The role of the RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium

Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a broad variety of functions. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium Even though hfq has turned out to have no severe influence on the growth or the viability of the pathogenic bacterium Salmonella Typhimurium under laboratory conditions, we could show that it is strongly involved in the regulation of pathogenicity. A Δhfq mutant leads to loss of effector protein expression and secretion and thereby to reduced invasion of non-phagocytic cells and to reduced ability of intracellular replication in macrophages. Based on these observations, loss of infectivity in a mouse-model of infection could be proven. Further studies revealed not only lack of secreted proteins in the Δhfq mutant, but showed severe changes in the overall protein pattern when compared to its isogenic wild type strain, with an overrepresentation of membrane and membrane-associated proteins. Concerning the virulence phenotype, we have been able to restore effector protein expression (even if not their secretion) by overexpression of one of the major transcription factors involved in expression of virulence genes encoded in Salmonella pathogenicity island 1 (SPI1), namely HilA. Additionally, we could show that alteration in mRNA stability is causing for example the increase of the major outer membrane protein, OmpD or the decrease in the flagellar protein, FliC. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq Our analysis represents a demonstration for usage of high throughput pyrosequencing (HTPS) in bacteria to determine the large regulon of the pleiotropic regulator, Hfq. The combination of transcriptomics with co-immunoprecipitation (coIP) of direct binding partners of Hfq and subsequent cDNA library synthesis and its sequencing allowed the dissection of genes directly influenced by Hfq and downstream effects based on deregulation of transcription factors. By analysis of RNA co-immunoprecipitated with Hfq compared to control coIPs in Salmonella Typhimurium lysates we were able to determine specific enrichment factors for a large set of mRNAs as well as sRNAs. Comparison with the transcriptomic data showed that Hfq regulates multiple major transcription factors, like a transcription factor of SPI1, HilD, and the major transcription factor, FlhD2C2, regulating the large class of flagellar genes in Salmonella and other bacterial species. By overexpression of these transcription factors we could restore phenotypes of a Δhfq mutant, e.g. loss of effector protein expression and secretion and reduced expression of the class III flagellar gene, FliC. Concerning sRNA expression in Salmonella, we found 10 new sRNAs in this pathogen and were able to verify the expression of a large set of sRNAs that have been known to be conserved in the model organism, Escherichia coli. Aside noncoding RNAs also two mRNAs encoding for small open reading frames (ORFs) in E. coli could be detected in the coIP RNA sample from Salmonella Typhimurium

Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria

The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium

The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Qβ RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression

Characterization of the role of ribonucleases in Salmonella small RNA decay

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

Comparative Omics-Driven Genome Annotation Refinement: Application across Yersiniae

Genome sequencing continues to be a rapidly evolving technology, yet most downstream aspects of genome annotation pipelines remain relatively stable or are even being abandoned. The annotation process is now performed almost exclusively in an automated fashion to balance the large number of sequences generated. One possible way of reducing errors inherent to automated computational annotations is to apply data from omics measurements (i.e. transcriptional and proteomic) to the un-annotated genome with a proteogenomic-based approach. Here, the concept of annotation refinement has been extended to include a comparative assessment of genomes across closely related species. Transcriptomic and proteomic data derived from highly similar pathogenic Yersiniae (Y. pestis CO92, Y. pestis Pestoides F, and Y. pseudotuberculosis PB1/+) was used to demonstrate a comprehensive comparative omic-based annotation methodology. Peptide and oligo measurements experimentally validated the expression of nearly 40% of each strain's predicted proteome and revealed the identification of 28 novel and 68 incorrect (i.e., observed frameshifts, extended start sites, and translated pseudogenes) protein-coding sequences within the three current genome annotations. Gene loss is presumed to play a major role in Y. pestis acquiring its niche as a virulent pathogen, thus the discovery of many translated pseudogenes, including the insertion-ablated argD, underscores a need for functional analyses to investigate hypotheses related to divergence. Refinements included the discovery of a seemingly essential ribosomal protein, several virulence-associated factors, a transcriptional regulator, and many hypothetical proteins that were missed during annotation

