Genome-wide Chromatin Profiling of Legionella pneumophila-Infected Human Macrophages Reveals Activation of the Probacterial Host Factor TNFAIP2.

BACKGROUND: Legionella pneumophila is a causative agent of severe pneumonia. Infection leads to a broad host cell response, as evident, for example, on the transcriptional level. Chromatin modifications, which control gene expression, play a central role in the transcriptional response to L. pneumophila METHODS: We infected human-blood-derived macrophages (BDMs) with L. pneumophila and used chromatin immunoprecipitation followed by sequencing to screen for gene promoters with the activating histone 4 acetylation mark. RESULTS: We found the promoter of tumor necrosis factor α-induced protein 2 (TNFAIP2) to be acetylated at histone H4. This factor has not been characterized in the pathology of L. pneumophila TNFAIP2 messenger RNA and protein were upregulated in response to L. pneumophila infection of human-BDMs and human alveolar epithelial (A549) cells. We showed that L. pneumophila-induced TNFAIP2 expression is dependent on the NF-κB transcription factor. Importantly, knock down of TNFAIP2 led to reduced intracellular replication of L. pneumophila Corby in A549 cells. CONCLUSIONS: Taken together, genome-wide chromatin analysis of L. pneumophila-infected macrophages demonstrated induction of TNFAIP2, a NF-κB-dependent factor relevant for bacterial replication.

Nomadic lifestyle of Lactobacillus plantarum revealed by comparative genomics of 54 strains isolated from different habitats.

The ability of bacteria to adapt to diverse environmental conditions is well-known. The process of bacterial adaptation to a niche has been linked to large changes in the genome content, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse habitats that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of habitat. With the aim of unravelling the link between evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Comparative genome analysis identified a high level of genomic diversity and plasticity among the strains analysed. Phylogenomic and functional divergence studies coupled with gene-trait matching analyses revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings revealed the absence of specific genomic signatures marking adaptations of L. plantarum towards the diverse habitats it is associated with. This suggests fundamentally similar trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.

The role of Ets2 transcription factor in the induction of microRNA-155 (miR-155) by lipopolysaccharide and its targeting by interleukin-10.

MicroRNA-155 (miR-155) is highly expressed in many cancers such as B cell lymphomas and myeloid leukemia and inflammatory disorders such as rheumatoid arthritis, atopic dermatitis, and multiple sclerosis. The role of miR-155 as both a promoter of inflammation and an oncogenic agent provides a clear need for miR-155 itself to be stringently regulated. We therefore investigated the transcriptional regulation of miR-155 in response to the respective pro- and anti-inflammatory mediators LPS and IL-10. Bioinformatic analysis revealed Ets binding sites on the miR-155 promoter, and we found that Ets2 is critical for miR-155 induction by LPS. Truncation and mutational analysis of the miR-155 promoter confirmed the role of the Ets2 binding site proximal to the transcription start site for LPS responsiveness. We observed increased binding of Ets2 to the miR-155 promoter and Ets2 deficient mice displayed decreased induction of miR-155 in response to LPS. IL-10 inhibited the induction of Ets2 mRNA and protein by LPS, thereby decreasing Ets2 function on the pri-155 promoter. We have thus identified Ets2 as a key novel regulator in both the positive and negative control of miR-155 in the inflammatory response.

Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase epsilon-dependent induction of Sp1 protein and interleukin 15.

In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.

Screening for eukaryotic motifs in Legionella pneumophila reveals Smh1 as bacterial deacetylase of host histones.

