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Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H(2)O(2)-induced killing
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
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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.

Place, publisher, year, edition, pages
2011. Vol. 79, no 3, 1218-1224 p.
Keyword [en]
Catalase, metabolism, Virulence
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-43210DOI: 10.1128/IAI.01116-10ISI: 000287700200024PubMedID: 21189323OAI: oai:DiVA.org:umu-43210DiVA: diva2:412379
Available from: 2011-04-22 Created: 2011-04-22 Last updated: 2016-05-24Bibliographically 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)
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Supervisors
Available from: 2016-02-05 Created: 2016-02-03 Last updated: 2016-02-04Bibliographically approved

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Lindgren, HelenaHonn, MarieForsberg, ÅkeSjöstedt, Anders
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Clinical BacteriologyMolecular Infection Medicine Sweden (MIMS)Department of Clinical MicrobiologyUmeå Centre for Microbial Research (UCMR)
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