The role of the RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium

Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a broad variety of functions. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium Even though hfq has turned out to have no severe influence on the growth or the viability of the pathogenic bacterium Salmonella Typhimurium under laboratory conditions, we could show that it is strongly involved in the regulation of pathogenicity. A Δhfq mutant leads to loss of effector protein expression and secretion and thereby to reduced invasion of non-phagocytic cells and to reduced ability of intracellular replication in macrophages. Based on these observations, loss of infectivity in a mouse-model of infection could be proven. Further studies revealed not only lack of secreted proteins in the Δhfq mutant, but showed severe changes in the overall protein pattern when compared to its isogenic wild type strain, with an overrepresentation of membrane and membrane-associated proteins. Concerning the virulence phenotype, we have been able to restore effector protein expression (even if not their secretion) by overexpression of one of the major transcription factors involved in expression of virulence genes encoded in Salmonella pathogenicity island 1 (SPI1), namely HilA. Additionally, we could show that alteration in mRNA stability is causing for example the increase of the major outer membrane protein, OmpD or the decrease in the flagellar protein, FliC. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq Our analysis represents a demonstration for usage of high throughput pyrosequencing (HTPS) in bacteria to determine the large regulon of the pleiotropic regulator, Hfq. The combination of transcriptomics with co-immunoprecipitation (coIP) of direct binding partners of Hfq and subsequent cDNA library synthesis and its sequencing allowed the dissection of genes directly influenced by Hfq and downstream effects based on deregulation of transcription factors. By analysis of RNA co-immunoprecipitated with Hfq compared to control coIPs in Salmonella Typhimurium lysates we were able to determine specific enrichment factors for a large set of mRNAs as well as sRNAs. Comparison with the transcriptomic data showed that Hfq regulates multiple major transcription factors, like a transcription factor of SPI1, HilD, and the major transcription factor, FlhD2C2, regulating the large class of flagellar genes in Salmonella and other bacterial species. By overexpression of these transcription factors we could restore phenotypes of a Δhfq mutant, e.g. loss of effector protein expression and secretion and reduced expression of the class III flagellar gene, FliC. Concerning sRNA expression in Salmonella, we found 10 new sRNAs in this pathogen and were able to verify the expression of a large set of sRNAs that have been known to be conserved in the model organism, Escherichia coli. Aside noncoding RNAs also two mRNAs encoding for small open reading frames (ORFs) in E. coli could be detected in the coIP RNA sample from Salmonella Typhimurium

MicroRNA Profiling as Tool for <i>In Vitro</i> Developmental Neurotoxicity Testing: The Case of Sodium Valproate

<div><p>Studying chemical disturbances during neural differentiation of murine embryonic stem cells (mESCs) has been established as an alternative <i>in vitro</i> testing approach for the identification of developmental neurotoxicants. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development and ESC differentiation and specification. Thus, neural differentiation of mESCs <i>in vitro</i> allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed changes in miRNome and transcriptome during neural differentiation of mESCs exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neurally differentiating mESCs upon VPA treatment. Based on miRNA profiling we observed that VPA shifts the lineage specification from neural to myogenic differentiation (upregulation of muscle-abundant miRNAs, <i>mir-206, mir-133a</i> and <i>mir-10a</i>, and downregulation of neural-specific <i>mir-124a, mir-128</i> and <i>mir-137</i>). These findings were confirmed on the mRNA level and via immunochemistry. Particularly, the expression of myogenic regulatory factors (MRFs) as well as muscle-specific genes (<i>Actc1, calponin</i>, <i>myosin light chain, asporin, decorin</i>) were found elevated, while genes involved in neurogenesis (e.g. <i>Otx1</i>, <i>2, and Zic3, 4, 5</i>) were repressed. These results were specific for valproate treatment and―based on the following two observations―most likely due to the inhibition of histone deacetylase (HDAC) activity: (i) we did not observe any induction of muscle-specific miRNAs in neurally differentiating mESCs exposed to the unrelated developmental neurotoxicant sodium arsenite; and (ii) the expression of muscle-abundant <i>mir-206</i> and <i>mir-10a</i> was similarly increased in cells exposed to the structurally different HDAC inhibitor trichostatin A (TSA). Based on our results we conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. The observed lineage shift into myogenesis, where miRNAs may play an important role, could be one of the developmental neurotoxic mechanisms of VPA.</p></div

Valproate effects on viability and expression of β-III-Tubulin.

<p>The cells were induced to differentiate into neurons for 16 days under continuous substance exposure. Cell viability was estimated using CellTiterBlue assay and is shown as a percentage of solvent control (A), expression of β-III-tubulin was analyzed by flow cytometry and is shown as a percentage of solvent control for each concentration tested (B). Results represent a mean of three independent differentiation experiments ± SEM.</p