Identification of an Essential Francisella tularensis subsp. tularensis Virulence Factor

Abstract

Francisella tularensis, the highly virulent etiologic agent of tularemia, is a low-dose intracellular pathogen that is able to escape from the phagosome and replicate in the cytosol. Although there has been progress in identifying loci involved in the pathogenicity of this organism, analysis of the genome sequence has revealed few obvious virulence factors. We previously reported isolation of an F. tularensis subsp. tularensis strain Schu S4 transposon insertion mutant with a mutation in a predicted hypothetical lipoprotein, FTT1103, that was deficient in intracellular replication in HepG2 cells. In this study, a mutant with a defined nonpolar deletion in FTT1103 was created, and its phenotype, virulence, and vaccine potential were characterized. A phagosomal integrity assay and lysosome-associated membrane protein 1 colocalization revealed that ⌬FTT1103 mutant bacteria were defective in phagosomal escape. FTT1103 mutant bacteria were maximally attenuated in the mouse model; mice survived, without visible signs of illness, challenge by more than 10 10 CFU when the intranasal route was used and challenge by 10 6 CFU when the intraperitoneal, subcutaneous, or intravenous route was used. The FTT1103 mutant bacteria exhibited dissemination defects. Mice that were infected by the intranasal route had low levels of bacteria in their livers and spleens, and these bacteria were cleared by 3 days postinfection. Mutant bacteria inoculated by the subcutaneous route failed to disseminate to the lungs. BALB/c or C57BL/6 mice that were intranasally vaccinated with 10 8 CFU of FTT1103 mutant bacteria were protected against subsequent challenge with wild-type strain Schu S4. These experiments identified the FTT1103 protein as an essential virulence factor and also demonstrated the feasibility of creating defined attenuated vaccines based on a type A strain.

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

“…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.…”

“…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].…”

Development of tularemic scFv antibody fragments using phage display

“…However, the function of the protein FTT1103 remains unknown. The FTT1103 mutant strain was found to be defective in intracellular growth and it was observed that in BALB/c monocyte/macrophage cell line J774A.1 has a decreased ability to escape the phagosome [26]. Despite its requirement for virulence, this protein is not an essential protective antigen.…”

“…According to the LipoP prediction program (http://www.cbs.dtu.dk/ services/LipoP/, January, 2010), FTT1103 seems to be a membrane lipoprotein [27]. It is highly probable that protein FTT1103 belongs to a group of periplasmatic proteins [26], because its N-terminal signal peptide is eliminated via signal peptidase II [27]. The locus FTT1103 is predicted to encode a hypothetical lipoprotein which shares some similarity to DsbA proteins that catalyze disulfide bond formation.…”

“…As few as 10 organisms injected subcutaneously or 25 inhaled organisms can lead to various manifestations of tularemic infection [24,25]. Recently, a novel locus, FTT1103, F. tularensis Schu S4 has been identified [26]. Conserved hypothetical protein FTT1103 is composed of 365 amino acids and specified by MW 38,72 kDa and pI 5,11.…”