| 1. |
- Desvars, Amélie, 1980-, et al.
(författare)
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Epidemiology and Ecology of Tularemia in Sweden, 1984-2012
- 2015
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Ingår i: Emerging Infectious Diseases. - : Centers for Disease Control and Prevention (CDC). - 1080-6040 .- 1080-6059. ; 21:1, s. 32-39
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Tidskriftsartikel (refereegranskat)abstract
- The zoonotic disease tularemia is endemic in large areas of the Northern Hemisphere, but research is lacking on patterns of spatial distribution and connections with ecologic factors. To describe the spatial epidemiology of and identify ecologic risk factors for tularemia incidence in Sweden, we analyzed surveillance data collected over 29 years (1984-2012). A total of 4,830 cases were notified, of which 3,524 met all study inclusion criteria. From the first to the second half of the study period, mean incidence increased 10-fold, from 0.26/100,000 persons during 1984-1998 to 2.47/100,000 persons during 1999 2012 (p<0.001). The incidence of tularemia was higher than expected in the boreal and alpine ecologic regions (p<0.001), and incidence was positively correlated with the presence of lakes and rivers (p<0.001). These results provide a comprehensive epidemiologic description of tularemia in Sweden and illustrate that incidence is higher in locations near lakes and rivers.
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| 2. |
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| 3. |
- Kelk, Peyman, 1976-, et al.
(författare)
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IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin
- 2008
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Ingår i: International Journal of Medical Microbiology. - : Elsevier BV. - 1438-4221 .- 1618-0607. ; 298:5/6, s. 529-541
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Tidskriftsartikel (refereegranskat)abstract
- Aggregatibacter (Actinobacillus) actinomycetemcomitans forms a leukotoxin that selectively lyses primate neutrophils, monocytes and triggers apoptosis in promyeloic cells and degranulation of human neutrophils. Recently, we showed that the leukotoxin causes activation of caspase-1 and abundant secretion of bio-active IL-1beta from human macrophages. In this study, we show that high levels of IL-beta correlated with a high proportion of A. actinomycetemcomitans in clinical samples from a patient with aggressive periodontitis. To determine the relative contribution of leukotoxin to the overall bacteria-induced IL-1beta secretion, macrophages were isolated from peripheral blood and exposed to different concentrations of live A. actinomycetemcomitans strains with either no, low or high production of leukotoxin. Cell lysis and levels of IL-1beta, IL-6, TNF-alpha and caspase-1 were measured by ELISA and flow cytometry. Leukotoxin was the predominant cause of IL-1beta secretion from macrophages, even in the A. actinomycetemcomitans strain with low leukotoxin production. Macrophages exposed to non-leukotoxic bacteria accumulated cytosolic pro-IL-1beta, which was secreted by a secondary exposure to leukotoxic bacteria. In conclusion, the present study shows for the first time that A. actinomycetemcomitans-induced IL-1beta secretion from human macrophages in vitro is mainly caused by leukotoxin.
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| 4. |
- Broman, T, et al.
(författare)
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Molecular Detection of Persistent Francisella tularensis Subspecies holarctica in Natural Waters
- 2011
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Ingår i: International Journal of Microbiology. - : Hindawi Publishing Corporation. - 1687-918X .- 1687-9198. ; 2011, s. Article ID 851946-
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Tidskriftsartikel (refereegranskat)abstract
- Tularemia, caused by the bacterium Francisella tularensis, where F. tularensis subspecies holarctica has long been the cause of endemic disease in parts of northern Sweden. Despite this, our understanding of the natural life-cycle of the organism is still limited. During three years, we collected surface water samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with endemic tularemia. Real-time PCR screening demonstrated the presence of F. tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively. The 16S rRNA sequences from those samples all grouped to the species F. tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from selected sampling points confirmed the presence of F. tularensis subspecies holarctica-specific sequences. These sequences were detected in water sampled during both outbreak and nonoutbreak years. Our results indicate that diverse F. tularensis-like organisms, including F. tularensis subsp. holarctica, persist in natural waters and sediments in the investigated areas with endemic tularemia.
