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  • 1.
    Alam, Athar
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Javed, Eram
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 11324Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The Delta clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both Delta clpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

  • 2.
    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.

  • 3.
    Broman, T
    et al.
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Thelaus, J
    Andersson, A-C
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Bäckman, S
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Wikström, P
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Larsson, E
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Granberg, M
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Karlsson, L
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Bäck, E
    Department of Infectious Diseases, Örebro University Hospital, Örebro.
    Eliasson, H
    Department of Infectious Diseases, Örebro University Hospital, Örebro.
    Mattsson, R
    National Veterinary Institute, Uppsala.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Forsman, M
    Department of CBRN Defence and Security, Swedish Defence Research Agency, Umeå.
    Molecular Detection of Persistent Francisella tularensis Subspecies holarctica in Natural Waters2011Ingår i: International Journal of Microbiology, ISSN 1687-918X, E-ISSN 1687-9198, Vol. 2011, s. Article ID 851946-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 4. Bruce, Michael G
    et al.
    Deeks, Shelley L
    Zulz, Tammy
    Bruden, Dana
    Navarro, Christine
    Lovgren, Marguerite
    Jette, Louise
    Kristinsson, Karl
    Sigmundsdottir, Gudrun
    Jensen, Knud Brinkløv
    Lovoll, Oistein
    Nuorti, J Pekka
    Herva, Elja
    Nystedt, Anders
    Sjostedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Koch, Anders
    Hennessy, Thomas W
    Parkinson, Alan J
    International Circumpolar Surveillance System for invasive pneumococcal disease, 1999-20052008Ingår i: Emerging Infectious Diseases, ISSN 1080-6040, E-ISSN 1080-6059, Vol. 14, nr 1, s. 25-33Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The International Circumpolar Surveillance System is a population-based surveillance network for invasive bacterial disease in the Arctic. The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced for routine infant vaccination in Alaska (2001), northern Canada (2002-2006), and Norway (2006). Data for invasive pneumococcal disease (IPD) were analyzed to identify clinical findings, disease rates, serotype distribution, and antimicrobial drug susceptibility; 11,244 IPD cases were reported. Pneumonia and bacteremia were common clinical findings. Rates of IPD among indigenous persons in Alaska and northern Canada were 43 and 38 cases per 100,000 population, respectively. Rates in children <2 years of age ranged from 21 to 153 cases per 100,000 population. In Alaska and northern Canada, IPD rates in children <2 years of age caused by PCV7 serotypes decreased by >80% after routine vaccination. IPD rates are high among indigenous persons and children in Arctic countries. After vaccine introduction, IPD caused by non-PCV7 serotypes increased in Alaska.

  • 5.
    Bröms, Jeanette E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ishikawa, Takahiko
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun N.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A15522013Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, s. 96-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens.

  • 6.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Meyer, Lena
    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).
    IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS2011Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 9, s. 3683-3696Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 7.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    A conserved α-helix essential for a type VI secretion-like system of Francisella tularensis2009Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 8, s. 2431-2446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 8.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Dissection of the Functions of the IglC Protein of Francisella tularensis2010Ingår i: The challenge of highly pathogenic microorganisms: mechanisms of virulence and novel medical countermeasures, Springer, 2010, s. 67-75Konferensbidrag (Refereegranskat)
    Abstract [en]

    Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS). These include iglA and iglB, the homologues of which are conserved in T6SSs. They are part of the igl operon, also encompassing the iglC and iglD genes. We have used a yeast two-hybrid system to study the interaction of the Igl proteins of E tularensis LVS. Previously, we identified a region of IglA necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth with an essential role for a conserved alpha-helical region. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens. Herein, the interaction of IglC with other members of the operon was investigated. It showed no binding to the other members in the yeast two-hybrid assay and we found also that two cysteine residues, C191 and C192, predicted to be putative prenylation sites, played no role for the important contribution of IglC to the intracellular replication of E tularensis although C191 was important for the stability of the protein.

  • 9.
    Bröms, Jeanette E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Meyer, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Larsson, Pär
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 4, artikel-id e34639Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both Delta vgrG and Delta dotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.

  • 10.
    Bröms, Jeanette E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Meyer, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Department of Experimental Medical Science, Section for Immunology, Lund University, Lund, Sweden.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control type VI system-mediated secretion2017Ingår i: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 8, nr 6, s. 821-847Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an Delta iglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, Delta iglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-alpha, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first similar to 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.

