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Identification of new virulence factors in Francisella tularensis
Umeå University, Faculty of Medicine, Molecular Biology.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq.

The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain.

In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.

Place, publisher, year, edition, pages
Umeå: Institutionen för Molekylärbiologi, Umeå universitet , 2010. , 55 p.
Keyword [en]
Francisella tularensis, regulation, virulence, Hfq, DsbA, type IV pili, membrane
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-30857ISBN: 978-91-7264-916-3 (print)OAI: oai:DiVA.org:umu-30857DiVA: diva2:288895
Public defence
2010-02-12, Major Groove,, byggnad 6L, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-01-22 Created: 2010-01-20 Last updated: 2010-01-22Bibliographically approved
List of papers
1. Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis.
Open this publication in new window or tab >>Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis.
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2009 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 5, 1866-80 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.

Place, publisher, year, edition, pages
American society for microbiology, 2009
Identifiers
urn:nbn:se:umu:diva-30849 (URN)10.1128/IAI.01496-08 (DOI)19223477 (PubMedID)
Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2017-12-12Bibliographically approved
2. Proteome analysis of an attenuated Francisella tularensis dsbA mutant: identification of potential DsbA substrate proteins.
Open this publication in new window or tab >>Proteome analysis of an attenuated Francisella tularensis dsbA mutant: identification of potential DsbA substrate proteins.
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2009 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 11, 5336-46 p.Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

Place, publisher, year, edition, pages
American chemical society, 2009
Keyword
Francisella tularensis, virulence factor, DsbA, VacJ, lipoprotein, oxidoreductase activity, 2-DE, shotgun proteomics
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30929 (URN)10.1021/pr900570b (DOI)19799467 (PubMedID)
Available from: 2010-01-22 Created: 2010-01-22 Last updated: 2017-12-12Bibliographically approved
3. 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: 2017-12-14Bibliographically approved
4. 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
5. Role of type IV pilin encoding genes in virulence of Francisella tularensis subspecies holarctica
Open this publication in new window or tab >>Role of type IV pilin encoding genes in virulence of Francisella tularensis subspecies holarctica
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The number of virulence factors identified in Francisella tularensis, the causative agent of tularemia, is so far relatively few. The F. tularensis genome contains some genes with homology to known virulence factors. One of these is the type IV pili system, which is known to have a key role in virulence of other bacterial species. When we compared different F. tularensis subspecies we could identify distinct differences in Type IV pilin genes between the highly virulent type A strains and the less pathogenic type B strains. In this work we addressed the role in virulence of the different pilin genes in a virulent type B strain. Of all the pilin genes only PilA and the pseudopilins FTT1621-1622 were proven to have a role in virulence. In addition we also verified that the gene encoding the PilT ATPase is non-functional due to a non-sense mutation and we also confirmed that the truncated pilT has no role in mouse virulence. Furthermore we also provide evidence that the F. tularensis pilins are posttranslationally modified presumably by glycosylation by a PilO dependent mechanism.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30932 (URN)
Available from: 2010-01-22 Created: 2010-01-22 Last updated: 2010-01-25

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