A 55 kDa hypothetical membrane protein is an iron-regulated virulence factor of Francisella tularensis subsp. novicida U112

Milne, Timothy S.; Michell, Stephen L.; Diaper, Helen; Wikström, Pernilla; Svensson, Kerstin; Oyston, Petra C. F.; Titball, Richard W.

doi:10.1099/jmm.0.47190-0

Cited by 24 publications

(26 citation statements)

References 32 publications

(32 reference statements)

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“…The median lethal dose (MLD) for F. novicida U112 has been previously calculated as 3.4610 2 c.f.u. by the subcutaneous route (Milne et al, 2007). These results demonstrate a significant increase in survival (P50.001) when mice were challenged with U112 DrelA : : cam, demonstrating that this mutant was attenuated .100 fold.…”

Section: Induction Of Rela Is Involved In Resistance Of Francisella Tmentioning

confidence: 57%

“…F. novicida strain U112 was obtained from the American Type Culture Collection. The strain was grown on blood cysteine glucose agar (BCGA) supplemented with 4 % cysteine, 4 % histidine, 5 % glucose and 10 % fresh filtered horse blood, or on Thayer-Martin agar (Milne et al, 2007). Where required, media were supplemented with 100 mg polymixin B ml 21 , 10 mg chloramphenicol ml 21 and 10 % (w/v) sucrose.…”

Section: Methodsmentioning

confidence: 99%

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RelA regulates virulence and intracellular survival of Francisella novicida

et al. 2009

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Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.

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Section: Induction Of Rela Is Involved In Resistance Of Francisella Tmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

RelA regulates virulence and intracellular survival of Francisella novicida

et al. 2009

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“…It was recently shown that the fslD and fslE homologs in a strain of the closely related novicida subspecies are cotranscribed and that transcription is induced by iron starvation (20). A strain with a mutation in the fslE homolog was competent for siderophore production.…”

Section: Discussionmentioning

confidence: 99%

fslE Is Necessary for Siderophore-Mediated Iron Acquisition in Francisella tularensis Schu S4

Ramakrishnan¹,

Meeker²,

Dragulev

2008

J Bacteriol

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Strains of Francisella tularensis secrete a siderophore in response to iron limitation. Siderophore production is dependent on fslA, the first gene in an operon that appears to encode biosynthetic and export functions for the siderophore. Transcription of the operon is induced under conditions of iron limitation. The fsl genes lie adjacent to the fur homolog on the chromosome, and there is a canonical Fur box sequence in the promoter region of fslA. We generated a ⌬fur mutant of the Schu S4 strain of F. tularensis tularensis and determined that siderophore production was now constitutive and no longer regulated by iron levels. Quantitative reverse transcriptase PCR analysis with RNA from Schu S4 and the mutant strain showed that Fur represses transcription of fslA under iron-replete conditions. We determined that fslE (locus FTT0025 in the Schu S4 genome), located downstream of the siderophore biosynthetic genes, is also under Fur regulation and is transcribed as part of the fslABCDEF operon. We generated a defined in-frame deletion of fslE and found that the mutant was defective for growth under iron limitation. Using a plate-based growth assay, we found that the mutant was able to secrete a siderophore but was defective in utilization of the siderophore. FslE belongs to a family of proteins that has no known homologs outside of the Francisella species, and the fslE gene product has been previously localized to the outer membrane of F. tularensis strains. Our data suggest that FslE may function as the siderophore receptor in F. tularensis.

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“…FTT0918 is highly similar to fslE, and the corresponding genes in F. novicida and in SCHU S4 were recently shown to be part of the fsl operon (11,20,32). Interestingly, using the TribeMCL method, a previous study identified that the two genes encoded proteins that belonged to a protein family with five members, also including the FTT0267, FTT0602, and FTT0919 proteins (17).…”

Section: Discussionmentioning

confidence: 99%

The 58-Kilodalton Major Virulence Factor of Francisella tularensis Is Required for Efficient Utilization of Iron

et al. 2009

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We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the ⌬FTT0918 and ⌬fslA isogenic mutants were analyzed. The gene product missing in the ⌬fslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The ⌬FTT0918 mutant, but not the ⌬fslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the ⌬fslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the ⌬fslA strain but not of the ⌬FTT0918 strain. The sensitivity of SCHU S4 and the ⌬FTT0918 and ⌬fslA strains to streptonigrin demonstrated that the ⌬FTT0918 strain contained a smaller free intracellular iron pool and that the ⌬fslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the ⌬FTT0918 strain, the ⌬fslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

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A 55 kDa hypothetical membrane protein is an iron-regulated virulence factor of Francisella tularensis subsp. novicida U112

Cited by 24 publications

References 32 publications

RelA regulates virulence and intracellular survival of Francisella novicida

RelA regulates virulence and intracellular survival of Francisella novicida

fslE Is Necessary for Siderophore-Mediated Iron Acquisition in Francisella tularensis Schu S4

The 58-Kilodalton Major Virulence Factor of Francisella tularensis Is Required for Efficient Utilization of Iron

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