  • 11.
    Bröms, Jeanette E.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Meyer, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sun, Kun
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Lavander, Moa
    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).
    Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 11, artikel-id e50473Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM beta-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native beta-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.

  • 12.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    The role of the Francisella Tularensis pathogenicity island in type VI secretion, intracellular survival, and modulation of host cell signaling2010Ingår i: Frontiers in microbiology, ISSN 1664-302X, Vol. 1, s. 136-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 13.
    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.

  • 14. Champion, Mia D
    et al.
    Zeng, Qiandong
    Nix, Eli B
    Nano, Francis E
    Keim, Paul
    Kodira, Chinnappa D
    Borowsky, Mark
    Young, Sarah
    Koehrsen, Michael
    Engels, Reinhard
    Pearson, Matthew
    Howarth, Clint
    Larson, Lisa
    White, Jared
    Alvarado, Lucia
    Forsman, Mats
    Totalförsvarets forskninginstitut, Umeå Sweden FOI.
    Bearden, Scott W
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Titball, Richard
    Michell, Stephen L
    Birren, Bruce
    Galagan, James
    Comparative genomic characterization of Francisella tularensis strains belonging to low and high virulence subspecies2009Ingår i: PLoS pathogens, ISSN 1553-7374, Vol. 5, nr 5, s. e1000459-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 15.
    Conlan, J Wayne
    et al.
    NRC, Kanada.
    Shen, Hua
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Zingmark, Carl
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Oyston, Petra CF
    Chen, Wangxue
    House, Robert V
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    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 immunization2010Ingår i: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 28, nr 7, s. 1824-1831Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 16. Conlan, J Wayne
    et al.
    Zhao, Xigeng
    Harris, Gregory
    Shen, Hua
    Bolanowski, Mark
    Rietz, Cecilia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Chen, Wangxue
    Molecular immunology of experimental primary tularemia in mice infected by respiratory or intradermal routes with type A Francisella tularensis.2008Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, nr 10, s. 2962-2969Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 17. De Pascalis, Roberto
    et al.
    Chou, Alicia Y.
    Ryden, Patrik
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Kennett, Nikki J.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Elkins, Karen L.
    Models Derived from In Vitro Analyses of Spleen, Liver, and Lung Leukocyte Functions Predict Vaccine Efficacy against the Francisella tularensis Live Vaccine Strain (LVS)2014Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, nr 2, artikel-id e00936Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e. g., tumor necrosis factor alpha [TNF-alpha], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and quantifying correlative T cell responses.

    IMPORTANCE

    Identifying and quantifying correlates of protection is especially challenging for intracellular bacteria, including Francisella tularensis. F. tularensis is classified as a category A bioterrorism agent, and no vaccines have been licensed in the United States, but tularemia is a rare disease. Therefore, clinical trials to test promising vaccines are impractical. In this report, we further evaluated a novel approach to developing correlates by assessing T cell immune responses in lungs and livers of differentially vaccinated mice; these nonprofessional immune tissues are colonized by Francisella. The relative degree of vaccine efficacy against systemic challenge was reflected by the ability of immune T cells, particularly liver T cells, to control the intramacrophage replication of bacteria in vitro and by relative gene expression of several immunological mediators. We therefore developed analytical models that combined bacterial replication data and gene expression data. Several resulting models provided excellent discrimination between vaccines of different efficacies.

