Proteome Analysis of an Attenuated Francisella tularensis dsbA Mutant: Identification of Potential DsbA Substrate Proteins

Abstract

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

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

“…The goal of present study was to confirm the proposed FTS_1067 proteinprotein interactions reported previously [5] using either immunopurification of FLAG-tagged FTS_1067 protein from trans-complemented F. tularensis FSC200/ ΔFTS_1067 strain or the B2H systems and also to confirm whether these systems are suitable for this purpose.…”

“…Unfortunately, the measurements of the β-galactosidase activity (Figure 2) did not prove the interaction of FTS_1067 with either of the two tested proteins. Similarly, all other proteins identified in the comparative proteome study [5] yielded negative results when assayed for the interactions as well (Pavkova and Schmidt, personal communications).…”

“…Previously, it was demonstrated that the deletion of gene coding for the FTT_1103 or FTS_1067 resulted in the attenuation in mouse model of infection and in the defect in proliferation in vitro [4,5]. Therefore, the effect of complementation was also studied by in vitro proliferation of the complemented strain in murine bone marrow-derived macrophages.…”

“…Using restriction sites for NdeI and SacI enzymes, the fragment was ligated to pKK289KmGFP shuttle vector instead of gfp gene. The final construct was introduced to F. tularensis deletion mutant strain FSC200/ΔFTS_1067 [5] by electroporation [16].…”

“…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]. Recently, it was presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are real virulence factors [2].…”