Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

Transcriptional profiling of human macrophages during infection with Bordetella pertussis.

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

RNA modifications in structure prediction - Status quo and future challenges.

Chemical modifications of RNA nucleotides change their identity and characteristics and thus alter genetic and structural information encoded in the genomic DNA. tRNA and rRNA are probably the most heavily modified genes, and often depend on derivatization or isomerization of their nucleobases in order to correctly fold into their functional structures. Recent RNomics studies, however, report transcriptome wide RNA modification and suggest a more general regulation of structuredness of RNAs by this so called epitranscriptome. Modification seems to require specific substrate structures, which in turn are stabilized or destabilized and thus promote or inhibit refolding events of regulatory RNA structures. In this review, we revisit RNA modifications and the related structures from a computational point of view. We discuss known substrate structures, their properties such as sub-motifs as well as consequences of modifications on base pairing patterns and possible refolding events. Given that efficient RNA structure prediction methods for canonical base pairs have been established several decades ago, we review to what extend these methods allow the inclusion of modified nucleotides to model and study epitranscriptomic effects on RNA structures.

RIsearch2: suffix array-based large-scale prediction of RNA-RNA interactions and siRNA off-targets.

Intermolecular interactions of ncRNAs are at the core of gene regulation events, and identifying the full map of these interactions bears crucial importance for ncRNA functional studies. It is known that RNA-RNA interactions are built up by complementary base pairings between interacting RNAs and high level of complementarity between two RNA sequences is a powerful predictor of such interactions. Here, we present RIsearch2, a large-scale RNA-RNA interaction prediction tool that enables quick localization of potential near-complementary RNA-RNA interactions between given query and target sequences. In contrast to previous heuristics which either search for exact matches while including G-U wobble pairs or employ simplified energy models, we present a novel approach using a single integrated seed-and-extend framework based on suffix arrays. RIsearch2 enables fast discovery of candidate RNA-RNA interactions on genome/transcriptome-wide scale. We furthermore present an siRNA off-target discovery pipeline that not only predicts the off-target transcripts but also computes the off-targeting potential of a given siRNA. This is achieved by combining genome-wide RIsearch2 predictions with target site accessibilities and transcript abundance estimates. We show that this pipeline accurately predicts siRNA off-target interactions and enables off-targeting potential comparisons between different siRNA designs. RIsearch2 and the siRNA off-target discovery pipeline are available as stand-alone software packages from http://rth.dk/resources/risearch.

Differential transcriptional responses to Ebola and Marburg virus infection in bat and human cells.

The unprecedented outbreak of Ebola in West Africa resulted in over 28,000 cases and 11,000 deaths, underlining the need for a better understanding of the biology of this highly pathogenic virus to develop specific counter strategies. Two filoviruses, the Ebola and Marburg viruses, result in a severe and often fatal infection in humans. However, bats are natural hosts and survive filovirus infections without obvious symptoms. The molecular basis of this striking difference in the response to filovirus infections is not well understood. We report a systematic overview of differentially expressed genes, activity motifs and pathways in human and bat cells infected with the Ebola and Marburg viruses, and we demonstrate that the replication of filoviruses is more rapid in human cells than in bat cells. We also found that the most strongly regulated genes upon filovirus infection are chemokine ligands and transcription factors. We observed a strong induction of the JAK/STAT pathway, of several genes encoding inhibitors of MAP kinases (DUSP genes) and of PPP1R15A, which is involved in ER stress-induced cell death. We used comparative transcriptomics to provide a data resource that can be used to identify cellular responses that might allow bats to survive filovirus infections.

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution.

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation.

Identification of new protein coding sequences and signal peptidase cleavage sites of Helicobacter pylori strain 26695 by proteogenomics.