  • 18.
    Desvars, Amélie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Furberg, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Epidemiologi och global hälsa.
    Hjertqvist, Marika
    Vidman, Linda
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Rydén, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Epidemiology and Ecology of Tularemia in Sweden2015Ingår i: International Journal of Epidemiology, ISSN 0300-5771, E-ISSN 1464-3685, Vol. 44, s. 58-58Artikel i tidskrift (Övrigt vetenskapligt)
  • 19.
    Desvars, Amélie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Furberg, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Epidemiologi och global hälsa. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Hjertqvist, Marika
    Vidman, Linda
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rydén, Patrik
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Epidemiology and Ecology of Tularemia in Sweden, 1984-20122015Ingår i: Emerging Infectious Diseases, ISSN 1080-6040, E-ISSN 1080-6059, Vol. 21, nr 1, s. 32-39Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 20.
    Desvars-Larrive, Amélie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Liu, X.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hjertqvist, M.
    Sjöstedt, A.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Johansson, A.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ryden, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    High-risk regions and outbreak modelling of tularemia in humans2017Ingår i: Epidemiology and Infection, ISSN 0950-2688, E-ISSN 1469-4409, Vol. 145, nr 3, s. 482-490Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sweden reports large and variable numbers of human tularemia cases, but the high-risk regions are anecdotally defined and factors explaining annual variations are poorly understood. Here, high-risk regions were identified by spatial cluster analysis on disease surveillance data for 1984-2012. Negative binomial regression with five previously validated predictors (including predicted mosquito abundance and predictors based on local weather data) was used to model the annual number of tularemia cases within the high-risk regions. Seven high-risk regions were identified with annual incidences of 3.8-44 cases/100 000 inhabitants, accounting for 56.4% of the tularemia cases but only 9.3% of Sweden's population. For all high-risk regions, most cases occurred between July and September. The regression models explained the annual variation of tularemia cases within most high-risk regions and discriminated between years with and without outbreaks. In conclusion, tularemia in Sweden is concentrated in a few high-risk regions and shows high annual and seasonal variations. We present reproducible methods for identifying tularemia high-risk regions and modelling tularemia cases within these regions. The results may help health authorities to target populations at risk and lay the foundation for developing an early warning system for outbreaks.

  • 21.
    Edin, Alicia
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Granholm, Susanne
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Koskiniemi, Satu
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Allard, Annika
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Development and Laboratory Evaluation of a Real-Time PCR Assay for Detecting Viruses and Bacteria of Relevance for Community-Acquired Pneumonia2015Ingår i: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, nr 3, s. 315-324Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Community-acquired pneumonia may present with similar clinical symptoms, regardless of viral or bacterial cause. Diagnostic assays are needed to rapidly discriminate between causes, because this will guide decisions on appropriate treatment. Therefore, a quantitative real-time PCR (qPCR) assay with duplex reactions targeting eight bacteria and six viruses was developed. Technical performance was examined with linear plasmids. Upper and Lower respiratory tract specimens were used to compare the qPCR assay with standard microbiological methods. The limit of detection was 5 to 20 DNA template copies with approximately 1000-fold differences in concentrations of the two competing templates. SDs for positive controls were <5%. The use of the qPCR assay resulted in 113 positive identifications in 94 respiratory specimens compared with 38 by using standard diagnostics. Diagnostic accuracy of the qPCR assay varied between 60% positive agreement with standard tests for Streptococcus pneumoniae and 100% for Mycoplasma pneumoniae, Moraxella catarrhalis, and Staphylococcus aureus. Negative percentage of agreement was >95% for M. pneumoniae, Streptococcus pyogenes, respiratory syncytial virus, and influenza A virus; whereas it was only 56% for Haemophilus influenzae. Multiple microbial agents were identified in 19 of 44 sputum and 19 of 50 nasopharynx specimens. We conclude that in parallel qPCR detection of the targeted respiratory bacteria and viruses is feasible. The results indicate good technical performance of the assay in clinical specimens.

  • 22. Elfving, Karin
    et al.
    Olsen, Björn
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Waldenström, Jonas
    Lundkvist, Åke
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Mejlon, Hans
    Nilsson, Kenneth
    Dissemination of spotted fever rickettsia agents in Europe by migrating birds2010Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, nr 1, s. e8572-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 23.
    Eliasson, Henrik
    et al.
    Universitetssjukhuset Örebro.
    Olcén, Per
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Jurstrand, Margareta
    Bäck, Erik
    Örebro.
    Andersson, Sören
    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.2008Ingår i: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 15, nr 8, s. 1238-43Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 24.
    Eneslätt, Kjell
    et al.
    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.
    Rydén, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system2018Ingår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, artikel-id 27Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis.

  • 25.
    Eneslätt, Kjell
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Normark, Monica
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Björk, Rafael
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rietz, Cecilia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Zingmark, Carl
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wolfraim, Lawrence A
    Stöven, Svenja
    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).
    Signatures of T cells as correlates of immunity to Francisella tularensis2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 3, s. e32367-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4(+) and/or CD8(+) T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naive). Significant differences were detected between either of the immune donor groups and naive individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-gamma, MCP-1, and MIP-1 beta. Expression of IFN-gamma, MIP-1 beta, and CD107a by CD4(+)CD45RO(+) or CD8(+) CD45RO(+) T cells correlated to antigen concentrations. In particular, IFN-gamma and MIP-1 beta strongly discriminated between immune and naive individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-alpha-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.

