In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

The Development of Thermoresponsive Polymeric Simvastatin Prodrug for the Treatment of Experimental Periodontitis in Rats.

Periodontitis (PD) is a severe inflammatory gum pathology that damages the periodontal soft tissue and bone. It is highly prevalent in the US, affecting more than 47% of adults. Besides routine scaling and root planing, there are few effective treatments for PD. Developed as an effective treatment for hyperlipidemia, simvastatin (SIM) is also known for its well-established anti-inflammatory and osteogenic properties, suggesting its potential utility in treating PD. Its clinical translation, however, has been impeded by its poor water-solubility, lack of osteotropicity, and side effects (e.g., hepatoxicity) associated with systemic exposure. To address these challenges, an N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-based thermoresponsive polymeric prodrug of SIM (ProGel-SIM) was developed as a local therapy for PD. Its aqueous solution is free-flowing at 4 °C and transitions into a hydrogel at ∼30 °C, allowing for easy local application and retention. After a thorough characterization of its physicochemical properties, ProGel-SIM was administered weekly into the periodontal pocket of an experimental rat model of PD. At 3 weeks post initiation of the treatment, the animals were euthanized with palate isolated for µ-CT and histological analyses. When compared to dose equivalent simvastatin acid (SMA, active form of SIM) treatment, the rats in the ProGel-SIM treated group showed significantly higher periodontal bone volume (0.34 mm3 vs 0.20 mm3, P = 0.0161) and less neutrophil (PMN) infiltration (P < 0.0001) and IL-1ß secretion (P = 0.0036). No measurable side effect was observed. Collectively, these results suggest that ProGel-SIM may be developed as a promising drug candidate for the effective clinical treatment of PD.

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1.

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different ß-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability.

The Systems Architecture of Molecular Memory in Poplar after Abiotic Stress.

Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar (Populus× canescens) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across five major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression profiles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis (Arabidopsis thaliana) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-specific components contributes to the coordination of post-recovery responses to stress in woody plants.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

The salmon louse genome: Copepod features and parasitic adaptations.

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

IL-4-BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy.

BACKGROUND: This study group has previously identified IL-9-producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. OBJECTIVES: This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. METHODS: An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow-derived MMC9 culture system was used to define IL-4-BATF signaling in MMC9 development. RESULTS: Epicutaneous sensitization- and bone marrow reconstitution-based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. Insilico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. CONCLUSIONS: IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts.

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

Thermoresponsive Hydrogel-Based Local Delivery of Simvastatin for the Treatment of Periodontitis.

Except for routine scaling and root planing, there are few effective nonsurgical therapeutic interventions for periodontitis and associated alveolar bone loss. Simvastatin (SIM), one of the 3-hydroxy-3-methylglutaryl-cosenzyme A reductase inhibitors, which is known for its capacity as a lipid-lowering medication, has been proven to be an effective anti-inflammatory and bone anabolic agent that has shown promising benefits in mitigating periodontal bone loss. The local delivery of SIM into the periodontal pocket, however, has been challenging due to SIM's poor water solubility and its lack of osteotropicity. To overcome these issues, we report a novel SIM formulation of a thermoresponsive, osteotropic, injectable hydrogel (PF127) based on pyrophosphorolated pluronic F127 (F127-PPi). After mixing F127-PPi with F127 at a 1:1 ratio, the resulting PF127 was used to dissolve free SIM to generate the SIM-loaded formulation. The thermoresponsive hydrogel's rheologic behavior, erosion and SIM release kinetics, osteotropic property, and biocompatibility were evaluated in vitro. The therapeutic efficacy of SIM-loaded PF127 hydrogel on periodontal bone preservation and inflammation resolution was validated in a ligature-induced periodontitis rat model. Given that SIM is already an approved medication for hyperlipidemia, the data presented here support the translational potential of the SIM-loaded PF127 hydrogel for better clinical management of periodontitis and associated pathologies.

Association of tracking oral hygiene with dental outcomesin a general dental practice.

