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The Francisella pathogenicity island: its role in type VI secretion and intracellular infection
Umeå University, Faculty of Medicine, Department of Clinical Microbiology. (Anders Sjöstedt)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 82 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1708
Keyword [en]
Francisella, FPI, Type VI Secretion, Igl, DotU, VgrG, Pdp, microinjection, phagosomal escape, intracellular replication
National Category
Microbiology in the medical area
Research subject
Clinical Bacteriology
Identifiers
URN: urn:nbn:se:umu:diva-101321ISBN: 978-91-7601-246-8 (print)OAI: oai:DiVA.org:umu-101321DiVA: diva2:798587
Public defence
2015-04-24, sal E04, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-04-01 Created: 2015-03-27 Last updated: 2015-05-08Bibliographically approved
List of papers
1. IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS
Open this publication in new window or tab >>IglG and IglI of the Francisella pathogenicity island are important virulence determinants of Francisella tularensis LVS
2011 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, no 9, 3683-3696 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
American Society for Microbiology, 2011
National Category
Immunology in the medical area Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-83830 (URN)10.1128/IAI.01344-10 (DOI)000293891000021 ()21690239 (PubMedID)
Funder
Swedish Research Council, 2006-3426, 2006-2877, 2009-5026
Available from: 2013-12-09 Created: 2013-12-09 Last updated: 2017-12-06Bibliographically approved
2. DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity
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, 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: 2017-12-07Bibliographically approved
3. A mutagenesisbased approach to map functional domain(s) within the N- terminus of Francisella tularensis IglE
Open this publication in new window or tab >>A mutagenesisbased approach to map functional domain(s) within the N- terminus of Francisella tularensis IglE
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-101519 (URN)
Available from: 2015-04-01 Created: 2015-04-01 Last updated: 2015-04-01Bibliographically approved
4. Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection
Open this publication in new window or tab >>Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e50473Article in journal (Refereed) Published
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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-63027 (URN)10.1371/journal.pone.0050473 (DOI)000311535700086 ()23185631 (PubMedID)
Available from: 2012-12-28 Created: 2012-12-27 Last updated: 2017-12-06Bibliographically approved
5. Microinjection of Francisella tularensis and Listeria monocytogenes reveals the importance of bacterial and host factors for successful replication
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, 3233-3242 p.Article 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: 2017-12-04Bibliographically approved
6. Link between intraphagosomal biotin and rapid phagosomal escape in Francisella
Open this publication in new window or tab >>Link between intraphagosomal biotin and rapid phagosomal escape in Francisella
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 44, 18084-18089 p.Article in journal (Refereed) Published
Abstract [en]

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens.

National Category
Infectious Medicine Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-62166 (URN)10.1073/pnas.1206411109 (DOI)000311149900081 ()23071317 (PubMedID)
Available from: 2012-12-11 Created: 2012-12-10 Last updated: 2017-12-07Bibliographically approved
7. The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
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, 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
Keyword
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: 2017-12-06Bibliographically approved

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