  • 26.
    Eneslätt, Kjell
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Rietz, Cecilia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Rydén, Patrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för matematik och matematisk statistik.
    Stöven, Svenja
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    House, Robert V
    Wolfraim, Lawrence A
    Tärnvik, Arne
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis2011Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 41, nr 4, s. 974-980Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here, lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1β, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4(+) CCR7(+) , CD4(+) CD62L(+) , CD8(+) CCR7(+) , and CD8(+) CD62L(+) cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline.

  • 27.
    Forslund, Anna-Lena
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Salomonsson, Emelie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Goloviov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Kuoppa, Kerstin
    FOI, Umeå (Swedish Defence Research Agency).
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI, Umeå (Swedish Defence Research Agency).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensisManuskript (Övrig (populärvetenskap, debatt, mm))
    Abstract [en]

    Background: All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.

    Results: In a previous study, we were able to show that one pilin gene, pilA, was essential for virulence of a type B strain in a mouse infection model. In this work we have examined the role of several pilin genes in the virulence of the pathogenic type A strain SCHU S4. pilA, pilC, pilQ, and pilT were mutated by in-frame deletion mutagenesis. Interestingly, when mice were infected with a mixture of each mutant strain and the wild-type strain, the pilA, pilC and pilQ mutants were out-competed, while the pilT mutant was equally competitive as the wild-type.

    Conclusions: This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

  • 28.
    Forslund, Anna-Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Salomonsson, Emelie Näslund
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kuoppa, Kerstin
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis2010Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, s. 227-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

  • 29. Fulton, Kelly M.
    et al.
    Zhao, Xigeng
    Petit, Mireille D.
    Kilmury, Sara L. N.
    Wolfraim, Lawrence A.
    House, Robert V.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Twine, Susan M.
    Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination2011Ingår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 301, nr 7, s. 591-601Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60% or more sera from vaccinees and convalescents. A further four proteins were recognised by 30-60% of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines. Crown Copyright (C) 2011 Published by Elsevier GmbH. All rights reserved.

  • 30.
    Golovlev, Igor
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Twine, Susan M.
    Shen, Hua
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Conlan, Wayne
    A Delta clpB Mutant of Francisella tularensis Subspecies holarctica Strain, FSC200, Is a More Effective Live Vaccine than F. tularensis LVS in a Mouse Respiratory Challenge Model of Tularemia2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 11, s. e78671-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis subsp. tularensis is a highly virulent pathogen for humans especially if inhaled. Consequently, it is considered to be a potential biothreat agent. An experimental vaccine, F. tularensis live vaccine strain, derived from the less virulent subsp. holarctica, was developed more than 50 years ago, but remains unlicensed. Previously, we developed a novel live vaccine strain, by deleting the chaperonin clpB gene from F. tularensis subsp. tularensis strain, SCHU S4. SCHU S4 Delta clpB was less virulent for mice than LVS and a more effective vaccine against respiratory challenge with wild type SCHU S4. In the current study, we were interested to determine whether a similar mutant on the less virulent subsp. holarctica background would also outperform LVS in terms of safety and efficacy. To this end, clpB was deleted from clinical holarctica strain, FSC200. FSC200 Delta clpB had a significantly higher intranasal LD50 than LVS for BALB/c mice, but replicated to higher numbers at foci of infection after dermal inoculation. Moreover, FSC200 Delta clpB killed SCID mice more rapidly than LVS. However, dermal vaccination of BALB/c mice with the former versus the latter induced greater protection against respiratory challenge with SCHU S4. This increased efficacy was associated with enhanced production of pulmonary IL-17 after SCHU S4 challenge.

  • 31.
    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.

  • 32.
    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.

  • 33.
    Honn, Marie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). MIMS.
    Lindgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). MIMS.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). MIMS.
    OxyR: an important regulator of the oxidative stress response of Francisella tularensis LVSManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

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

  • 34.
    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.

