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  • 1.
    Binesse, Johan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindgren, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Conlan, Wayne
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Roles of Reactive Oxygen Species-Degrading Enzymes of Francisella tularensis SCHU S42015Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, nr 6, s. 2255-2263Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO-) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO- but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.

  • 2.
    Bönquist, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Guina, Tina
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages2008Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, nr 8, s. 3502-3510Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Francisella tularensis live vaccine strain (LVS), in contrast to its iglC mutant, replicates in the cytoplasm of macrophages. We studied the outcome of infection of the murine macrophagelike cell line J774A.1 with LVS and with iglC, iglD, and mglA mutants, the latter of which is deficient in a global regulator. Compared to LVS, all of the mutants showed impaired intracellular replication up to 72 h, and the number of the mglA mutant bacteria even decreased. Colocalization with LAMP-1 was significantly increased for all mutants compared to LVS, indicating an impaired ability to escape into the cytoplasm. A lysosomal acidity-dependent dye accumulated in approximately 40% of the vacuoles containing mutant bacteria but not at all in vacuoles containing LVS. Preactivation of the macrophages with gamma interferon inhibited the intracellular growth of all strains and significantly increased acidification of phagosomes containing the mutants, but it only slightly increased the LAMP-1 colocalization. The intracellular replication and phagosomal escape of the iglC and iglD mutants were restored by complementation in trans. In conclusion, the IglC, IglD, and MglA proteins each directly or indirectly critically contribute to the virulence of F. tularensis LVS, including its intracellular replication, cytoplasmic escape, and inhibition of acidification of the phagosomes.

  • 3.
    Engdahl, Cecilia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Näslund, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Lindgren, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Ahlm, Clas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Bucht, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    The Rift Valley Fever virus protein NSm and putative cellular protein interactions2012Ingår i: Virology Journal, ISSN 1743-422X, Vol. 9, s. 139-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and beta-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cistrans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection.

  • 4.
    Golovliov, Igor
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Eneslätt, Kjell
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Conlan, Wayne
    Mosnier, Amandine
    Henry, Thomas
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy2016Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikel-id 152Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naive, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, Delta clpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naive splenocytes, Delta clpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the Delta clpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the Delta clpB-immune splenocytes also contained higher levels of IFN-gamma, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-gamma, TNF-alpha, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS(-/-) mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

  • 5.
    Honn, Marie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bharath, Gurram Kumar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lack of OxyR and KatG Results in Extreme Susceptibility of Francisella tularensis LVS to Oxidative Stress and Marked Attenuation In vivo2017Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 7, artikel-id 14Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is an intracellular bacterium and as such is expected to encounter a continuous attack by reactive oxygen species (ROS) in its intracellular habitat and efficiently coping with oxidative stress is therefore essential for its survival. The oxidative stress response system of F tularensis is complex and includes multiple antioxidant enzymes and pathways, including the transcriptional regulator OxyR and the H2O2-decomposing enzyme catalase, encoded by katG. The latter is regulated by OxyR. A deletion of either of these genes, however, does not severely compromise the virulence of F tularensis and we hypothesized that if the bacterium would be deficient of both catalase and OxyR, then the oxidative defense and virulence of F tularensis would become severely hampered. To test this hypothesis, we generated a double deletion mutant, Delta oxyR/Delta katG, of F tularensis LVS and compared its phenotype to the parental LVS strain and the corresponding single deletion mutants. In accordance with the hypothesis, Delta oxyR/Delta katG was distinctly more susceptible than Delta oxyR and Delta katG to H2O2, ONOO-, and O-2(-), moreover, it hardly grew in mouse-derived BMDM or in mice, whereas Delta katG and Delta oxyR grew as well as F tularensis LVS in BMDM and exhibited only slight attenuation in mice. Altogether, the results demonstrate the importance of catalase and OxyR for a robust oxidative stress defense system and that they act cooperatively. The lack of both functions render F tularensis severely crippled to handle oxidative stress and also much attenuated for intracellular growth and virulence.

  • 6.
    Honn, Marie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS2012Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, s. 14-Artikel i tidskrift (Refereegranskat)
    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.

