Long Non-coding RNAs in Bacterial Infections

Bacterial pathogens have coevolved with their hosts and acquired strategies to circumvent defense mechanisms of host cells. It was shown that bacteria interfere with the expression of mammalian microRNAs to modify immune signaling, autophagy, or the apoptotic machinery. Recently, a new class of regulatory RNAs, long non-coding RNAs (lncRNAs), was reported to have a pivotal role in the regulation of eukaryotic gene expression. A growing body of literature reports on specific involvement of lncRNAs in the host cell response toward bacterial infections. This mini review summarizes recent data that focuses on lncRNA function in host cells during bacterial infection and provides a perspective where future research in this regard may be going

Mycobacterium smegmatis But Not Mycobacterium avium subsp. hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β

Pathogenic mycobacteria are able to persist intracellularly in macrophages, whereas non-pathogenic mycobacteria are effectively combated and eliminated after their phagocytosis. It is known that TGF-β plays an important role in this context. Infection with pathogenic mycobacteria such as Mycobacterium tuberculosis or M. avium leads to production of active TGF-β, which blocks the ability of IFN-γ and TNF-α to inhibit intracellular replication. On the other hand, it is known that the long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is involved in the regulation of TGF-β. In this study, we show how the infection of THP-1-derived human macrophages with the saprophytic M. smegmatis but not with the facultatively pathogenic M. avium subsp. hominissuis leads to increased MEG3 expression. This is associated with the downregulation of DNA methyltransferases (DNMT) 1 and 3b, which are known to regulate MEG3 expression via promoter hypermethylation. Consequently, we observe a significant downregulation of TGF-β in M. smegmatis-infected macrophages but not in M. avium subsp. hominissuis pointing to lncRNAs as novel mediators of host cell response during mycobacterial infections

Structural Analysis of microRNA-Target Interaction by Sequential Seed Mutagenesis and Stem-Loop 3' RACE

As a consequence of recent RNAseq efforts, miRNAomes of diverse tissues and species are available. However, most interactions between microRNAs and regulated mRNAs are still to be deciphered. While in silico analysis of microRNAs results in prediction of hundreds of potential targets, bona-fide interactions have to be verified e.g. by luciferase reporter assays using fused target sites as well as controls incorporating mutated seed sequences. The aim of this study was the development of a straightforward approach for sequential mutation of multiple target sites within a given 3' UTR. The established protocol is based on Seed Mutagenesis Assembly PCR (SMAP) allowing for rapid identification of microRNA target sites. Based on the presented approach, we were able to determine the transcription factor NKX3.1 as a genuine target of miR-155. The sequential mutagenesis of multiple microRNA target sites was examined by miR-29a mediated CASP7 regulation, which revealed one of two predicted target sites as the predominant site of interaction. Since 3' UTR sequences of non-model organisms are either lacking in databases or computationally predicted, we developed a Stem-Loop 3' UTR RACE PCR (SLURP) for efficient generation of required 3' UTR sequence data. The stem-loop primer allows for first strand cDNA synthesis by nested PCR amplification of the 3' UTR. Besides other applications, the SLURP method was used to gain data on porcine CASP7 3'UTR evaluating evolutionary conservation of the studied interaction. Sequential seed mutation of microRNA targets based on the SMAP approach allows for rapid structural analysis of several target sites within a given 3' UTR. The combination of both methods (SMAP and SLURP) enables targeted analysis of microRNA binding sites in hitherto unknown mRNA 3' UTRs within a few days

Microrna response of primary human macrophages to Arcobacter Butzleri infection

The role of microRNAs (miRNAs) in infectious diseases is becoming more and more apparent, and the use of miRNAs as a diagnostic tool and their therapeutic application has become the major focus of investigation. The aim of this study was to identify miRNAs involved in the immune signaling of macrophages in response to Arcobacter (A.) butzleri infection, an emerging foodborne pathogen causing gastroenteritis. Therefore, primary human macrophages were isolated and infected, and miRNA expression was studied by means of RNAseq. Analysis of the data revealed the expression of several miRNAs, which were previously associated with bacterial infections such as miR-155, miR-125, and miR-212. They were shown to play a key role in Toll-like receptor signaling where they act as fine-tuners to establish a balanced immune response. In addition, miRNAs which have yet not been identified during bacterial infections such as miR-3613, miR-2116, miR-671, miR-30d, and miR-629 were differentially regulated in A. butzleri-infected cells. Targets of these miRNAs accumulated in pathways such as apoptosis and endocytosis — processes that might be involved in A. butzleri pathogenesis. Our study contributes new findings about the interaction of A. butzleri with human innate immune cells helping to understand underlying regulatory mechanisms in macrophages during infection

Rapid identification of 3’ UTR sequences by means of Stem-Loop 3’ UTR RACE PCR (SLURP).

<p>Section A: Figure shows stem-loop primer (SLURP rt) binding and priming of the reverse transcription reaction. As highlighted in green and yellow, the 'SLURP rt' provides two primer binding sites for a nested PCR that are hidden by the secondary structure. Section B: The use of two batched gene specific primers (GSP rev i and GSP rev ii) together with 'fw i and fw ii' binding to the sites provided by the 'SLURP rt' allowed for a nested PCR. Section C: Identified miR-29a target sites within the 3’ UTR of porcine CASP7. SLURP based sequence revealed that target site 1 was highly conserved between humans and pigs. The predicted target site 2 suggested non-canonical binding of miR-29a as also shown in humans.</p

SMAP reveals the transcription factor NKX3.1 as a miR-155 target.