  • 35.
    Ishikawa, Takahiko
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sabharwal, Dharmesh
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Milton, Debra L
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Pathoadaptive conditional regulation of the type VI secretion system in Vibrio cholerae O1 strains2012Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, nr 2, s. 575-584Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The most recently discovered secretion pathway in gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.

  • 36.
    Johansson, Anders
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Celli, Jean
    Conlan, Wayne
    NRC, Canada.
    Elkins, Karen L
    Forsman, Mats
    Swedish Defense Research Agency, Umea, Sweden.
    Keim, Paul S
    Larsson, Pär
    Swedish Defense Research Agency, Umea, Sweden.
    Manoil, Colin
    Nano, Francis E
    Petersen, Jeannine M
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Objections to the transfer of Francisella novicida to the subspecies rank of Francisella tularensis2010Ingår i: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 60, nr 8, s. 1717-1718Artikel i tidskrift (Refereegranskat)
  • 37.
    Johansson, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Farlow, Jason
    Larsson, Pär
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Dukerich, Meghan
    Chambers, Elias
    Byström, Mona
    Fox, James
    Chu, May
    Forsman, Mats
    FOI, Umeå.
    Sjöstedt, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    Keim, Paul
    Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis.2004Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, nr 17, s. 5808-5818Artikel i tidskrift (Refereegranskat)
  • 38.
    Johansson, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Forsman, Mats
    FOI, Umeå.
    Sjöstedt, Anders
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi. Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Klinisk bakteriologi.
    The development of tools for diagnosis of tularemia and typing of Francisella tularensis.2004Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 112, nr 11-12, s. 898-907Artikel i tidskrift (Refereegranskat)
  • 39.
    Kadzhaev, Konstantin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Zingmark, Carl
    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).
    Bolanowski, Mark
    Shen, Hua
    Conlan, Wayne
    NRC/ Canada.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Identification of genes contributing to the virulence of Francisella tularensis SCHU S4 in a mouse intradermal infection model2009Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 4, nr 5, s. e5463-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 40.
    Kauppi, Anna M.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edin, Alicia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ziegler, Ingrid
    Mölling, Paula
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gylfe, Åsa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Strålin, Kristoffer
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Metabolites in Blood for Prediction of Bacteremic Sepsis in the Emergency Room2016Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, nr 1, artikel-id e0147670Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A metabolomics approach for prediction of bacteremic sepsis in patients in the emergency room (ER) was investigated. In a prospective study, whole blood samples from 65 patients with bacteremic sepsis and 49 ER controls were compared. The blood samples were analyzed using gas chromatography coupled to time-of-flight mass spectrometry. Multivariate and logistic regression modeling using metabolites identified by chromatography or using conventional laboratory parameters and clinical scores of infection were employed. A predictive model of bacteremic sepsis with 107 metabolites was developed and validated. The number of metabolites was reduced stepwise until identifying a set of 6 predictive metabolites. A 6-metabolite predictive logistic regression model showed a sensitivity of 0.91(95% CI 0.69-0.99) and a specificity 0.84 (95% CI 0.58-0.94) with an AUC of 0.93 (95% CI 0.89-1.01). Myristic acid was the single most predictive metabolite, with a sensitivity of 1.00 (95% CI 0.85-1.00) and specificity of 0.95 (95% CI 0.74-0.99), and performed better than various combinations of conventional laboratory and clinical parameters. We found that a metabolomics approach for analysis of acute blood samples was useful for identification of patients with bacteremic sepsis. Metabolomics should be further evaluated as a new tool for infection diagnostics.

  • 41.
    Kelk, Peyman
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Abd, H
    Claesson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sandström, G
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.2011Ingår i: Cell death & disease, ISSN 2041-4889, Vol. 2, s. e126-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.