PURPOSE: To track plaque scores on a subset of teeth in general dental practice patients to determine if plaque scores could improve along with periodontal, restorative and extraction outcomes. METHODS: Percentage of surfaces with subgingival plaque were recorded and graphed on five teeth (#3, 8, 14, 19, 30) at each appointment, followed by focused oral hygiene instructions, in 343 patients over a 5-10-year period. Patient age, gender, prophylaxes/year, and experimental teeth periodontitis stage, % 4-5 and ≥ 6 mm pockets, % bleeding on probing, % surfaces restored and patients with extractions were recorded. Relationships among average plaque scores and the longitudinal periodontal, restorative and extraction changes were analyzed using Chi-Square, Kruskal-Wallis, and Wilcoxon Rank Sum tests. RESULTS: Plaque scores improved from median 40% to 25% (P< 0.0001) over the 5-10 years. Plaque scores and periodontitis stages were associated (P= 0.03) with few periodontally healthy patients (9%) having poor plaque scores (> 50% plaque surfaces). Furthermore, good plaque scores (≤ 25%) and periodontal health (Stage I) were linked to the need for few restorations (P< 0.0001), while prophylaxes/year had no significant relationship. Extractions were related more with Stage III/IV (advanced) periodontitis (P< 0.0001) than with plaque score (NS). CLINICAL SIGNIFICANCE: In a general dental practice, tracking plaque scores at each appointment on a subset of representative teeth can be time-efficient, and is associated with improved oral hygiene, stable periodontal status and reduced restorative needs.

Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo.

The molecular roles of many RNA-binding proteins in bacterial post-transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP-seq) have begun to revolutionize the transcriptome-wide mapping of eukaryotic RNA-binding protein target sites. We have applied CLIP-seq to chart the target landscape of two major bacterial post-transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single-nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3'-located Rho-independent terminators as a universal motif involved in Hfq-RNA interactions. Additionally, Hfq preferentially binds 5' to sRNA-target sites in mRNAs, and 3' to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA-mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA-target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP-seq approach will bring new insights into post-transcriptional gene regulation by RNA-binding proteins in diverse bacterial species.

Engineering Biomimetic Nanofiber Microspheres with Tailored Size, Predesigned Structure, and Desired Composition via Gas Bubble-Mediated Coaxial Electrospray.

Minimally invasive therapies avoiding surgical complexities evoke great interest in developing injectable biomedical devices. Herein, a versatile approach is reported for engineering injectable and biomimetic nanofiber microspheres (NMs) with tunable sizes, predesigned structures, and desired compositions via gas bubble-mediated coaxial electrospraying. The sizes and structures of NMs are controlled by adjusting processing parameters including air flow rate, applied voltage, distance, and spinneret configuration in the coaxial setup. Importantly, unlike the self-assembly method, this technique can be used to fabricate NMs from any material feasible for electrospinning or other nanofiber fabrication techniques. To demonstrate the versatility, open porous NMs are successfully fabricated that consist of various short nanofibers made of poly(ε-caprolactone), poly(lactic-co-glycolic acid), gelatin, methacrylated gelatin, bioglass, and magneto-responsive polymer composites. Open porous NMs support human neural progenitor cell growth in 3D with a larger number and more neurites than nonporous NMs. Additionally, highly open porous NMs show faster cell infiltration and host tissue integration than nonporous NMs after subcutaneous injection to rats. Such a novel class of NMs holds great potential for many biomedical applications such as tissue filling, cell and drug delivery, and minimally invasive tissue regeneration.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

The DNMT3A R882H mutant displays altered flanking sequence preferences.

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

Tethering peptides onto biomimetic and injectable nanofiber microspheres to direct cellular response.

Biomimetic and injectable nanofiber microspheres (NMs) could be ideal candidate for minimally invasive tissue repair. Herein, we report a facile approach to fabricate peptide-tethered NMs by combining electrospinning, electrospraying, and surface conjugation techniques. The composition and size of NMs can be tuned by varying the processing parameters during the fabrication. Further, bone morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) mimicking peptides have been successfully tethered onto poly(ε-caprolactone) (PCL):gelatin:(gelatin-methacryloyl) (GelMA)(1:0.5:0.5) NMs through photocrosslinking of the methacrylic group in GelMA and octenyl alanine (OCTAL) in the modified peptides. The BMP-2-OCTAL peptide-tethered NMs significantly promote osteogenic differentiation of bone marrow-derived stem cells (BMSCs). Moreover, human umbilical vein endothelial cells (HUVECs) seeded on VEGF mimicking peptide QK-OCTAL-tethered NMs significantly up-regulated vascular-specific proteins, leading to microvascularization. The strategy developed in this work holds great potential in developing a biomimetic and injectable carrier to efficiently direct cellular response (Osteogenesis and Angiogenesis) for tissue repair.

Grad-seq guides the discovery of ProQ as a major small RNA-binding protein.

The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation.

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.