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

Abstract

FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.

Components of the type six secretion system are substrates ofFrancisella tularensisSchu S4 DsbA-like FipB protein

“…However, a separate DsbC protein has not been identified in Francisella strains. We, and others have found that FipB has both oxidoreductase and isomerase activities 13,14 . Recombinant FipB has also been demonstrated to have chaperone activity using an in vitro assay 15 …”

Identification of two substrates of FTS_1067 protein – An essential virulence factor of Francisella tularensis

“…One should argue that the cytoplasm, where interactions in B2H assays take place is reducing environment and that the FTS_1067 must be in oxidized state to act as an oxidoreductase or isomerase. But as was mentioned above, the results of our group [3] and the results by Qin et al [1] indicate that both oxidoreductase and isomerase activities of FTS_1067 or FTT_1103 are dependent on their reduced state and thus, the reducing environment in cytoplasm should not be limiting.…”

“…Recently, it was shown that both FTS_1067 and FTT_1103 proteins need to be reduced to act as an isomerase or oxidoreductase [1,3]. Due to this fact, we decided to further validate the observed protein-protein interactions by using an independent approach, the B2H systems where the interactions take place in reducing cytoplasm.…”

“…In fact, the role of DsbA protein in virulence is indirect and depends on its correct oxidoreductase [1,5,23] and recently confirmed isomerase activities [1][2][3]. Hence, it is highly desirable to identify the FTS_1067 substrates that might be responsible for the FTS_1067 contribution to F. tularensis subsp.…”

“…holarctica strain FSC200, FTL_1096 in F. tularensis subsp. holarctica strain LVS, also referred to as FipB) with proved oxidoreductase and isomerase activities [1][2][3]. It was shown that the deletion of the gene encoding DsbA led to the high attenuation in vivo associated with the ability to induce host-protective immunity [4,5].…”