  • 7.
    Lai, Xin-He
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Luan, Shi-Lu
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Biomedicinsk laboratorievetenskap.
    Stenman, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Infection with Francisella novicida leads to killing of murine macrophages by necrosisManuskript (preprint) (Övrigt vetenskapligt)
  • 8.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Honn, Marie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Golovlev, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Kadzhaev, Konstantin
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Conlan, Wayne
    NRC Canada.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    The 58-kilodalton major virulence factor of Francisella tularensis is required for efficient utilization of iron2009Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, nr 10, s. 4429-4436Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the Delta FTT0918 and Delta fslA isogenic mutants were analyzed. The gene product missing in the Delta fslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The Delta FTT0918 mutant, but not the Delta fslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the Delta fslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the Delta fslA strain but not of the Delta FTT0918 strain. The sensitivity of SCHU S4 and the Delta FTT0918 and Delta fslA strains to streptonigrin demonstrated that the Delta FTT0918 strain contained a smaller free intracellular iron pool and that the Delta fslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the Delta FTT0918 strain, the Delta fslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

  • 9.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Honn, Marie
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Salomonsson, Emelie
    Kuoppa, Kerstin
    Forsberg, Åke
    Swedish Def Res Agcy, Div CBRN Def & Secur, SE-90182 Umea, Sweden .
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Iron content differs between Francisella tularensis subspecies tularensis and subspecies holarctica strains and correlates to their susceptibility to H(2)O(2)-induced killing2011Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 3, s. 1218-1224Artikel i tidskrift (Refereegranskat)
    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.

  • 10.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindgren, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Mechanisms of heme utilization by Francisella tularensis2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 3, artikel-id e0119143Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

  • 11.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis2016Ingår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, nr 1, s. 288-295Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a Delta fslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a Delta feoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 mu M gallium and 10 mu g/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo.

  • 12.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Stenman, Linda
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Tärnvik, Arne
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Sjöstedt, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages.2005Ingår i: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 7, nr 3, s. 467-475Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.

  • 13.
    Lindgren, Helena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Stenmark, Stephan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Chen, Wangxue
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Tärnvik, Arne
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis.2004Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 72, nr 12, s. 7172-7182Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.

  • 14. Siebert, Claire
    et al.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ferre, Sabrina
    Villers, Corinne
    Boisset, Sandrine
    Perard, Julien
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Maurin, Max
    Brochier-Armanet, Celine
    Coute, Yohann
    Renesto, Patricia
    Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation2019Ingår i: Emerging Microbes & Infections, ISSN 2222-1751, Vol. 8, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is the causative agent in tularemia for which the high prevalence of treatment failure and relapse is a major concern. Directed-evolution experiments revealed that acquisition of fluoroquinolone (FQ) resistance was linked to factors in addition to mutations in DNA gyrase. Here, using F. tularensis live vaccine strain (LVS) as a model, we demonstrated that FupA/B (Fer-Utilization Protein) expression is linked to FQ susceptibility, and that the virulent strain F. tularensis subsp. tularensisSCHU S4 deleted for the homologous FupA protein exhibited even higher FQ resistance. In addition to an increased FQ minimal inhibitory concentration, LVSΔfupA/B displayed tolerance toward bactericidal compounds including ciprofloxacin and gentamicin. Interestingly, the FupA/B deletion was found to promote increased secretion of outer membrane vesicles (OMVs). Mass spectrometry-based quantitative proteomic characterization of vesicles from LVS and LVS∆fupA/B identified 801 proteins, including a subset of 23 proteins exhibiting differential abundance between both strains which may therefore contribute to the reduced antibiotic susceptibility of the FupA/B-deleted strain. We also demonstrated that OMVs are key structural elements of LVSΔfupA/Bbiofilms providing protection against FQ. These results provide a new basis for understanding and tackling antibiotic resistance and/or persistence of Francisella and other pathogenic members of the Thiotrichales class.

  • 15.
    Stenmark, Stephan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Tärnvik, Arne
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    T-cell-dependent and independent protection against Francisella tularensisManuskript (preprint) (Övrigt vetenskapligt)
  • 16.
    Tancred, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Binesse, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Inhibition of the oxidative burst by Francisella tularensis LVS is dependent on expression of MglA and the pathogenicity island proteins IglB, IglC, and IglD, but is not affected by IFN-γManuskript (preprint) (Övrigt vetenskapligt)
  • 17.
    Tancred, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Telepnev, Maxim
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Golovlev, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Andersson, Blanka
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Andersson, Henrik
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Administration of a donor of nitric oxide inhibits mglA expression of intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion2011Ingår i: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 30, nr 11, s. 1570-1583Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide or 3-morpholinosydnonimine hydrochloride (SIN-1), which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50- to 100-fold. F. tularensis-infected J774 cells are incapable of secreting TNF-alpha in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-alpha secretion of J774 cells. Strong staining for nitrotyrosine was observed in SNAP-treated bacteria and mass spectrometry identified nitration of two ribosomal 50S proteins, a CBS domain pair protein, and bacterioferritin. The results demonstrate that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in nitration of several F. tularensis proteins.