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| 5. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
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A conserved α-helix essential for a type VI secretion-like system of Francisella tularensis
- 2009
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 191:8, s. 2431-2446
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These include iglA and iglB, the homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33-132, necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth. In particular, residues 103-122, overlapping with a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in yeast, but markedly impaired intra-macrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to T6S, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium and Escherichia coli were also shown to interact in yeast and the interaction was dependent on the preservation of the same alpha-helix. Heterologous interactions formed between non-native IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens.
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| 6. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
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IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS
- 2011
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 79:9, s. 3683-3696
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.
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| 7. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
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The role of the Francisella Tularensis pathogenicity island in type VI secretion, intracellular survival, and modulation of host cell signaling
- 2010
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Ingår i: Frontiers in microbiology. - : Frontiers Media SA. - 1664-302X. ; 1, s. 136-
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them.
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| 8. |
- Bönquist, Linda, 1974-, et al.
(författare)
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MglA and Igl proteins contribute to the modulation of Francisella tularensis live vaccine strain-containing phagosomes in murine macrophages
- 2008
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:8, s. 3502-3510
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Champion, Mia D, et al.
(författare)
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Comparative genomic characterization of Francisella tularensis strains belonging to low and high virulence subspecies
- 2009
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Ingår i: PLoS pathogens. - : Public Library of Science (PLoS). - 1553-7374. ; 5:5, s. e1000459-
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Tidskriftsartikel (refereegranskat)abstract
- Tularemia is a geographically widespread, severely debilitating, and occasionally lethal disease in humans. It is caused by infection by a gram-negative bacterium, Francisella tularensis. In order to better understand its potency as an etiological agent as well as its potential as a biological weapon, we have completed draft assemblies and report the first complete genomic characterization of five strains belonging to the following different Francisella subspecies (subsp.): the F. tularensis subsp. tularensis FSC033, F. tularensis subsp. holarctica FSC257 and FSC022, and F. tularensis subsp. novicida GA99-3548 and GA99-3549 strains. Here, we report the sequencing of these strains and comparative genomic analysis with recently available public Francisella sequences, including the rare F. tularensis subsp. mediasiatica FSC147 strain isolate from the Central Asian Region. We report evidence for the occurrence of large-scale rearrangement events in strains of the holarctica subspecies, supporting previous proposals that further phylogenetic subdivisions of the Type B clade are likely. We also find a significant enrichment of disrupted or absent ORFs proximal to predicted breakpoints in the FSC022 strain, including a genetic component of the Type I restriction-modification defense system. Many of the pseudogenes identified are also disrupted in the closely related rarely human pathogenic F. tularensis subsp. mediasiatica FSC147 strain, including modulator of drug activity B (mdaB) (FTT0961), which encodes a known NADPH quinone reductase involved in oxidative stress resistance. We have also identified genes exhibiting sequence similarity to effectors of the Type III (T3SS) and components of the Type IV secretion systems (T4SS). One of the genes, msrA2 (FTT1797c), is disrupted in F. tularensis subsp. mediasiatica and has recently been shown to mediate bacterial pathogen survival in host organisms. Our findings suggest that in addition to the duplication of the Francisella Pathogenicity Island, and acquisition of individual loci, adaptation by gene loss in the more recently emerged tularensis, holarctica, and mediasiatica subspecies occurred and was distinct from evolutionary events that differentiated these subspecies, and the novicida subspecies, from a common ancestor. Our findings are applicable to future studies focused on variations in Francisella subspecies pathogenesis, and of broader interest to studies of genomic pathoadaptation in bacteria.
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| 10. |
- Conlan, J Wayne, et al.
(författare)
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Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria : effects of host background and route of immunization
- 2010
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1873-2518. ; 28:7, s. 1824-1831
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4DeltaclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4DeltaclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Delta0918DeltacapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4DeltaclpB, or SCHU S4Delta0918DeltacapB provided no obvious correlate to their relative efficacies.
