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Proteome analysis of an attenuated Francisella tularensis dsbA mutant: identification of potential DsbA substrate proteins.
Center of Advanced Studies and Institute of Molecular Pathology, Faculty of Military Health Science UO, Czech Republic.
Center of Advanced Studies and Institute of Molecular Pathology, Faculty of Military Health Science UO, Czech Republic.
Center of Advanced Studies and Institute of Molecular Pathology, Faculty of Military Health Science UO, Czech Republic.
Umeå University, Faculty of Medicine, Molecular Biology.
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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. Vol. 8, no 11, 5336-46 p.
Keyword [en]
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: urn:nbn:se:umu:diva-30929DOI: 10.1021/pr900570bPubMedID: 19799467OAI: oai:DiVA.org:umu-30929DiVA: diva2:288877
Available from: 2010-01-22 Created: 2010-01-22 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Identification of new virulence factors in Francisella tularensis
Open this publication in new window or tab >>Identification of new virulence factors in Francisella tularensis
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
Francisella tularensis, regulation, virulence, Hfq, DsbA, type IV pili, membrane
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-30857 (URN)978-91-7264-916-3 (ISBN)
Public defence
2010-02-12, Major Groove,, byggnad 6L, Norrlands universitetssjukhus, Umeå, 10:00 (English)
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Available from: 2010-01-22 Created: 2010-01-20 Last updated: 2010-01-22Bibliographically approved

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Forslund, Anna-LenaForsberg, Åke

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