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The role of the Type IV pili system in the virulence of Francisella tularensis
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2008. , 73 p.
Keyword [en]
Francisella tularensis, tularemia, bacterial pathogenesis, Type IV pili, Type II secretion, Neisseria gonorrhoeae, PilA, RD18, RD19
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1656ISBN: 978-91-7264-553-0 (print)OAI: oai:DiVA.org:umu-1656DiVA: diva2:141727
Public defence
2008-06-05, Major Groove, 6L NUS, Umeå Universitet 901 87, Umeå, 09:00
Opponent
Supervisors
Available from: 2008-05-15 Created: 2008-05-15Bibliographically approved
List of papers
1. Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis
Open this publication in new window or tab >>Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis
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2006 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, no 6, 1818-1830 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.

Place, publisher, year, edition, pages
Blackwell Publishing Ltd, 2006
Identifiers
urn:nbn:se:umu:diva-3217 (URN)10.1111/j.1365-2958.2006.05061.x (DOI)
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2016-03-01Bibliographically approved
2. The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis
Open this publication in new window or tab >>The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis
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(English)Manuscript (Other (popular science, discussion, etc.))
Abstract [en]

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

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

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

Keyword
Francisella tularensis, SCHU S4, type IV pili, virulence
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-3218 (URN)
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2010-01-22Bibliographically approved
3. Heterologous expression of Francisella tularensis type IV pilin genes in Neisseria gonorrhoeae confirms that PilA can form functional pili
Open this publication in new window or tab >>Heterologous expression of Francisella tularensis type IV pilin genes in Neisseria gonorrhoeae confirms that PilA can form functional pili
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3219 (URN)
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2010-01-13Bibliographically approved
4. Reintroduction of two deleted virulence loci restores full virulence of the live vaccine strain (LVS) of Francisella tularensis
Open this publication in new window or tab >>Reintroduction of two deleted virulence loci restores full virulence of the live vaccine strain (LVS) of Francisella tularensis
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(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-3220 (URN)
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2010-01-14Bibliographically approved

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