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| 11. |
- Conlan, J Wayne, et al.
(författare)
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Molecular immunology of experimental primary tularemia in mice infected by respiratory or intradermal routes with type A Francisella tularensis.
- 2008
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 45:10, s. 2962-2969
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Tidskriftsartikel (refereegranskat)abstract
- The type A subspecies of Francisella tularensis is a highly virulent facultative intracellular bacterial pathogen, and a potential biological weapon. Recently, there has been renewed interest in developing new vaccines and therapeutics against this bacterium. Natural cases of disease, tularemia, caused by the type A subspecies are very rare. Therefore, the United States Food and Drug Administration will rely on the so-called Animal Rule for efficacy testing of anti-Francisella medicines. This requires the human disease to be modeled in one or more animal species in which the pathogenicity of the agent is reasonably well understood. Mice are natural hosts for F. tularensis, and might be able to satisfy this requirement. Tularemia pathogenesis appears to be primarily due to the host inflammatory response which is poorly understood at the molecular level. Additionally, the extent to which this response varies depending on host and pathogen genetic background, or by pathogen challenge route or dose is unknown. Therefore, the present study examined sera and infected tissues from C57BL/6 and BALB/c mice challenged by natural intradermal (ID) and respiratory routes with one of two distinct type A strains of the pathogen for cytokine and chemokine responses that might help to explain the morbidity associated with tularemia. The results show that the molecular immune response was mostly similar regardless of the variables examined. For instance, mRNA for the proinflammatory cytokine IL-6, and chemokines KC, and IP-10 was consistently upregulated at all sites of infection. Upregulation of mRNA for several other cytokines and chemokines occurred in a more tissue restricted manner. For instance, IFN-gamma was highly upregulated in the skin of BALB/c, but not C57BL/6 mice after ID inoculation of the pathogen, whilst IL-10 mRNA upregulation was only consistently seen in the skin and lungs.
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| 12. |
- Elfving, Karin, et al.
(författare)
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Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp. IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.
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| 13. |
- Eliasson, Henrik, et al.
(författare)
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Kinetics of the immune response associated with tularemia : comparison of an enzyme-linked immunosorbent assay, a tube agglutination test, and a novel whole-blood lymphocyte stimulation test
- 2008
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Ingår i: Clinical and Vaccine Immunology. - 1556-6811 .- 1556-679X. ; 15:8, s. 1238-1243
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Tidskriftsartikel (refereegranskat)abstract
- We have developed and evaluated a novel and simplified whole-blood lymphocyte stimulation assay that focuses on the measurement of gamma interferon after 24 h of stimulation with whole-cell tularemia antigen and a tularemia enzyme-linked immunosorbent assay (ELISA) based on highly purified lipopolysaccharide antigen. Comparison of the kinetics of the two assays and those of the traditional tube agglutination test shows that the cellular immune response can be detected earlier by the lymphocyte stimulation assay. This test already shows a high proportion of positive results during the first week after the onset of the disease, may be applicable in everyday laboratory practice, and has the potential of changing routine diagnostics for tularemia. The new ELISA has a high sensitivity and becomes positive to a high degree during the second week of disease.
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| 14. |
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| 15. |
- Honn, Marie, 1982-, et al.
(författare)
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The role of MglA for adaptation to oxidative stress of Francisella tularensis LVS
- 2012
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Ingår i: BMC Microbiology. - London : BioMed Central. - 1471-2180. ; 12, s. 14-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 16. |
- Kadzhaev, Konstantin, et al.
(författare)
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Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model
- 2009
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:5, s. e5463-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis.METHODOLOGY/PRINCIPAL FINDINGS: The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD(50) of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably Delta pyrB and Delta recA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD(50) of > 10(3) CFU. In fact, the latter seven mutants showed very marked attenuation with an LD(50) of > or = 10(7) CFU.CONCLUSIONS/SIGNIFICANCE: The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.
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| 17. |
- Lampe, Elisabeth O., et al.