<p>Section A: Luciferase reporter assays after mutagenesis proved NKX3.1 as a miR-155 target. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-155 mimic compared with a nonsense miRNA control together with the mutated seed (pTKGhNKX3.1m) as well as wild type control (pTKGhNKX3.1) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (****: P < 0.001, unpaired t test). Section B: mRNA degradation assays by means of RT-qPCR experiments are shown detecting relative NKX3.1 levels (reference gene: GAPDH). U937 were transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. RNA was isolated at 24 and 48 h post transfection. Columns show mean relative NKX3.1 transcript levels (± SD) of three biological replicates each measured in triplicates compared with nonsense transfected controls. Section C: Western Blots detecting NKX3.1 and GAPDH (reference protein) are shown using the monocytic U937 cells transfected with miR-155, nonsense miRNA and NKX3.1 siRNA. Intrinsic NKX3.1 levels are decreased after miR-155 transfection compared with nonsense controls and siRNA (48 h post transfection). The bar graph shows the luminescence-based relative quantification of protein (NKX3.1:GAPDH) of three individual biological replicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05, unpaired t test). Transfection efficiency was evaluated by transfecting Cy3 labelled nonsense siRNA and fluorescence microscopy. While NKX3.1 translation is markedly inhibited by miR-155, cellular NKX3.1 mRNA levels remained stable after miR-155 at 24 h and decreased after 48 h suggesting slow cellular turnover rates of miR-155 directed NKX3.1 inhibition. Section D: Endogenous miR-155 expression leads to decreased luciferase activity of transfected reporters. The bar graph on the left hand side shows relative miR-155 expression in U937 cells at 5 and 24 h after LPS stimulation. The other bar graph shows that endogenous miR-155 expression results in clear repression of luciferase activity (nonsense transfected) while ectopically introduced miR-155 results in elevated repression and mutated controls remain unaffected. The columns show means of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (**: P < 0.01,***: P < 0.001, unpaired t test).</p

Concept of Seed Mutagenesis Assembly PCR (SMAP).

<p>Figure shows seed mutagenesis of miR-155 targets SHIP1 and NKX3.1. Gel images show amplicons obtained after each step applying a gradient PCR, while NC represents the no template control at the lowest annealing temperature. Section A: 3’ UTR amplicons were generated harbouring the predicted target site. Section B: Oligonucleotides were used for seed mutagenesis and generation of overlapping termini that possessed approximately 12 nucleotides at the 3’ as well as 5’ ends flanking the mutated site. Section C: Assembly PCR of amplicons with mutated termini provided 3’ UTRs including the mutated seed. The amplicon was fused to a luciferase gene for reporter gene assays. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-155 mimic compared with a nonsense miRNA control together with the mutated seed (pTKGhSHIP1m) as well as wild type control (pTKGhSHIP1) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (****: P < 0.001, unpaired t test).</p

Sequential mutagenesis of multiple miR-29a target sites within the 3’ UTR of CASP7 by means of SMAP.

<p>Section A: Full length 3’ UTR of CASP7 was generated harbouring both identified target sites of miR-29a. Section B: For individual but also combined target site mutagenesis, the 3’ UTR was theoretically divided in three segments defined by locations of target sites. Sections C-E: Modular assembly of amplicons resulted in sequential mutagenesis of target sites. Section F: Gel image shows five amplicons after mutagenesis as well as assembled products for reporter gene fusion. NC represents the no template control of the assembly PCR. Relative luciferase activity (Luc _Gaussia : Luc _Cypridina) was determined using a miR-29a mimic compared with a nonsense miRNA control together with the mutated target site 1 (pTKGhCASP7m1), mutated target site 2 (pTKGhCASP7m2), mutated target sites 1 and 2 (pTKGhCASP7m1+2) as well as the wild type control (pTKGhCASP7) . The columns show means of normalised luciferase activity of three biological replicates each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between samples (*: P < 0.05; ***: P < 0.001, paired t test).</p

Analysis of long non-coding RNA and mRNA expression in bovine macrophages brings up novel aspects of Mycobacterium avium subspecies paratuberculosis infections

Abstract Paratuberculosis is a major disease in cattle that severely affects animal welfare and causes huge economic losses worldwide. Development of alternative diagnostic methods is of urgent need to control the disease. Recent studies suggest that long non-coding RNAs (lncRNAs) play a crucial role in regulating immune function and may confer valuable information about the disease. However, their role has not yet been investigated in cattle with respect to infection towards Paratuberculosis. Therefore, we investigated the alteration in genomic expression profiles of mRNA and lncRNA in bovine macrophages in response to Paratuberculosis infection using RNA-Seq. We identified 397 potentially novel lncRNA candidates in macrophages of which 38 were differentially regulated by the infection. A total of 820 coding genes were also significantly altered by the infection. Co-expression analysis of lncRNAs and their neighbouring coding genes suggest regulatory functions of lncRNAs in pathways related to immune response. For example, this included protein coding genes such as TNIP3, TNFAIP3 and NF-κB2 that play a role in NF-κB2 signalling, a pathway associated with immune response. This study advances our understanding of lncRNA roles during Paratuberculosis infection