Abstract
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.
Highlights
Francisella tularensis subspecies tularensis, known as type A Francisella, causes a potentially life-threatening disease called tularemia
We verified that fipA and fipB are co-transcribed by reverse transcription PCR (RT-PCR) (Figure 1)
When the complemented strains were compared to the DfipAB mutant, the number of colony form units (CFU) recovered from the CXXA strain was statistically higher (p value,0.007) than the DfipAB mutant, while there was no statistical difference between the AXXC and DfipAB mutants. These results indicated that the CXXC motif was not required for uptake, but was important for FipB mediated intracellular replication, the C164 amino acid was more critical for function
Summary
Francisella tularensis subspecies tularensis, known as type A Francisella, causes a potentially life-threatening disease called tularemia. Concerns over use of F. tularensis as a biological weapon have arisen due to its documented use as a bioweapon in WWII, and reports of the development of weaponized strains that are resistant to antibiotics and vaccines [1,2]. These concerns have led to increased interest in defining the mechanisms of virulence, and immunity as means towards identifying new targets for therapy and immune protection. Far only a few loci have been directly implicated in phagosome survival or escape Most of these loci are located on the Francisella pathogenicity island (FPI) [11,12,13]
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