  • 42.
    Kelk, Peyman
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Parodontologi. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Abd, Hadi
    Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden.
    Claesson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi.
    Sandström, Gunnar
    Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden ; Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Parodontologi.
    Inflammatory cell death of human macrophages in response to Aggregatibacter actinomycetemcomitans leukotoxinManuskript (Övrigt vetenskapligt)
    Abstract [en]

    Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the Repeats in Toxin (RTX) family, is believed to be one of its virulence factors and to play an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. Interestingly, this process resembled pyroptosis, and resulted in an extensive leukotoxin-induced interleukin-1β (IL-1β) secretion. This activation was mainly mediated by caspase-1 activation, while the levels of mRNA for IL-1β were not affected by the leukotoxin. A similar pattern was seen for IL-18, but the level of that cytokine was about 30 times lower. Both of these cytokines are synthesized as biologically inactive precursors and need active caspase-1 for their activation and secretion. In conclusion, A. actinomycetemcomitans leukotoxin induces a pyroptosis-like cell death in human macrophages and that leads to a specific and excessive pro-inflammatory response. This novel virulence mechanism of the leukotoxin may play an important role in the pathogenic potential of this bacterium.

  • 43.
    Kelk, Peyman
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Parodontologi. Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Claesson, Rolf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Chen, Casey
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi. Parod.
    IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin2008Ingår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, nr 5/6, s. 529-541Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 44.
    Kroca, Michal
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Tärnvik, Arne
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Vγ9Vδ2 T cells from individuals undergoing Pontiac fever or tularemia respond at a similar extent to phosphoantigen from Francisella tularensis or Legionella micdadei.Manuskript (Övrig (populärvetenskap, debatt, mm))
  • 45.
    Lai, Xin-He
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Expression of iglC is mecessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis2004Ingår i: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 37, nr 5, s. 225-230Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. In a previous study, an iglC null mutant of F. tularensis live vaccine strain LVS was generated by allelic replacement and in the current study this iglC mutant was successfully complemented in trans. We characterized the capacity of this iglC mutant and the complemented strain to induce macrophage apoptosis. The iglC mutant did not induce apoptosis in the infected cells. In contrast, the complemented iglC strain was able to multiply in the murine macrophage-like cell line J774A.1 and induced apoptosis similar to that of the wild-type strain. It is the first successful example of complementation in trans of a F. tularensis mutant strain and more importantly this work provides direct evidence that the intracellular growth ability is essential for F. tularensis to induce macrophage apoptosis.

  • 46.
    Lai, Xin-He
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Fancisella tularensis induces cytopathogenicity and apaptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication2001Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 69, nr 7, s. 4691-4694Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth of Francisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly located F. tularensis bacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.

  • 47.
    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)
  • 48.
    Lai, Xin-He
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Delineation of the molecular mechanisms of Francisella tularensis-induced apoptosis in murine macrophages2003Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 71, nr 8, s. 4642-4646Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection with F. tularensis strain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochrome c into the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, the F. tularensis infection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.

  • 49.
    Lai, Xin-He
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Wang, S-Y
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edebro, Helene
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Francisella strains express hemolysins of distinct characteristics2003Ingår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 224, nr 1, s. 91-95Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain.

  • 50. Lampe, E. O.
    et al.
    Zingmark, Carl
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Hemnann, L.
    Brudal, E.
    Rishovd, A. L.
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Hagedorn, M.
    Griffiths, G. W.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Winther-Larsen, H. C.
    A study of virulence factors in the fish pathogen F. noatunensis ssp noatunensis2013Ingår i: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 34, nr 6, s. 1716-1716Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The bacterium Francisella noatunensis ssp. noatunensis (in text: F. noatunensis) is the ethiological agent of the disease francisellosis in Atlantic cod. Francisellosis has been one of the major limiting factors in the development of Norwegian aquaculture industry based on Atlantic cod. Lacking an effective treatment or vaccine there is urgent need for studies related to the pathogenesis of the disease.

    The closely related human pathogen F. tularensis is more extensively studied and due to relatively high sequence similarity with F. noatunensis, indirect evidence on important virulence factors can be obtained by reverse genetics. The Francisella Pathogenicity Island (FPI) has been identified in all sequenced genomes of Francisella sp. and contains genes associated with the ability of the bacterium to survive and replicate within macrophages.

    To elucidate the pathogenesis of F. noatunensis, infection assays have been performed on primary cells extracted from the head kidney of Atlantic cod. Disruptive mutations of the potential virulence factors IglC, IglD (important for intracellular growth in F. tularensis subsp.) and ClpB (a heat shock protein identified in F. tularensis), have been constructed in F. noatunensis and the infection pattern is in the process of characterization. Model systems that are utilized in the characterization are the amoebae and professional phagocyte Dictyostelium discoideum, zebrafish and macrophages extracted from head kidney of Atlantic cod.

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