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Regulatory pathways and virulence inhibition in Listeria monocytogenes
Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).ORCID-id: 0000-0001-5702-9742
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Listeria monocytogenes is a rod-shaped Gram positive bacterium. It generally exist ubiquitously in nature, where it lives as a saprophyte. Occasionally it however enters the food chain, from where it can be ingested by humans and cause gastro-intestinal distress. In immunocompetent individuals L. monocytogenes is generally cleared within a couple of weeks, but in immunocompromised patients it can progress to listeriosis, a potentially life-threatening infection in the central nervous system. If the infected individual is pregnant, the bacteria can cross the placental barrier and infect the fetus, possibly leading to spontaneous abortion.

The infectivity of L. monocytogenes requires a certain set of genes, and the majority of them is dependent on the transcriptional regulator PrfA. The expression and activity of PrfA is controlled at several levels, and has traditionally been viewed to be active at 37 °C (virulence conditions) where it bind as a homodimer to a “PrfA-box” and induces the expression of the downstream gene.

One of these genes is ActA, which enables intracellular movement by recruiting an actin polymerizing protein complex. When studying the effects of a blue light receptor we surprisingly found an effect of ActA at non-virulent conditions, where it is required for the bacteria to properly react to light exposure.

To further study the PrfA regulon we tested deletion mutants of several PrfA-regulated virulence genes in chicken embryo infection studies. Based on these studies we could conclude that the chicken embryo model is a viable complement to traditional murine models, especially when investigating non-traditional internalin pathogenicity pathways. We have also studied the effects of small molecule virulence inhibitors that, by acting on PrfA, can inhibit L. monocytogenes infectivity in cell cultures with concentrations in the low micro-molar range.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå Universitet , 2016. , s. 37
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1772
Nyckelord [en]
Listeria monocytogenes, PrfA, ActA, infection
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-114085ISBN: 978-91-7601-397-7 (tryckt)OAI: oai:DiVA.org:umu-114085DiVA, id: diva2:893603
Disputation
2016-02-04, KB3B1, KBC-huset, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-01-14 Skapad: 2016-01-12 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
Delarbeten
1. Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes
Öppna denna publikation i ny flik eller fönster >>Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes
2013 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 87, nr 4, s. 909-924Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Recently, several light receptors have been identified in non-phototrophic bacteria, but their physiological roles still remain rather elusive. Here we show that colonies of the saprophytic bacterium Listeria monocytogenes undergo synchronized multicellular behaviour on agar plates, in response to oscillating light/dark conditions, giving rise to alternating ring formation (opaque and translucent rings). On agar plates, bacteria from opaque rings survive increased levels of reactive oxygen species (ROS), as well as repeated cycles of light and dark, better than bacteria from translucent rings. The ring formation is strictly dependent on a blue-light receptor, Lmo0799, acting through the stress-sigma factor, sigma B. A transposon screening identified 48 mutants unable to form rings at alternating light conditions, with several of them showing a decreased sigma B activity/level. However, some of the tested mutants displayed a varied sigma B activity depending on which of the two stress conditions tested (light or H2O2 exposure). Intriguingly, the transcriptional regulator PrfA and the virulence factor ActA were shown to be required for ring formation by a mechanism involving activation of sigma B. All in all, this suggests a distinct pathway for Lmo0799 that converge into a common signalling pathway for sigma B activation. Our results show that night and day cycles co-ordinate a reversible differentiation of a L.monocytogenes colony at room temperature, by a process synchronized by a blue-light receptor and sigma B.

Nationell ämneskategori
Mikrobiologi inom det medicinska området
Identifikatorer
urn:nbn:se:umu:diva-67399 (URN)10.1111/mmi.12140 (DOI)000314925700015 ()
Tillgänglig från: 2013-04-09 Skapad: 2013-03-18 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
2. Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
Öppna denna publikation i ny flik eller fönster >>Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
Visa övriga...
2016 (Engelska)Ingår i: Cell chemical biology, ISSN 2451-9448, Vol. 23, nr 3, s. 404-414Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-114083 (URN)10.1016/j.chembiol.2016.02.013 (DOI)000381508300013 ()26991105 (PubMedID)
Anmärkning

Originally published in manuscipt form in thesis.

Tillgänglig från: 2016-01-12 Skapad: 2016-01-12 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
3. Exploring the chicken embryo as a possible model for studying Listeria monocytogenes pathogenicity
Öppna denna publikation i ny flik eller fönster >>Exploring the chicken embryo as a possible model for studying Listeria monocytogenes pathogenicity
2014 (Engelska)Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 4, artikel-id 170Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections in humans, often with fatal outcomes. Many different animal models exist to study L. monocytogenes pathogenicity, and we have investigated the chicken embryo as an infection model: What are the benefits and possible drawbacks? We have compared a defined wild-type strain with its isogenic strains lacking well-characterized virulence factors. Our results show that wild-type L. monocytogenes, already at a relatively low infection dose (similar to 5 x 10(2) cfu), caused death of the chicken embryo within 36 h, in contrast to strains lacking the main transcriptional activator of virulence, PrfA, or the cytolysin LLO. Surprisingly, strains lacking the major adhesins InIA and InIB caused similar mortality as the wild-type strain. In conclusion, our results suggest that the chicken embryo is a practical model to study L. monocytogenes infections, especially when analyzing alternative virulence pathways independent of the InIA and InIB adhesins. However, the route of infection might be different from a human infection. The chicken embryo model and other Listeria infection models are discussed.

Nyckelord
Listeria monocytogenes, chicken embryo, PrfA, virulence: InIA, LLO, InIB
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:umu:diva-100984 (URN)10.3389/fcimb.2014.00170 (DOI)000349152600003 ()25540772 (PubMedID)
Tillgänglig från: 2015-03-16 Skapad: 2015-03-16 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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