Requirement of the CXXC Motif of Novel Francisella Infectivity Potentiator Protein B FipB, and FipA in Virulence of F. tularensis subsp. tularensis

The lipoprotein encoded by the Francisella tularensis subsp. tularensis locus FTT1103 is essential for virulence; an FTT1103 deletion mutant is defective in uptake and intracellular survival, and mice survive high dose challenges of greater than 108 bacteria. This protein has two conserved domains; one is found in a class of virulence proteins called macrophage infectivity potentiator (Mip) proteins, and the other in oxidoreductase Disulfide Bond formation protein A (DsbA)-related proteins. We have designated the protein encoded by FTT1103 as FipB for Francisella infectivity potentiator protein B. The locus FTT1102 (fipA), which is upstream of fipB, also has similarity to same conserved Mip domain. Deletion and site-specific mutants of fipA and fipB were constructed in the Schu S4 strain, and characterized with respect to intracellular replication and in vivo virulence. A nonpolar fipA mutant demonstrated reduced survival in host cells, but was only slightly attenuated in vivo. Although FipB protein was present in a fipA mutant, the abundance of the three isoforms of FipB was altered, suggesting that FipA has a role in post-translational modification of FipB. Similar to many DsbA homologues, FipB contains a cysteine-any amino acid-any amino acid-cysteine (CXXC) motif. This motif was found to be important for FipB's role in virulence; a deletion mutant complemented with a gene encoding a FipB protein in which the first cysteine was changed to an alanine residue (AXXC) failed to restore intracellular survival or in vivo virulence. Complementation with a gene that encoded a CXXA containing FipB protein was significantly defective in intracellular growth; however, only slightly attenuated in vivo

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Δ<i>fipA</i> bacteria are defective in intracellular growth.

<p>J774A.1 cells were infected at an MOI of 50∶1 with the indicated strains of bacteria as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024611#s4" target="_blank">materials and methods</a>; cells were thoroughly washed, lysed at the indicated time points, and then diluted and plated to determine CFU/ml.</p

<i>fipA</i> and <i>fipB</i> are co-transcribed.

<p>RNA was isolated from wild-type Schu S4, and then converted to cDNA. (<b>A</b>) Overview of chromosomal region containing <i>fipA</i> and <i>fipB</i>. Short arrows indicate the location of the PCR primers. The wavy arrow indicates the direction of transcription. The numbers between the genes indicate the size of the intergenic region. The hatched boxes (FTT1101 and FTT1104) indicate predicted pseudogenes. (<b>B</b>) PCR was performed using the indicated primers (RT-RNA). Controls were: No reverse transcriptase added to the cDNA synthesis reactions (No RT-RNA), and PCR using Schu S4 DNA as template and the indicated primers (DNA). Expected sizes of PCR products were as follows A–B: 314 bps, C–E: 311 bps, and D–F: 392 bps.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Detection of FipB in the Δ<i>fipA</i> bacteria.

<p>On Western blots FipB migrates as three bands. In the Δ<i>fipA</i> mutant the lower band was diminished. Western blot of bacterial lysates of indicated strains with anti-FipB antibody; antibody to <i>E. coli</i> GroEL, which cross-reacts with the <i>Francisella</i> protein, was used as a loading control. Recombinant His-FipB was used to generate the anti-FipB antibody, and serves as a positive control.</p

Effect of CXXC mutations on uptake and intracellular replication in J774A1.

<p>Monolayers of J774A.1 cells were infected with indicated strains at an MOI of 100∶1. The number of intracellular bacteria per well after infection for 2 (<b>A</b>) or 24 (<b>B</b>) hrs was determined as described in methods; cells were thoroughly washed, lysed at the indicated time points, and then diluted and plated to determine CFU/ml. Bars represent the mean ±SD of a representative experiment performed in triplicate. This experiment was repeated two times with similar results. For panel A “*” indicates p<0. 003 compared to Schu S4. For panel B “*” indicates p<0.00001 compared to Schu S4 and “**”<0.007 compared to <b>Δ</b><i>fipAB</i>.</p

Survival of <i>fipA</i>, and Δ<i>fipAB</i> mice after intranasal inoculation.

1<p>C57/BL6 mice were intranasally challenged with the indicated inoculum dose, which was confirmed by plating the inoculum.</p>2<p>Indicates the number of days after challenge that mice showed the first signs of irreversible mortality, and were euthanized. Mice were followed for a minimum of 20 days. Similar to the Δ<i>fipAB</i> mutant, mice similarly challenged with the Δ<i>fipB</i> mutant survive for more than 30 days without any signs of infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024611#pone.0024611-Qin1" target="_blank">[14]</a>.</p