  • 18. Twine, Susan
    et al.
    Byström, Mona
    Chen, Wangxue
    Forsman, Mats
    FOI, Umeå (Swedish Defence Research Agency).
    Golovliov, Igor
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Johansson, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Kelly, John
    Lindgren, Helena
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Svensson, Kerstin
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Zingmark, Carl
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Conlan, Wayne
    NRC, Kanada.
    Sjöstedt, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine2005Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 73, nr 12, s. 8345-8352Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis subsp. tularensis (type A) strain SCHU S4 is a prototypic strain of the pathogen that is highly virulent for humans and other mammals. Its intradermal (i.d.) 50% lethal dose (LD50) for mice is <10 CFU. We discovered a spontaneous mutant, designated FSC043, of SCHU S4 with an i.d. LD50 of >10(8) CFU. FSC043 effectively vaccinated mice against challenge with a highly virulent type A strain, and the protective efficacy was at least as good as that of F. tularensis LVS, an empirically attenuated strain which has been used as an efficacious human vaccine. Comparative proteomics was used to identify two proteins of unknown function that were identified as defective in LVS and FSC043, and deletion mutants of SCHU S4 were created for each of the two encoding genes. One mutant, the DeltaFTT0918 strain, failed to express a 58-kDa protein, had an i.d. LD50 of approximately 10(5) CFU, and was found to be less capable than SCHU S4 of growing in peritoneal mouse macrophages. Mice that recovered from sublethal infection with the DeltaFTT0918 mutant survived when challenged 2 months later with >100 LD50s of the highly virulent type A strain FSC033. This is the first report of the generation of defined mutants of F. tularensis subsp. tularensis and their use as live vaccines.

  • 19. Twine, Susan M
    et al.
    Vinogradov, Evguenii
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Conlan, J. Wayne
    Roles for wbtC, wbtI, and kdtA genes in lipopolysaccharide biosynthesis, protein glycosylation, virulence, and immunogenicity in Francisella tularensis strain SCHU S42012Ingår i: Pathogens, ISSN 2076-0817, Vol. 1, nr 1, s. 12-29Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Using a strategy of gene deletion mutagenesis, we have examined the roles of genes putatively involved in lipopolysaccharide biosynthesis in the virulent facultative intracellular bacterial pathogen, Francisella tularensis subspecies tularensis, strain SCHU S4 in LPS biosynthesis, protein glycosylation, virulence and immunogenicity. One mutant, ∆wbtI, did not elaborate a long chain O-polysaccharide (OPS), was completely avirulent for mice, and failed to induce a protective immune response against challenge with wild type bacteria. Another mutant, ∆wbtC, produced a long chain OPS with altered chemical and electrophoretic characteristics. This mutant showed markedly reduced glycosylation of several known glycoproteins. Additionally this mutant was highly attenuated, and elicited a protective immune response against systemic, but not respiratory challenge with wild type SCHU S4. A third mutant, ∆kdtA, produced an unconjugated long chain OPS, lacking a detectable core structure, and which was not obviously expressed at the surface. It was avirulent and elicited partial protection against systemic challenge only.

  • 20. Wallet, Pierre
    et al.
    Benaoudia, Sacha
    Mosnier, Amandine
    Lagrange, Brice
    Martin, Amandine
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Michal, Fanny
    Basso, Pauline
    Djebali, Sophia
    Provost, Angelina
    Allatif, Omran
    Meunier, Etienne
    Broz, Petr
    Yamamoto, Masahiro
    Py, Benedicte F.
    Faudry, Eric
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Henry, Thomas
    IFN-gamma extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection2017Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, nr 10, artikel-id e1006630Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cellintrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-gamma induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-gamma priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-gamma-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners.

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