| 1. |
- Andresen, Liis, et al.
(författare)
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Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy) alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors-a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 - showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries.
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| 2. |
- Andresen, Liis, et al.
(författare)
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Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p) ppGpp (de la Fuente-Nunez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p) ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p) ppGpp synthesis moderately sensitizes-rather than protects-E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p) ppGpp.
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| 3. |
- Andresen, Liis, et al.
(författare)
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CLIP-Seq in Bacteria : Global Recognition Patterns of Bacterial RNA-Binding Proteins
- 2018
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Ingår i: High-Density Sequencing Applications in Microbial Molecular Genetics. - : ELSEVIER ACADEMIC PRESS INC. - 9780128159934 ; , s. 127-145
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Bokkapitel (refereegranskat)abstract
- RNA-protein interactions are at the heart of many central cellular processes, and RNA-binding proteins (RBPs) associate with virtually all RNA molecules in a cell. In bacteria, global RBPs, often in conjunction with small regulatory RNAs, affect physiology and virulence by controlling transcription, translation, and RNA decay. To understand how these regulatory proteins orchestrate global gene expression, detailed maps of their cellular RNA binding sites are required. To this end, cross-linking and immunoprecipitation followed by deep sequencing (CLIP-seq) has revolutionized RBP studies by providing knowledge about global recognition patterns of RBPs in both eukaryotic and bacterial cells. In this chapter, we provide a step-by-step protocol for global mapping of bona fide RBP binding sites using CLIP-seq in bacteria. This protocol has been successfully applied for charting the binding sites of Hfq, CsrA, and ProQ, three global regulatory RBPs in Salmonella enterica and Escherichia coli, and should be readily applicable to other RBPs and bacterial species.
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| 4. |
- Andresen, Liis, et al.
(författare)
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The Small Toxic Salmonella Protein TimP Targets the Cytoplasmic Membrane and Is Repressed by the Small RNA TimR
- 2020
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Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Small proteins are gaining increased attention due to their important functions in major biological processes throughout the domains of life. However, their small size and low sequence conservation make them difficult to identify. It is therefore not surprising that enterobacterial ryfA has escaped identification as a small protein coding gene for nearly 2 decades. Since its identification in 2001, ryfA has been thought to encode a noncoding RNA and has been implicated in biofilm formation in Escherichia coli and pathogenesis in Shigella dysenteriae. Although a recent ribosome profiling study suggested ryfA to be translated, the corresponding protein product was not detected. In this study, we provide evidence that ryfA encodes a small toxic inner membrane protein, TimP, overexpression of which causes cytoplasmic membrane leakage. TimP carries an N-terminal signal sequence, indicating that its membrane localization is Sec-dependent. Expression of TimP is repressed by the small RNA (sRNA) TimR, which base pairs with the timP mRNA to inhibit its translation. In contrast to overexpression, endogenous expression of TimP upon timR deletion permits cell growth, possibly indicating a toxicity-independent function in the bacterial membrane. IMPORTANCE Next-generation sequencing (NGS) has enabled the revelation of a vast number of genomes from organisms spanning all domains of life. To reduce complexity when new genome sequences are annotated, open reading frames (ORFs) shorter than 50 codons in length are generally omitted. However, it has recently become evident that this procedure sorts away ORFs encoding small proteins of high biological significance. For instance, tailored small protein identification approaches have shown that bacteria encode numerous small proteins with important physiological functions. As the number of predicted small ORFs increase, it becomes important to characterize the corresponding proteins. In this study, we discovered a conserved but previously overlooked small enterobacterial protein. We show that this protein, which we dubbed TimP, is a potent toxin that inhibits bacterial growth by targeting the cell membrane. Toxicity is relieved by a small regulatory RNA, which binds the toxin mRNA to inhibit toxin synthesis.
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| 5. |
- Beljantseva, Jelena, et al.
(författare)
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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:14, s. 3726-3731
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Tidskriftsartikel (refereegranskat)abstract
- The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.
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| 6. |
- Berggren, Sofia, et al.
(författare)
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ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
- 2024
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Ingår i: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3 ' UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
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| 7. |
- Jevtusevskaja, Jekaterina, et al.
(författare)
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Combination with antimicrobial peptide lyses improves loop-mediated isothermal amplification based method for Chlamydia trachomatis detection directly in urine sample
- 2016
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Ingår i: BMC Infectious Diseases. - : Springer Science and Business Media LLC. - 1471-2334. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chlamydia trachomatis is an obligate intracellular human pathogen and is the most common cause of sexually transmitted diseases affecting both men and women. The pathogen can cause prostatitis and epididymitis in men. In women, cervicitis, pelvic inflammatory disease, ectopic pregnancy and acute or chronic pelvic pain are frequent complications. More than half of C. trachomatis-positive patients have minimal or no symptoms, providing an ongoing reservoir for the infection. The lack of sensitive large-scale applicable point-of-care (POC) tests for C. trachomatis detection makes it difficult to diagnose chlamydia infection efficiently in resource-limited environments. Methods: A rapid and sensitive assay based on loop-mediated isothermal amplification method (LAMP) was combined with antimicrobial peptide lysis, which is able to detect at least 7 C. trachomatis pathogens per reaction directly from urine samples. Results: Our study comprising 91 first-void urine samples showed that specificity of the assay is 100 % and sensitivity 73 % when using antimicrobial peptide lysis mix. Additionally we demonstrate that our assay does not give any cross-reactivity with 30 pathogen's DNA potentially present in the urine samples. Furthermore, the assay's novel approach does not require purification or extraction of DNA from clinical sample prior to amplification, so the need for specialized equipment is eliminated. Conclusions: The whole procedure is significantly less laborious, less time-consuming and consequently less expensive for early detection and identification of infectious disease. C. trachomatis specific LAMP assay is relatively simple to perform and could therefore be applied in numerous POC settings.
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| 8. |
- Krõlov, Katrin, et al.
(författare)
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Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings
- 2017
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Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 17:12, s. 1117-1125
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Tidskriftsartikel (refereegranskat)abstract
- Background: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Methods: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. Results: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. Conclusion: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.
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