Legionella pneumophila (L.p.) is a bacterial pathogen which is a common causative agent of pneumonia. In humans, it infects alveolar macrophages and transfers hundreds of virulence factors that interfere with cellular signalling pathways and the transcriptomic landscape to sustain its own replication. By this interaction, it has acquired eukaryote-like protein motifs by gene transfer events that partake in the pathogenicity of Legionella. In a computational screening approach for eukaryotic motifs in the transcriptome of Legionella, we identified the L.p. strain Corby protein ABQ55614 as putative histone-deacetylase and named it "suppressing modifier of histones 1" (Smh1). During infection, Smh1 is translocated from the Legionella vacuole into the host cytosol. When expressed in human macrophage THP-1 cells, Smh1 was localized predominantly in the nucleus, leading to broad histone H3 and H4 deacetylation, blunted expression of a large number of genes (e.g. IL-1ß and IL-8), and fostered intracellular bacterial replication. L.p. with a Smh1 knockdown grew normally in media but showed a slight growth defect inside the host cell. Furthermore, Smh1 showed a very potent histone deacetylation activity in vitro, e.g. at H3K14, that could be inhibited by targeted mutation of the putative catalytic center inferred by analogy with eukaryotic HDAC8, and with the deacetylase inhibitor trichostatin A. In summary, Smh1 displays functional homology with class I/II type HDACs. We identified Smh1 as a new Legionella virulence factor with a eukaryote-like histone-deacetylase activity that moderates host gene expression and might pave the way for further histone modifications.IMPORTANCELegionella pneumophila (L.p.) is a prominent bacterial pathogen, which is a common causative agent of pneumonia. In order to survive inside the host cell, the human macrophage, it profoundly interacts with host cell processes to advance its own replication. In this study, we identify a bacterial factor, Smh1, with yet unknown function as a host histone deacetylase. The activity of this factor in the host cell leads to attenuated gene expression and increased intracellular bacterial replication.

A MicroRNA Network Controls Legionella pneumophila Replication in Human Macrophages via LGALS8 and MX1.

Legionella pneumophila is an important cause of pneumonia. It invades alveolar macrophages and manipulates the immune response by interfering with signaling pathways and gene transcription to support its own replication. MicroRNAs (miRNAs) are critical posttranscriptional regulators of gene expression and are involved in defense against bacterial infections. Several pathogens have been shown to exploit the host miRNA machinery to their advantage. We therefore hypothesize that macrophage miRNAs exert positive or negative control over Legionella intracellular replication. We found significant regulation of 85 miRNAs in human macrophages upon L. pneumophila infection. Chromatin immunoprecipitation and sequencing revealed concordant changes of histone acetylation at the putative promoters. Interestingly, a trio of miRNAs (miR-125b, miR-221, and miR-579) was found to significantly affect intracellular L. pneumophila replication in a cooperative manner. Using proteome-analysis, we pinpointed this effect to a concerted downregulation of galectin-8 (LGALS8), DExD/H-box helicase 58 (DDX58), tumor protein P53 (TP53), and then MX dynamin-like GTPase 1 (MX1) by the three miRNAs. In summary, our results demonstrate a new miRNA-controlled immune network restricting Legionella replication in human macrophages.IMPORTANCE Cases of Legionella pneumophila pneumonia occur worldwide, with potentially fatal outcome. When causing human disease, Legionella injects a plethora of virulence factors to reprogram macrophages to circumvent immune defense and create a replication niche. By analyzing Legionella-induced changes in miRNA expression and genomewide chromatin modifications in primary human macrophages, we identified a cell-autonomous immune network restricting Legionella growth. This network comprises three miRNAs governing expression of the cytosolic RNA receptor DDX58/RIG-I, the tumor suppressor TP53, the antibacterial effector LGALS8, and MX1, which has been described as an antiviral factor. Our findings for the first time link TP53, LGALS8, DDX58, and MX1 in one miRNA-regulated network and integrate them into a functional node in the defense against L. pneumophila.

The long noncoding RNA CHROME regulates cholesterol homeostasis in primate.

The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular and systemic cholesterol homeostasis. LncRNA CHROME expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

Identification of 170 New Long Noncoding RNAs in Schistosoma mansoni.

Long noncoding RNAs (lncRNAs) are transcripts generally longer than 200 nucleotides with no or poor protein coding potential, and most of their functions are also poorly characterized. Recently, an increasing number of studies have shown that lncRNAs can be involved in various critical biological processes such as organism development or cancer progression. Little, however, is known about their effects in helminths parasites, such as Schistosoma mansoni. Here, we present a computational pipeline to identify and characterize lncRNAs from RNA-seq data with high confidence from S. mansoni adult worms. Through the utilization of different criteria such as genome localization, exon number, gene length, and stability, we identified 170 new putative lncRNAs. All novel S. mansoni lncRNAs have no conserved synteny including human and mouse. These closest protein coding genes were enriched in 10 significant Gene Ontology terms related to metabolism, transport, and biosynthesis. Fifteen putative lncRNAs showed differential expression, and three displayed sex-specific differential expressions in praziquantel sensitive and resistant adult worm couples. Together, our method can predict a set of novel lncRNAs from the RNA-seq data. Some lncRNAs are shown to be differentially expressed suggesting that those novel lncRNAs can be given high priority in further functional studies focused on praziquantel resistance.

