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Regulatory roles of two small RNAs in the human pathogen Listeria monocytogenes and the evaluation of an alternative infection model
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth.

Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites.

To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins.

L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes.

Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2012. , 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1507
Keyword [en]
small RNA, sRNA, Riboswitch, Listeria monocytogenes, ncRNA, PrfA
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-55432ISBN: 978-91-7459-434-8 (print)OAI: oai:DiVA.org:umu-55432DiVA: diva2:526720
Public defence
2012-06-15, Major groove, NUS, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2012-05-25 Created: 2012-05-14 Last updated: 2012-05-25Bibliographically approved
List of papers
1. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes
Open this publication in new window or tab >>A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes
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2009 (English)In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 139, no 4, 770-779 p.Article in journal (Refereed) Published
Abstract [en]

Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5´-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.

Keyword
Listeria, riboswitch, SreA, PrfA
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-33105 (URN)10.1016/j.cell.2009.08.046 (DOI)19914169 (PubMedID)
Available from: 2010-04-12 Created: 2010-04-12 Last updated: 2017-12-12Bibliographically approved
2. The Listeria transcriptional landscape from saprophytism to virulence
Open this publication in new window or tab >>The Listeria transcriptional landscape from saprophytism to virulence
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2009 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 459, no 7249, 950-956 p.Article in journal (Refereed) Published
Abstract [en]

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

Keyword
11-beta-Hydroxysteroid Dehydrogenase Type 1/*genetics/metabolism, Animals, Cerebral Cortex/enzymology/metabolism, Circadian Rhythm, Corticosterone/blood, Gene Expression Regulation; Enzymologic, Hippocampus/enzymology/*metabolism, Male, Obesity/blood/enzymology/*genetics, RNA; Messenger/metabolism, Rats, Rats; Sprague-Dawley, Rats; Zucker
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-33159 (URN)10.1038/nature08080 (DOI)19448609 (PubMedID)
Available from: 2010-04-13 Created: 2010-04-13 Last updated: 2017-12-12Bibliographically approved
3. Rli38, a novel stress induced small RNA required for Listeria monocytogenes virulence
Open this publication in new window or tab >>Rli38, a novel stress induced small RNA required for Listeria monocytogenes virulence
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-55429 (URN)
Available from: 2012-05-14 Created: 2012-05-14 Last updated: 2012-05-25Bibliographically approved
4. Evaluating the chicken embryo as a model for studying Listeria monocytogenes pathogenesis: a role for the PrfA pathway
Open this publication in new window or tab >>Evaluating the chicken embryo as a model for studying Listeria monocytogenes pathogenesis: a role for the PrfA pathway
(English)Manuscript (preprint) (Other academic)
National Category
Basic Medicine Microbiology
Identifiers
urn:nbn:se:umu:diva-55425 (URN)
Available from: 2012-05-14 Created: 2012-05-14 Last updated: 2012-05-25Bibliographically approved

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