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The oxidative stress response of Francisella tularensis
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. (Anders Sjöstedt)ORCID iD: 0000-0001-5254-3873
2016 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
The oxidative stress response of Francisella tularensis (Swedish)
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 [en]
Francisella tularensis, FupA, MglA, OxyR, ROS, oxidative stress
National Category
Microbiology in the medical area
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-115635ISBN: 978-91-7601-415-8 (print)OAI: oai:DiVA.org:umu-115635DiVA: diva2:900224
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
List of papers
1. Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H(2)O(2)-induced killing
Open this publication in new window or tab >>Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H(2)O(2)-induced killing
Show others...
2011 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, no 3, 1218-1224 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is classified as a category A select agent and a facultative intracellular bacterium. Why F. tularensis subsp. tularensis causes a more severe form of tularemia than F. tularensis subsp. holarctica does is not known. In this study, we have identified prominent phenotypic differences between the subspecies, since we found that F. tularensis subsp. tularensis strains contained less iron than F. tularensis subsp. holarctica strains. Moreover, strain SCHU S4 of F. tularensis subsp. tularensis was less susceptible than FSC200 and the live vaccine strain (LVS) of F. tularensis subsp. holarctica to H(2)O(2)-induced killing. The activity of the H(2)O(2)-degrading enzyme catalase was similar between the strains, whereas the iron content affected their susceptibility to H(2)O(2), since iron starvation rendered F. tularensis subsp. holarctica strains more resistant to H(2)O(2). Complementing LVS with fupA, which encodes an important virulence factor that regulates iron uptake, reduced its iron content and increased the resistance to H(2)O(2)-mediated killing. By real-time PCR, it was demonstrated that FSC200 and LVS expressed higher levels of gene transcripts related to iron uptake and storage than SCHU S4 did, and this likely explained their high iron content. Together, the results suggest that F. tularensis subsp. tularensis strains have restricted iron uptake and storage, which is beneficial for their resistance to H(2)O(2)-induced killing. This may be an important factor for the higher virulence of this subspecies of F. tularensis, as reactive oxygen species, such as H(2)O(2), are important bactericidal components during tularemia.

Keyword
Catalase, metabolism, Virulence
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-43210 (URN)10.1128/IAI.01116-10 (DOI)000287700200024 ()21189323 (PubMedID)
Available from: 2011-04-22 Created: 2011-04-22 Last updated: 2017-12-11Bibliographically approved
2. The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS
Open this publication in new window or tab >>The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS
2012 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, 14- p.Article in journal (Refereed) Published
Abstract [en]

Background: The Francisella tularensis protein MglA performs complex regulatory functions since it influences the expression of more than 100 genes and proteins in F. tularensis. Besides regulating the igl operon, it has been suggested that it also regulates several factors such as SspA, Hfq, CspC, and UspA, all important to stress adaptation. Therefore, it can be hypothesized that MglA plays an important role for Francisella stress responses in general and for the oxidative stress response specifically.

Results: We investigated the oxidative stress response of the Delta mglA mutant of the live vaccine strain (LVS) of F. tularensis and found that it showed markedly diminished growth and contained more oxidized proteins than the parental LVS strain when grown in an aerobic milieu but not when grown microaerobically. Moreover, the Delta mglA mutant exhibited an increased catalase activity and reduced expression of the fsl operon and feoB in the aerobic milieu. The mutant was also found to be less susceptible to H2O2. The aberrant catalase activity and gene expression was partially normalized when the Delta mglA mutant was grown in a microaerobic milieu.

Conclusions: Altogether the results show that the Delta mglA mutant exhibits all the hallmarks of a bacterium subjected to oxidative stress under aerobic conditions, indicating that MglA is required for normal adaptation of F. tularensis to oxidative stress and oxygen-rich environments.

Place, publisher, year, edition, pages
London: BioMed Central, 2012
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-53946 (URN)10.1186/1471-2180-12-14 (DOI)000301483900001 ()22264342 (PubMedID)
Available from: 2012-04-12 Created: 2012-04-10 Last updated: 2017-12-07Bibliographically approved
3. OxyR: an important regulator of the oxidative stress response of Francisella tularensis LVS
Open this publication in new window or tab >>OxyR: an important regulator of the oxidative stress response of Francisella tularensis LVS
(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
Francisella tularensis, OxyR, oxidative stress
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-115632 (URN)
Available from: 2016-02-03 Created: 2016-02-03 Last updated: 2016-02-04Bibliographically approved

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