25 research outputs found
Rapid assessment of tetanus vaccine-induced immunity in Bangladesh and the Gambia.
We have developed recombinant fragment C based rapid point of care dipstick devices to assess tetanus immunization status using plasma or whole blood. The devices demonstrated specificity of 0.90 and sensitivity of 0.90 (whole blood)/0.94 (plasma) at field sites in Bangladesh and The Gambia when compared to a commercial ELISA with the immune cut-off titer set as ≥0.1IU/mL
The reduced genome of the Francisella tularensis live vaccine strain (LVS) encodes two iron acquisition systems essential for optimal growth and virulence.
Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB'), which led to a growth defect on iron-limited modified Muller Hinton agar plates. 55Fe uptake assays verified that the ΔfeoB' mutant had lost the capacity for ferrous iron uptake but was still competent for 55Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB' nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB' mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB' mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence
The fslE Homolog, FTL_0439 (fupA/B), Mediates Siderophore-Dependent Iron Uptake in Francisella tularensis LVS â–¿
The Gram-negative pathogen Francisella tularensis secretes a siderophore to obtain essential iron by a TonB-independent mechanism. The fslABCDE locus, encoding siderophore-related functions, is conserved among different Francisella strains. In the virulent strain Schu S4, fslE is essential for siderophore utilization and for growth under conditions of iron limitation. In contrast, we found that deletion of fslE did not affect siderophore utilization by the attenuated live vaccine strain (LVS). We found that one of the fslE paralogs encoded in the LVS genome, FTL_0439 (fupA/B), was able to partially complement a Schu S4 ΔfslE mutant for siderophore utilization. We generated a deletion of fupA/B in LVS and in the LVS ΔfslE background. The ΔfupA/B mutant showed reduced growth under conditions of iron limitation. It was able to secrete but was unable to utilize siderophore. Mutation of both fupA/B and fslE resulted in a growth defect of greater severity. The ΔfupA/B mutants showed a replication defect in J774.1A cells and decreased virulence following intraperitoneal infection in mice. Complementation of the ΔfupA/B mutation in cis restored the ability to utilize siderophore and concomitantly restored virulence. Our results indicate that fupA/B plays a significant role in the siderophore-mediated iron uptake mechanism of LVS whereas fslE appears to play a secondary role. Variation in iron acquisition mechanisms may contribute to virulence differences between the strains
fslE Is Necessary for Siderophore-Mediated Iron Acquisition in Francisella tularensis Schu S4â–¿
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 Δfur 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
Growth of LVS Δ<i>feoB</i>′ in liquid and on agar.
<p>Bacteria were inoculated in either iron rich TSB/c (A) or in che-CDM supplemented with FePPi at 2.5 μg/mL (High Fe) or 0.125 μg/mL (Low Fe)(B) and growth was followed as change in optical density at 600 nm (OD<sub>600</sub>). Values were plotted as the means ± S.E. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093558#pone-0093558-g001" target="_blank">Figure 1</a> C, bacterial strains were ten-fold serially diluted in che-CDM, spotted on MHA agar with (MHA+) and without (MHA-) iron supplementation and grown for 3 days at 37°C under aerobic conditions.</p
The \u3ci\u3eCaulobacter crescentus\u3c/i\u3e FlbD protein acts at \u3ci\u3eftr\u3c/i\u3e sequence elements both to activate and to repress transcription of cell cycle-regulated flagellar genes
The flagellar genes (fla genes) in Caulobacter crescentus are categorized into a regulatory hierarchy of four levels, I-IV, in which transcription of the class III and class IV genes late in the cell cycle from σ54-dependent promoters depends on expression of the class II genes above them. Timing of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. Here we report that purified FlbD activates transcription in vitro from the σ54-dependent class III flbG promoter and represses transcription from the class II fliF promoter by binding to ftr (flagellar transcription regulator) sequence elements required for their transcriptional regulation in vivo. The FlbD protein makes symmetrical base-specific contacts at three highly conserved guanine nucleotides in each half site of ftr1 and ftr1* at flbG and the single ftr4 site at fliF. The dual function of FlbD in activation of class III genes and repression of the class II fliF promoter is consistent with a central role of FlbD as a switch protein mediating the transition from level II to level III fla gene expression
The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain
Iron acquisition by LVS single and double mutants and complementing strains.
<p>LVS, the Δ<i>fslA</i> and Δ<i>feoB</i>′ mutants and in <i>cis</i> complements as indicated were grown overnight on fresh MHA+ plates (with additional siderophore supplementation to the Δ<i>fslA</i> Δ<i>feoB</i>′ mutant) at 37°C and further inoculated into che-CDM without FePPi for three hours with shaking. Single mutants and the Δ<i>feoB</i>′ complement were assessed in A and B, and the complement of the double deletion mutant was tested in C and D. The rate of <sup>55</sup>Fe uptake was determined at (A,C) 0.1 and (B,D) 3 μM [<sup>55</sup>Fe<sup>2+</sup>] in the presence of ascorbate. E, Strains were incubated with <sup>55</sup>Fe bound siderophore and rate of <sup>55</sup>Fe uptake was determined. F, Western blotting to determine expression levels of FslE, FupA/B, and the loading control GroEL in bacteria grown on MHA+ plates in comparison to LVS grown under iron-limiting conditions. In A–E, values are expressed as the means ± S.D. Significance was calculated relative to LVS values. * p<0.05, ***p<0.001.</p
LVS Δ<i>feoB</i>′ is defective for ferrous uptake but is capable of siderophore production and utilization.
<p>A and B, <sup>55</sup>Fe ferrous iron uptake. LVS and Δ<i>feoB</i>′ strains were grown overnight to mid-logarithmic phase in iron-limiting liquid che-CDM and rates of ferrous iron uptake were determined at 0.1 and 3 μM [<sup>55</sup>Fe<sup>2+</sup>] in the presence of ascorbate. C, Siderophore production. Culture supernatants of LVS, Δ<i>fslA</i>, Δ<i>feoB</i>′ strains grown overnight under iron-limitation were tested in the CAS assay and siderophore activity was normalized to OD<sub>600</sub>. D, Siderophore-mediated <sup>55</sup>Fe uptake. <sup>55</sup>Fe-bound siderophore was incubated with bacteria grown in iron-limiting che-CDM and the rate of <sup>55</sup>Fe uptake was determined. Values were expressed as the means ± S.D. Significance was calculated relative to LVS values. ***p<0.001.</p