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Bröms, Jeanette E
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Publications (10 of 28) Show all publications
Bröms, J. E., Meyer, L. & Sjöstedt, A. (2017). A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control type VI system-mediated secretion. Virulence, 8(6), 821-847
Open this publication in new window or tab >>A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control type VI system-mediated secretion
2017 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 8, no 6, p. 821-847Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
TAYLOR & FRANCIS INC, 2017
Keywords
Francisella pathogenicity island, Francisella tularensis, IglE, type VI secretion
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-141228 (URN)10.1080/21505594.2016.1258507 (DOI)000412306100029 ()27830989 (PubMedID)
Available from: 2017-10-27 Created: 2017-10-27 Last updated: 2018-06-09Bibliographically approved
Rigard, M., Bröms, J. E., Mosnier, A., Hologne, M., Martin, A., Lindgren, L., . . . Henry, T. (2016). Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence. PLoS Pathogens, 12(9), Article ID e1005821.
Open this publication in new window or tab >>Francisella tularensis IglG Belongs to a Novel Family of PAAR-Like T6SS Proteins and Harbors a Unique N-terminal Extension Required for Virulence
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2016 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 12, no 9, article id e1005821Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-127634 (URN)10.1371/journal.ppat.1005821 (DOI)000385621900013 ()
Available from: 2016-11-23 Created: 2016-11-16 Last updated: 2018-06-09Bibliographically approved
Ekestubbe, S., Bröms, J. E., Edgren, T., Fällman, M., Francis, M. S. & Forsberg, Å. (2016). The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence. Frontiers in Cellular and Infection Microbiology, 6, Article ID 175.
Open this publication in new window or tab >>The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence
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2016 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed) Published
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.

Keywords
LcrV, type III secretion system, YopH, translocation, pore formation, Yersinia pseudotuberculosis, virulence
National Category
Microbiology in the medical area Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-130149 (URN)10.3389/fcimb.2016.00175 (DOI)000389194200001 ()
Available from: 2017-01-12 Created: 2017-01-12 Last updated: 2018-06-09Bibliographically approved
Meyer, L., Bröms, J., Liu, X., Rottenberg, M. & Sjöstedt, A. (2015). Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication. Infection and Immunity, 83(8), 3233-3242
Open this publication in new window or tab >>Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication
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2015 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, no 8, p. 3233-3242Article in journal (Other academic) Published
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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-101520 (URN)10.1128/IAI.00416-15 (DOI)000357618300023 ()
Note

Originally included in thesis in manuscript form.

Available from: 2015-04-01 Created: 2015-04-01 Last updated: 2018-06-07Bibliographically approved
Sun, K., Bröms, J., Lavander, M., Gurram, B. K., Enquist, P.-A., Andersson, C. D., . . . Sjöstedt, A. (2014). Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion. Antimicrobial Agents and Chemotherapy, 58(7), 4123-4130
Open this publication in new window or tab >>Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion
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2014 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 7, p. 4123-4130Article in journal (Refereed) Published
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-91844 (URN)10.1128/AAC.02819-13 (DOI)000338846500069 ()24798289 (PubMedID)
Available from: 2014-08-27 Created: 2014-08-18 Last updated: 2018-06-07Bibliographically approved
Bröms, J. E., Ishikawa, T., Wai, S. N. & Sjöstedt, A. (2013). A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552. BMC Microbiology, 13, 96
Open this publication in new window or tab >>A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552
2013 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, p. 96-Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
London, England: BioMed Central, 2013
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-73578 (URN)10.1186/1471-2180-13-96 (DOI)000319046300001 ()23642157 (PubMedID)
Available from: 2013-06-25 Created: 2013-06-25 Last updated: 2018-06-08Bibliographically approved
Lampe, E. O., Zingmark, C., Hemnann, L., Brudal, E., Rishovd, A. L., Bröms, J., . . . Winther-Larsen, H. C. (2013). A study of virulence factors in the fish pathogen F. noatunensis ssp noatunensis. Fish and Shellfish Immunology, 34(6), 1716-1716
Open this publication in new window or tab >>A study of virulence factors in the fish pathogen F. noatunensis ssp noatunensis
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2013 (English)In: Fish and Shellfish Immunology, ISSN 1050-4648, E-ISSN 1095-9947, Vol. 34, no 6, p. 1716-1716Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2013
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-76254 (URN)10.1016/j.fsi.2013.03.244 (DOI)000319646100267 ()
Available from: 2013-07-08 Created: 2013-07-08 Last updated: 2018-06-08Bibliographically approved
Lindgren, M., Eneslätt, K., Bröms, J. & Sjöstedt, A. (2013). Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis. Infection and Immunity, 81(6), 2076-2084
Open this publication in new window or tab >>Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis
2013 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 6, p. 2076-2084Article in journal (Refereed) Published
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.

Keywords
Immunology, Infectious Diseases
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-74503 (URN)10.1128/IAI.00275-13 (DOI)000318855100023 ()23529623 (PubMedID)
Available from: 2013-07-02 Created: 2013-07-01 Last updated: 2018-06-08Bibliographically approved
Lindgren, M., Bröms, J. E., Meyer, L., Golovliov, I. & Sjöstedt, A. (2013). The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection. BMC Microbiology, 13, Article ID 20.
Open this publication in new window or tab >>The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
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2013 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, article id 20Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
BioMed Central, 2013
Keywords
Francisella tularensis, type VI secretion, cytopathogenicity, intracellular replication, PdpC
National Category
Microbiology Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-66129 (URN)10.1186/1471-2180-13-20 (DOI)000314827600002 ()23356941 (PubMedID)
Note

The Francisella tularensis LVS Delta pdpC mutant exhibits a unique phenotype during intracellular infection

Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2018-06-08Bibliographically approved
Bröms, J. E., Meyer, L., Lavander, M., Larsson, P. & Sjöstedt, A. (2012). DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity. PLoS ONE, 7(4), Article ID e34639.
Open this publication in new window or tab >>DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, article id e34639Article in journal (Refereed) Published
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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-57392 (URN)10.1371/journal.pone.0034639 (DOI)000305341600057 ()
Available from: 2012-07-17 Created: 2012-07-16 Last updated: 2018-06-08Bibliographically approved
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