(författare)
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Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum
- 2016
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 82:5, s. 1586-1598
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Tidskriftsartikel (refereegranskat)abstract
- Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis Delta iglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis Delta iglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Delta atg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.
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| 18. |
- Lindgren, Helena, et al.
(författare)
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Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis
- 2016
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 60:1, s. 288-295
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 19. |
- Lindgren, Helena, 1969-, et al.
(författare)
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The 58-kilodalton major virulence factor of Francisella tularensis is required for efficient utilization of iron
- 2009
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 77:10, s. 4429-4436
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 20. |
- Meibom, Karin L, et al.
(författare)
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The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice
- 2008
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Ingår i: Molecular Microbiology. - Oxford : Blackwells. - 0950-382X .- 1365-2958. ; 67:6, s. 1384-1401
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular bacterial pathogens generally express chaperones such as Hsp100s during multiplication in host cells, allowing them to survive potentially hostile conditions. Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. The ability of F. tularensis to multiply and survive in macrophages is considered essential for its virulence. Although previous mutant screens in Francisella have identified the Hsp100 chaperone ClpB as important for intracellular survival, no detailed study has been performed. We demonstrate here that ClpB of F. tularensis live vaccine strain (LVS) is important for resistance to cellular stress. Promoter analysis shows that the transcriptional start is preceded by a sigma32-like promoter sequence and we demonstrate that expression of clpB is induced by heat shock. This indicates that expression of clpB is dependent on the heat-shock response mediated by sigma32, the only alternative sigma-factor present in Francisella. Our studies demonstrate that ClpB contributes to intracellular multiplication in vitro, but is not essential. However, ClpB is absolutely required for Francisella to replicate in target organs and induce disease in mice. Proteomic analysis of membrane-enriched fractions shows that five proteins are recovered at lower levels in the mutant strain. The crucial role of ClpB for in vivo persistence of Francisella may be linked to its assumed function in reactivation of aggregated proteins under in vivo stress conditions.
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| 21. |
- Meyer, Lena, et al.
(författare)
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Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication
- 2015
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 83:8, s. 3233-3242
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., Delta iglA, Delta iglC, Delta iglG, Delta iglI, and Delta pdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, Delta pdpE and Delta iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the Delta iglA and Delta iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.
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| 22. |
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| 23. |
- Salomonsson, Emelie, et al.
(författare)
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Reintroduction of two deleted virulence loci restores full virulence to the live vaccine strain of Francisella tularensis
- 2009
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 77:8, s. 3424-3431
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Tidskriftsartikel (refereegranskat)abstract
- A disadvantage of several old vaccines is that the genetic events resulting in the attenuation are often largely unknown and reversion to virulence cannot be excluded. In the 1950s, a live vaccine strain, LVS, was developed from a type B strain of Francisella tularensis, the causative agent of tularemia. LVS, which is highly attenuated for humans but still virulent for mice by some infection routes, has been extensively studied and found to protect staff from laboratory-acquired tularemia. The efforts to improve biopreparedness have identified a demand for a vaccine against tularemia. Recently the rapid progress in genomics of different Francisella strains has led to identification of several regions of differences (RDs). Two genes carried within RDs, pilA, encoding a putative type IV pilin, and FTT0918, encoding an outer membrane protein, have been linked to virulence. Interestingly, LVS has lost these two genes via direct repeat-mediated deletions. Here we show that reintroduction of the two deleted regions restores virulence of LVS in a mouse infection model to a level indistinguishable from that of virulent type B strains. The identification of the two attenuating deletion events could facilitate the licensing of LVS for use in humans.
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| 24. |
- Shen, Hua, et al.