PFAAT version 2.0: a tool for editing, annotating, and analyzing multiple sequence alignments.

BACKGROUND: By virtue of their shared ancestry, homologous sequences are similar in their structure and function. Consequently, multiple sequence alignments are routinely used to identify trends that relate to function. This type of analysis is particularly productive when it is combined with structural and phylogenetic analysis. RESULTS: Here we describe the release of PFAAT version 2.0, a tool for editing, analyzing, and annotating multiple sequence alignments. Support for multiple annotations is a key component of this release as it provides a framework for most of the new functionalities. The sequence annotations are accessible from the alignment and tree, where they are typically used to label sequences or hyperlink them to related databases. Sequence annotations can be created manually or extracted automatically from UniProt entries. Once a multiple sequence alignment is populated with sequence annotations, sequences can be easily selected and sorted through a sophisticated search dialog. The selected sequences can be further analyzed using statistical methods that explicitly model relationships between the sequence annotations and residue properties. Residue annotations are accessible from the alignment viewer and are typically used to designate binding sites or properties for a particular residue. Residue annotations are also searchable, and allow one to quickly select alignment columns for further sequence analysis, e.g. computing percent identities. Other features include: novel algorithms to compute sequence conservation, mapping conservation scores to a 3D structure in Jmol, displaying secondary structure elements, and sorting sequences by residue composition. CONCLUSION: PFAAT provides a framework whereby end-users can specify knowledge for a protein family in the form of annotation. The annotations can be combined with sophisticated analysis to test hypothesis that relate to sequence, structure and function.

Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps.

Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.

Functional conservation of an ancestral Pellino protein in helminth species.

The immune system of H. sapiens has innate signaling pathways that arose in ancestral species. This is exemplified by the discovery of the Toll-like receptor (TLR) pathway using free-living model organisms such as Drosophila melanogaster. The TLR pathway is ubiquitous and controls sensitivity to pathogen-associated molecular patterns (PAMPs) in eukaryotes. There is, however, a marked absence of this pathway from the plathyhelminthes, with the exception of the Pellino protein family, which is present in a number of species from this phylum. Helminth Pellino proteins are conserved having high similarity, both at the sequence and predicted structural protein level, with that of human Pellino proteins. Pellino from a model helminth, Schistosoma mansoni Pellino (SmPellino), was shown to bind and poly-ubiquitinate human IRAK-1, displaying E3 ligase activity consistent with its human counterparts. When transfected into human cells SmPellino is functional, interacting with signaling proteins and modulating mammalian signaling pathways. Strict conservation of a protein family in species lacking its niche signalling pathway is rare and provides a platform to examine the ancestral functions of Pellino proteins that may translate into novel mechanisms of immune regulation in humans.

Proteome-wide analysis of functional divergence in bacteria: exploring a host of ecological adaptations.

Functional divergence is the process by which new genes and functions originate through the modification of existing ones. Both genetic and environmental factors influence the evolution of new functions, including gene duplication or changes in the ecological requirements of an organism. Novel functions emerge at the expense of ancestral ones and are generally accompanied by changes in the selective forces at constrained protein regions. We present software capable of analyzing whole proteomes, identifying putative amino acid replacements leading to functional change in each protein and performing statistical tests on all tabulated data. We apply this method to 750 complete bacterial proteomes to identify high-level patterns of functional divergence and link these patterns to ecological adaptations. Proteome-wide analyses of functional divergence in bacteria with different ecologies reveal a separation between proteins involved in information processing (Ribosome biogenesis etc.) and those which are dependent on the environment (energy metabolism, defense etc.). We show that the evolution of pathogenic and symbiotic bacteria is constrained by their association with the host, and also identify unusual events of functional divergence even in well-studied bacteria such as Escherichia coli. We present a description of the roles of phylogeny and ecology in functional divergence at the level of entire proteomes in bacteria.