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OxyR: an important regulator of the oxidative stress response of Francisella tularensis LVS
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS. (Anders Sjöstedt)ORCID iD: 0000-0001-5254-3873
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS. (Anders Sjöstedt)
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). MIMS. (Anders Sjöstedt)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

An essential part of the oxidative stress response in Gram-negative bacteria is the H2O2-activated transcriptional regulator OxyR. Although it is much studied in common bacteria such as Escherichia coli, little is known about it about its role in the facultative intracellular bacterium Francisella tularensis. Here, we studied the role of OxyR in the strain F. tularensis LVS. We studied the effects of ROS on the LVS, ΔoxyR, ΔkatG and ΔoxyRkatG. The latter mutants lack expression of catalase, the function of which is important for degradation of reactive oxygen species, especially H2O2. The in vitro response of these mutants to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS, as was ΔkatG, with the exception of O2-. Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. Also the catalase activity was assessed and whereas LVS significantly upregulated the enzymatic activity in response to H2O2, this did not occur in the ΔoxyR mutant. Gene expression by ΔoxyR was compared to LVS and it was found that there was down-regulation of fur, katG, sodB, sodC, furA, and in particular of ahpC, in the mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages, whereas ΔoxyRkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis. The combined mutation of ΔoxyRkatG led to severely impaired handling of oxidative stress.

Keyword [en]
Francisella tularensis, OxyR, oxidative stress
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-115632OAI: oai:DiVA.org:umu-115632DiVA: diva2:900199
Available from: 2016-02-03 Created: 2016-02-03 Last updated: 2016-02-04Bibliographically approved
In thesis
1. The oxidative stress response of Francisella tularensis
Open this publication in new window or tab >>The oxidative stress response of Francisella tularensis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
The oxidative stress response of Francisella tularensis
Abstract [en]

Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host.

This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS.

We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica.

We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci.

We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyRkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2016. 49 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1781
Keyword
Francisella tularensis, FupA, MglA, OxyR, ROS, oxidative stress
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-115635 (URN)978-91-7601-415-8 (ISBN)
Public defence
2016-02-26, E04, Byggnad 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-02-05 Created: 2016-02-03 Last updated: 2016-02-04Bibliographically approved

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Honn, MarieLindgren, HelenaSjöstedt, Anders
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