(författare)
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Molecular immune responses to aerosol challenge with Francisella tularensis in mice inoculated with live vaccine candidates of varying efficacy
- 2010
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:10, s. e13349-
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Tidskriftsartikel (refereegranskat)abstract
- This is the first report characterizing local and systemic cytokine and chemokine responses in mice immunized with vaccines with different efficacies against aerosol challenge with virulent F. tularensis subsp. tularensis. It shows that increases in the levels of most of these immunomodulators, including those known to be critical for protective immunity, do not superficially correlate with protection unless adjusted for the effects of bacterial burden. Additionally, several cytokines were selectively suppressed in the lungs of naïve mice, suggesting that one mechanism of vaccine action is to overcome this pathogen-induced immunosuppression.
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| 25. |
- Tancred, Linda, 1974-, et al.
(författare)
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Administration of a donor of nitric oxide inhibits mglA expression of intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion
- 2011
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Ingår i: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 30:11, s. 1570-1583
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 26. |
- Tano, Krister, et al.
(författare)
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Alloiococcus otitidis--otitis media pathogen or normal bacterial flora?
- 2008
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - 0903-4641 .- 1600-0463. ; 116:9, s. 785-90
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Tidskriftsartikel (refereegranskat)abstract
- During the last decade a new potential otitis media pathogen, Alloiococcus otitidis, has been studied. It is still not clear whether this bacterium really is a pathogen, although it has been found in a high percentage of middle ear effusions in children. The present study aimed to investigate the presence of A. otitidis in the nasopharynx and outer ear canals, and to develop a culture method that would make it possible to isolate A. otitidis from these locations. Nasopharyngeal samples (n = 129) from children below 6 years were investigated by conventional culture on blood agar plates with 6% saline and rabbit antisera against A. otitidis, and by a PCR method. In the same way, we investigated 10 samples from vestibulum nasi of healthy persons, 68 samples from outer ear canals of patients with acute or chronic ear problems, and 24 samples from outer ear canals of healthy persons. In a rat model of acute otitis media, we instilled living A. otitidis into rat middle ears through the tympanic bulla and evaluated the outcome clinically by otomicroscopy at days 3, 6 and 14. Of the 129 nasopharyngeal cultures, 9 were positive for A. otitidis by PCR, but none by the culture method. Of the 68 samples from patients with running ears, 4 were positive for A. otitidis by PCR, but none by the culture method. Of the 24 healthy ear canals, 7 were positive for A. otitidis by PCR and 3 of them also by the culture method. No A. otitidis could be found from the vestibulum nasi. The rat experiment showed that the reactions in the middle ears were mild; we could not provoke a purulent acute otitis media in any of the rats. There was a 7% prevalence of A. otitidis in children below 6 years. The highest prevalence (29%) was found in outer ear canals of healthy persons, which strongly suggests that A. otitidis is part of the normal bacterial flora of the outer ear canal. The doubtful pathogenicity is also confirmed by the fact that--in the rat model--A. otitidis elicited only a mild response in the middle ear. It was possible to isolate A. otitidis using a blood agar plate with 6% saline.
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| 27. |
- Vonkavaara, Malin, 1981-, et al.
(författare)
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Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis
- 2008
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 10:6, s. 1327-1338
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Tidskriftsartikel (refereegranskat)abstract
- Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.
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| 28. |
- Waldenström, Jonas, et al.
(författare)
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Migrating birds and tickborne encephalitis virus
- 2007
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Ingår i: Emerging Infectious Diseases. - : Centers for Disease Control and Prevention (CDC). - 1080-6040 .- 1080-6059. ; 13:8, s. 1215-8
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Tidskriftsartikel (refereegranskat)abstract
- During spring and autumn 2001, we screened 13,260 migrating birds at Ottenby Bird Observatory, Sweden, and found 3.4% were infested with ticks. Four birds, each a different passerine species, carried tickborne encephalitis virus (TBEV)-infected ticks (Ixodes ricinus). Migrating birds may play a role in the geographic dispersal of TBEV-infected ticks.
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| 29. |
- Åhlund, Monika K, et al.
(författare)
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Directed screen of Francisella novicida virulence determinants using Drosophila melanogaster
- 2010
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Ingår i: Infection and Immunity. - : ASM International. - 0019-9567 .- 1098-5522. ; 78:7, s. 3118-3128
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.
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