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  • 1.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Åhlund, Monika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bröms, Jeanette
    Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
    Forsberg, Åke
    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).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6683-6700Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

  • 2.
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Type III secretion- the various functions of the translocon operon in bacterial pathogenesis2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.

    Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.

    The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.

    Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

  • 3.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra
    Forsberg, Åke
    Francis, Matthew
    Mapping of an YscY binding regulatory domain within the type III secretion chaperone LcrH of Yersinia pseudotuberculosisManuskript (Annet vitenskapelig)
  • 4.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Katrin E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion2005Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, nr 22, s. 7738-7752Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

  • 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 A15522013Inngår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, s. 96-Artikkel i tidsskrift (Fagfellevurdert)
    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 LVS2011Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 9, s. 3683-3696Artikkel i tidsskrift (Fagfellevurdert)
    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 tularensis2009Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 8, s. 2431-2446Artikkel i tidsskrift (Fagfellevurdert)
    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 tularensis2010Inngår i: The challenge of highly pathogenic microorganisms: mechanisms of virulence and novel medical countermeasures, Springer, 2010, s. 67-75Konferansepaper (Fagfellevurdert)
    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 pathogenicity2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 4, artikkel-id e34639Artikkel i tidsskrift (Fagfellevurdert)
    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 secretion2017Inngår i: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 8, nr 6, s. 821-847Artikkel i tidsskrift (Fagfellevurdert)
    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 infection2012Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 11, artikkel-id e50473Artikkel i tidsskrift (Fagfellevurdert)
    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 signaling2010Inngår i: Frontiers in microbiology, ISSN 1664-302X, Vol. 1, s. 136-Artikkel i tidsskrift (Fagfellevurdert)
    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.
    Bröms, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forslund, Anna-Lena
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis2003Inngår i: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 149, nr 9, s. 2615-2626Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a yopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia yopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.

  • 14.
    Bröms, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Medical Countermeasures, Swedish Defence Research Agency.
    Forslund, Anna-Lena
    Department of Medical Countermeasures, Swedish Defence Research Agency.
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Medical Countermeasures, Swedish Defence Research Agency.
    Francis, Matthew
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    PcrH of Pseudomonas aeruginosa is essential for secretion and assembly of the type III translocon2003Inngår i: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, nr 12, s. 1909-1921Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pseudomonas aeruginosa harbors a type III secretion system that translocates antihost effectors into an infected eukaryotic cell. PcrH is a key component of type III secretion in this essential virulence strategy. In the absence of PcrH, P. aeruginosa is translocation deficient because of a specific reduction in presecretory stability and subsequent secretion of PopB and PopD, 2 proteins essential for the translocation process. PcrH exerts this chaperone function by binding directly to PopB and PopD. Consistent with the genetic relatedness of PcrH with LcrH of pathogenic Yersinia species, these proteins are functionally interchangeable with respect to their ability to complement the translocation defect associated with either a lcrH or pcrH null mutant, respectively. Thus, the translocator class of chaperones performs a critical function in ensuring the assembly of a translocation competent type III secreton. Finally, unlike the regulatory roles of other translocator-class chaperones (e.g., LcrH, SicA of Salmonella enterica, and IpgC of Shigella species), in vitro regulation of P. aeruginosa type III secretion does not involve PcrH.

  • 15.
    Bröms, Jeanette
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Sundin, Charlotta
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Comparative analysis of type III effector translocation by Yersinia pseudotuberculosis expressing native LcrV or PcrV from Pseudomonas aeruginosa2003Inngår i: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 188, nr 2, s. 239-249Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The homologues LcrV of Yersinia species and PcrV of Pseudomonas aeruginosa are pore-forming components. When expressed in a Yersinia lcrV background, PcrV formed smaller pores in infected erythrocyte membranes, correlating to a lowered translocation of Yersinia effectors. To understand this phenomenon, cytotoxins exoenzyme S of P. aeruginosa and YopE of Yersinia were introduced into a Yersinia background without Yop effectors but expressing LcrV or PcrV. Comparable translocation of each substrate indicated that substrate recognition by LcrV/PcrV is not a regulator of translocation. Yersinia harboring pcrV coexpressed with its native operon efficiently translocated effectors into HeLa cell monolayers and formed large LcrV-like pores in erythrocyte membranes. Thus, a PcrV complex with native P. aeruginosa translocon components is required to form fully functional pores for complete complementation of effector translocation in Yersinia.

  • 16.
    Costa, Tiago R D
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edqvist, Petra J
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Åhlund, Monika K
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis2010Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 33, s. 25269-25284Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

  • 17. Edqvist, Petra
    et al.
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Steggo, Peter
    Forsberg, Åke
    Francis, Matthew
    Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificityManuskript (Annet vitenskapelig)
  • 18.
    Ekestubbe, Sofie
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Edgren, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    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).
    Francis, Matthew S.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    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).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, artikkel-id 175Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