Correct annotation of protein coding genes is the basis of conventional data analysis in proteomic studies. Nevertheless, most protein sequence databases almost exclusively rely on gene finding software and inevitably also miss protein annotations or possess errors. Proteogenomics tries to overcome these issues by matching MS data directly against a genome sequence database. Here we report an in-depth proteogenomics study of Helicobacter pylori strain 26695. MS data was searched against a combined database of the NCBI annotations and a six-frame translation of the genome. Database searches with Mascot and X! Tandem revealed 1115 proteins identified by at least two peptides with a peptide false discovery rate below 1%. This represents 71% of the predicted proteome. So far this is the most extensive proteome study of Helicobacter pylori. Our proteogenomic approach unambiguously identified four previously missed annotations and furthermore allowed us to correct sequences of six annotated proteins. Since secreted proteins are often involved in pathogenic processes we further investigated signal peptidase cleavage sites. By applying a database search that accommodates the identification of semi-specific cleaved peptides, 63 previously unknown signal peptides were detected. The motif LXA showed to be the predominant recognition sequence for signal peptidases. BIOLOGICAL SIGNIFICANCE: The results of MS-based proteomic studies highly rely on correct annotation of protein coding genes which is the basis of conventional data analysis. However, the annotation of protein coding sequences in genomic data is usually based on gene finding software. These tools are limited in their prediction accuracy such as the problematic determination of exact gene boundaries. Thus, protein databases own partly erroneous or incomplete sequences. Additionally, some protein sequences might also be missing in the databases. Proteogenomics, a combination of proteomic and genomic data analyses, is well suited to detect previously not annotated proteins and to correct erroneous sequences. For this purpose, the existing database of the investigated species is typically supplemented with a six-frame translation of the genome. Here, we studied the proteome of the major human pathogen Helicobacter pylori that is responsible for many gastric diseases such as duodenal ulcers and gastric cancer. Our in-depth proteomic study highly reliably identified 1115 proteins (FDR<0.01%) by at least two peptides (FDR<1%) which represent 71% of the predicted proteome deposited at NCBI. The proteogenomic data analysis of our data set resulted in the unambiguous identification of four previously missed annotations, the correction of six annotated proteins as well as the detection of 63 previously unknown signal peptides. We have annotated proteins of particular biological interest like the ferrous iron transport protein A, the coiled-coil-rich protein HP0058 and the lipopolysaccharide biosynthesis protein HP0619. For instance, the protein HP0619 could be a drug target for the inhibition of the LPS synthesis pathway. Furthermore it has been proven that the motif "LXA" is the predominant recognition sequence for the signal peptidase I of H. pylori. Signal peptidases are essential enzymes for the viability of bacterial cells and are involved in pathogenesis. Therefore signal peptidases could be novel targets for antibiotics. The inclusion of the corrected and new annotated proteins as well as the information of signal peptide cleavage sites will help in the study of biological pathways involved in pathogenesis or drug response of H. pylori.

Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation.

For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.

RNAcode: robust discrimination of coding and noncoding regions in comparative sequence data.

With the availability of genome-wide transcription data and massive comparative sequencing, the discrimination of coding from noncoding RNAs and the assessment of coding potential in evolutionarily conserved regions arose as a core analysis task. Here we present RNAcode, a program to detect coding regions in multiple sequence alignments that is optimized for emerging applications not covered by current protein gene-finding software. Our algorithm combines information from nucleotide substitution and gap patterns in a unified framework and also deals with real-life issues such as alignment and sequencing errors. It uses an explicit statistical model with no machine learning component and can therefore be applied "out of the box," without any training, to data from all domains of life. We describe the RNAcode method and apply it in combination with mass spectrometry experiments to predict and confirm seven novel short peptides in Escherichia coli and to analyze the coding potential of RNAs previously annotated as "noncoding." RNAcode is open source software and available for all major platforms at http://wash.github.com/rnacode.

RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Unorthodox mRNA start site to extend the highly structured leader of retrotransposon Tto1 mRNA increases transposition rate.

Retroelement RNAs serve as templates for both translation and reverse transcription into extrachromosomal DNA. DNA copies may be inserted into the host genome to multiply element sequences. This transpositional activity of retroelements is usually restricted to specific conditions, particularly to conditions that impose stress on the host organism. In this work, we examined how the mRNA initiation point, and features of primary and secondary structure, of tobacco retrotransposon Tto1 RNA influence its transpositional activity. We found that the most abundant Tto1 RNA is not a substrate for reverse transcription. It is poorly translated, and its 5'-end does not contain a region of redundancy with the most prominent 3'-end. In contrast, expression of an mRNA with the 5'-end extended by 28 nucleotides allows translation and gives rise to transposition events in the heterologous host, Arabidopsis thaliana. In addition, the presence of extended hairpins and of two short open reading frames in the 5'-leader sequence of Tto1 mRNA suggests that translation does not involve ribosome scanning from the mRNA 5'-end to the translation initiation site.

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.