proliferation assays showed that these conjugates have potent antimalarial activity, blocking early development, maturation, and replication of the parasite. The conjugates were equally effective against drug sensitive and resistant P. falciparum strains. The potency, selectivity, and predicted safety of PMO conjugates make this approach attractive for the development of a unique class of target-specific antimalarials and for large-scale functional analysis of the malarial genome.

Identification and functional analysis of the primary pantothenate transporter, PfPAT, of the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti.

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

RNAIII is linked with the pentose phosphate pathway through the activation of RpiRc in Staphylococcus aureus.

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.

Identification of gene encoding Plasmodium knowlesi phosphatidylserine decarboxylase by genetic complementation in yeast and characterization of in vitro maturation of encoded enzyme.

The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ∼5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.

PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity.

In the human malaria parasite Plasmodium falciparum, the synthesis of the major and essential membrane phospholipid, phosphatidylcholine, occurs via the CDP-choline and the serine decarboxylase phosphoethanolamine methylation (SDPM) pathways, which are fueled by host choline, serine, and fatty acids. Both pathways share the final two steps catalyzed by two essential enzymes, P. falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) and choline-phosphate transferase (PfCEPT). We identified a novel class of phospholipid mimetics, which inhibit the growth of P. falciparum as well as Leishmania and Trypanosoma species. Metabolic analyses showed that one of these compounds, PG12, specifically blocks phosphatidylcholine biosynthesis from both the CDP-choline and SDPM pathways via inhibition of PfCCT. In vitro studies using recombinant PfCCT showed a dose-dependent inhibition of the enzyme by PG12. The potent antimalarial of this compound, its low cytotoxicity profile, and its established mode of action make it an excellent lead to advance for further drug development and efficacy in vivo.

Forecasting Staphylococcus aureus Infections Using Genome-Wide Association Studies, Machine Learning, and Transcriptomic Approaches.

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.

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens.

Bacterial gene expression is under the control of a large set of molecules acting at multiple levels. In addition to the transcription factors (TFs) already known to be involved in global regulation of gene expression, small regulatory RNAs (sRNAs) are emerging as major players in gene regulatory networks, where they allow environmental adaptation and fitness. Developments in high-throughput screening have enabled their detection in the entire bacterial kingdom. These sRNAs influence a plethora of biological processes, including but not limited to outer membrane synthesis, metabolism, TF regulation, transcription termination, virulence, and antibiotic resistance and persistence. Almost always noncoding, they regulate target genes at the post-transcriptional level, usually through base-pair interactions with mRNAs, alone or with the help of dedicated chaperones. There is growing evidence that sRNA-mediated mechanisms of actions are far more diverse than initially thought, and that they go beyond the so-called cis- and trans-encoded classifications. These molecules can be derived and processed from 5' untranslated regions (UTRs), coding or non-coding sequences, and even from 3' UTRs. They usually act within the bacterial cytoplasm, but recent studies showed sRNAs in extracellular vesicles, where they influence host cell interactions. In this review, we highlight the various functions of sRNAs in bacterial pathogens, and focus on the increasing examples of widely diverse regulatory mechanisms that might compel us to reconsider what constitute the sRNA.

sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide.

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.

Small RNAs in vancomycin-resistant Enterococcus faecium involved in daptomycin response and resistance.

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.

Genome Sequence of the Clinical Isolate Staphylococcus aureus subsp. aureus Strain UAMS-1.

We report here the draft genome sequence of Staphylococcus aureus subsp. aureus strain UAMS-1. UAMS-1 is a virulent oxacillin-susceptible clinical isolate. Its genome is composed of 2,763,963 bp and will be useful for further gene expression analysis using RNA sequencing (RNA-seq) technology.

Structure, function and inhibition of the phosphoethanolamine methyltransferases of the human malaria parasites Plasmodium vivax and Plasmodium knowlesi.

Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.

Draft Genome Sequence of Staphylococcus aureus subsp. aureus Strain HG003, an NCTC8325 Derivative.

We report the draft genome sequence of a Staphylococcus aureus NCTC8325 derivative, strain HG003. HG003 contains functional global regulators rsbU and tcaR and is therefore considered as a reference for studies of regulation and virulence. The genome is composed of 2,797,898 bp and will be essential for subsequent RNAseq analysis.

Listeria monocytogenes differential transcriptome analysis reveals temperature-dependent Agr regulation and suggests overlaps with other regulons.

Listeria monocytogenes is a ubiquitous, opportunistic pathogenic organism. Environmental adaptation requires constant regulation of gene expression. Among transcriptional regulators, AgrA is part of an auto-induction system. Temperature is an environmental cue critical for in vivo adaptation. In order to investigate how temperature may affect AgrA-dependent transcription, we compared the transcriptomes of the parental strain L. monocytogenes EGD-e and its ΔagrA mutant at the saprophytic temperature of 25°C and in vivo temperature of 37°C. Variations of transcriptome were higher at 37°C than at 25°C. Results suggested that AgrA may be involved in the regulation of nitrogen transport, amino acids, purine and pyrimidine biosynthetic pathways and phage-related functions. Deregulations resulted in a growth advantage at 37°C, but affected salt tolerance. Finally, our results suggest overlaps with PrfA, σB, σH and CodY regulons. These overlaps may suggest that through AgrA, Listeria monocytogenes integrates information on its biotic environment.

Phosphoethanolamine methyltransferases in phosphocholine biosynthesis: functions and potential for antiparasite therapy.

S-adenosyl-L-methionine (SAM)-dependent methyltransferases represent a diverse group of enzymes that catalyze the transfer of a methyl group from a methyl donor SAM to nitrogen, oxygen, sulfur or carbon atoms of a large number of biologically active large and small molecules. These modifications play a major role in the regulation of various biological functions such as gene expression, signaling, nuclear division and metabolism. The three-step SAM-dependent methylation of phosphoethanolamine to form phosphocholine catalyzed by phosphoethanolamine N-methyltransferases (PMTs) has emerged as an important biochemical step in the synthesis of the major phospholipid, phosphatidylcholine, in some eukaryotes. PMTs have been identified in nematodes, plants, African clawed frogs, zebrafish, the Florida lancelet, Proteobacteria and human malaria parasites. Data accumulated thus far suggest an important role for these enzymes in growth and development. This review summarizes published studies on the biochemical and genetic characterization of these enzymes, and discusses their evolution and their suitability as targets for the development of therapies against parasitic infections, as well as in bioengineering for the development of nutritional and stress-resistant plants.