  • 19.
    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 strains2012Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, nr 2, s. 575-584Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 20. 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 noatunensis2013Inngår i: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 34, nr 6, s. 1716-1716Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 21.
    Lavander, Moa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Medical Countermeasures, Division of NBC Defense, Swedish Defense Research Agency, SE-901 82 Umeå, Sweden.
    Ericsson, Solveig K.
    Bröms, Jeanette E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Medical Countermeasures, Division of NBC Defense, Swedish Defense Research Agency, SE-901 82 Umeå, Sweden.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Medical Countermeasures, Division of NBC Defense, Swedish Defense Research Agency, SE-901 82 Umeå, Sweden.
    The Twin Arginine Translocation System is Essential for Virulence of Yersinia pseudotuberculosis2006Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, nr 3, s. 1768-1776Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Yersinia species pathogenic to humans have been extensively characterized with respect to type III secretion and its essential role in virulence. This study concerns the twin arginine translocation (Tat) pathway utilized by gram-negative bacteria to secrete folded proteins across the bacterial inner membrane into the periplasmic compartment. We have shown that the Yersinia Tat system is functional and required for motility and contributes to acid resistance. A Yersinia pseudotuberculosis mutant strain with a disrupted Tat system (tatC) was, however, not affected in in vitro growth or more susceptible to high osmolarity, oxidative stress, or high temperature, nor was it impaired in type III secretion. Interestingly, the tatC mutant was severely attenuated via both the oral and intraperitoneal routes in the systemic mouse infection model and highly impaired in colonization of lymphoid organs like Peyer's patches and the spleen. Our work highlights that Tat secretion plays a key role in the virulence of Y. pseudotuberculosis.

  • 22.
    Lindgren, Marie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bröms, Jeanette E.
    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).
    Golovliov, Igor
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection2013Inngår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, artikkel-id 20Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

    Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

    Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

  • 23.
    Lindgren, Marie
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Eneslätt, Kjell
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis2013Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, nr 6, s. 2076-2084Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Modulation of host cell death pathways appears to be a prerequisite for the successful lifestyles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic, and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand the prerequisites of this cell death, macrophages were infected with the F. tularensis live vaccine strain (LVS), and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes, which encode components of the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and, after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the Delta iglI or Delta iglC mutants, although the former strain replicated. The Delta iglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the Delta pdpC mutant showed no replication but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to those obtained with LVS. The unique phenotypes of the mutants provide insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.

  • 24.
    Meyer, Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Alam, A.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    A mutagenesisbased approach to map functional domain(s) within the N- terminus of Francisella tularensis IglEManuskript (preprint) (Annet vitenskapelig)
  • 25.
    Meyer, Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bröms, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Liu, Xijia
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rottenberg, M.E.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication2015Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, nr 8, s. 3233-3242Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    Certain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., Delta iglA, Delta iglC, Delta iglG, Delta iglI, and Delta pdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC(-/-) BMDM, MyD88(-/-) BMDM, J774 cells, or HeLa cells, LVS, Delta pdpE and Delta iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the Delta iglA and Delta iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC(-/-) BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88(-/-) BMDM and LVS showed no replication in either BMDM or MyD88(-/-) BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection.

  • 26. Rigard, Melanie
    et al.
    Bröms, Jeanette E.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Mosnier, Amandine
    Hologne, Maggy
    Martin, Amandine
    Lindgren, Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Punginelli, Claire
    Lays, Claire
    Walker, Olivier
    Charbit, Alain
    Telouk, Philippe
    Conlan, Wayne
    Terradot, Laurent
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
    Henry, Thomas
    Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence2016Inngår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 12, nr 9, artikkel-id e1005821Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The virulence of Francisella tularensis, the etiological agent of tularemia, relies on an atypical type VI secretion system ( T6SS) encoded by a genomic island termed the Francisella Pathogenicity Island ( FPI). While the importance of the FPI in F. tularensis virulence is clearly established, the precise role of most of the FPI-encoded proteins remains to be deciphered. In this study, using highly virulent F. tularensis strains and the closely related species F. novicida, IglG was characterized as a protein featuring a unique alpha-helical N-terminal extension and a domain of unknown function ( DUF4280), present in more than 250 bacterial species. Three dimensional modeling of IglG and of the DUF4280 consensus protein sequence indicates that these proteins adopt a PAAR-like fold, suggesting they could cap the T6SS in a similar way as the recently described PAAR proteins. The newly identified PAAR-like motif is characterized by four conserved cysteine residues, also present in IglG, which may bind a metal atom. We demonstrate that IglG binds metal ions and that each individual cysteine is required for T6SS-dependent secretion of IglG and of the Hcp homologue, IglC and for the F. novicida intracellular life cycle. In contrast, the Francisella-specific N-terminal alpha-helical extension is not required for IglG secretion, but is critical for F. novicida virulence and for the interaction of IglG with another FPI-encoded protein, IglF. Altogether, our data suggest that IglG is a PAAR-like protein acting as a bi-modal protein that may connect the tip of the Francisella T6SS with a putative T6SS effector, IglF.

  • 27.
    Sun, Kun
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bröms, Jeanette
    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).
    Gurram, Bharat Kumar
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Enquist, Per-Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Andersson, C. David
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Sjöstedt, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion2014Inngår i: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, nr 7, s. 4123-4130Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an alpha-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A(1) activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.

  • 28.
    Sundin, Charlotta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Thelaus, Johanna
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Polarisation of type III translocation by Pseudomonas aeruginosa requires PcrG, PcrV and PopN2004Inngår i: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 37, nr 6, s. 313-322Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell. We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PerV, PopB and PopD. To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants. In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased. When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium. This confirms that ExoS translocation by P. aeruginosa occurs by a polarised mechanism. In contrast, high levels of ExoS were recovered in the tissue Culture medium when cells were infected with pcrG, pcrV and popN mutants. Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions. This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells.

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