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

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.

FipB, an Essential Virulence Factor of Francisella tularensis subsp. tularensis, Has Dual Roles in Disulfide Bond Formation

“…Site-directed mutation, plasmid construction, and in cis complementation by homologous integration into the blaB locus were performed as described previously (9). Disruption of blaB, which encodes beta-lactamase resistance, is otherwise phenotypically neutral and provides a convenient screening mechanism (13).…”

“…Mip proteins are typically lipoproteins that form homodimers via their alpha-helical amino-terminal FKBP_N domain and also contain a peptidylprolyl cis/trans isomerase (FKBP_C) domain (7,8). The fipB gene is transcribed in an operon with fipA, which encodes a short polypeptide of 96 amino acids that also has similarity to FKBP_N, though it has only ϳ37% identity to the FKBP_N-related region of FipB (2,9).…”

Production of Outer Membrane Vesicles and Outer Membrane Tubes by Francisella novicida

“…(see Table S6 in the supplemental material) (11,20,36,(58)(59)(60)(61)(62)(63). Four of these proteins-PepO, BglX, ChiA, and Fsp53-are secreted by strain U112 in a type IV pilus-dependent manner (20).…”

Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

“…Disordered regions were observed in each of the eight 222-amino acid polypeptide chains in the asymmetric unit and indicate the localized flexibility of the FTT258 protein structure. The residues that were not well defined include [22][23] (37)(38)(39)(40) and shows 94% of the residues in the most favored / regions and no outliers, with the exception of the catalytic residue Ser-116 in four molecules (A, D, F, and G) of the structure. This unique conformation "a nucleophile elbow" around the catalytic serine has been observed in other ␣/␤ hydrolases (41).…”