| 1. |
- Bröms, Jeanette E, et al.
(författare)
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Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion
- 2005
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 187:22, s. 7738-7752
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Lampe, E. O., et al.
(författare)
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A study of virulence factors in the fish pathogen F. noatunensis ssp noatunensis
- 2013
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Ingår i: Fish and Shellfish Immunology. - : Elsevier. - 1050-4648 .- 1095-9947. ; 34:6, s. 1716-1716
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 3. |
- Alam, Athar, et al.
(författare)
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The Role of ClpB in Bacterial Stress Responses and Virulence
- 2021
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Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
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Forskningsöversikt (refereegranskat)abstract
- Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.
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| 4. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
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A conserved α-helix essential for a type VI secretion-like system of Francisella tularensis
- 2009
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 191:8, s. 2431-2446
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Tidskriftsartikel (refereegranskat)abstract
- Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These include iglA and iglB, the homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33-132, necessary for efficient binding to IglB as well as for IglAB protein stability and intra-macrophage growth. In particular, residues 103-122, overlapping with a highly conserved alpha-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in yeast, but markedly impaired intra-macrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to T6S, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhimurium and Escherichia coli were also shown to interact in yeast and the interaction was dependent on the preservation of the same alpha-helix. Heterologous interactions formed between non-native IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity and the same interaction is conserved in other human pathogens.
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| 5. |
- Bröms, Jeanette E., et al.
(författare)
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A functional VipA-VipB interaction is required for the type VI secretion system activity of Vibrio cholerae O1 strain A1552
- 2013
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Ingår i: BMC Microbiology. - London, England : BioMed Central. - 1471-2180. ; 13, s. 96-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Bröms, Jeanette E., et al.
(författare)
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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
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Ingår i: Virulence. - : TAYLOR & FRANCIS INC. - 2150-5594 .- 2150-5608. ; 8:6, s. 821-847
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Bröms, Jeanette E, et al.
(författare)
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Diminished LcrV secretion attenuates Yersinia pseudotuberculosis virulence.
- 2007
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Ingår i: J Bacteriol. - 0021-9193.
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Tidskriftsartikel (refereegranskat)abstract
- Many Gram negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion; anti-host effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the tranclocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside the bacteria and immunomodulation via interaction with TLR2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.
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| 8. |
- Bröms, Jeanette E, et al.
(författare)
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Dissection of the Functions of the IglC Protein of Francisella tularensis
- 2010
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Ingår i: The challenge of highly pathogenic microorganisms. - Dordrecht : Springer. - 9789048190539 - 9789048190546 ; , s. 67-75
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Konferensbidrag (refereegranskat)abstract
- 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.
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| 9. |
- Bröms, Jeanette E., et al.
(författare)
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DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Bröms, Jeanette E, 1974-, et al.
(författare)
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IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS
- 2011
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 79:9, s. 3683-3696
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ΔpdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ΔiglG and ΔiglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.
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