Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates

Abstract

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Keywords: Franscisella tularensis; immunity; inner membrane proteins; outer membrane proteins; proteomics.

FIG 1

Representative results of silver-stained SDS-PAGE analysis of LVS outer and inner membrane fractions. LPS bands, shown in the red box in the outer membrane fraction panel, assisted in differentiating between the inner and outer membrane fractions.

FIG 2

Mice were intramuscularly vaccinated with a physical mixture of 25 μg LVS membrane (LVS mem) preparations and 5 μg poly(I·C) or the same amount of LVS membranes and poly(I·C) encapsulated in PLGA nanoparticles. (A) LVS-specific IgG titers from serum 30 days postvaccination. (B and C) At 40 days postvaccination, mice were challenged with 2,000 CFU LVS intranasally and morbidity (B) and mortality (C) levels were assessed. **, P < 0.01 (determined by one-way ANOVA). LOD, limit of detection.

FIG 3

Mice were intramuscularly vaccinated with a mixture of 10 μg Schu S4 membrane preparations and 5 μg poly(I·C) or the same amount of Schu S4 membranes and poly(I·C) in PLGA nanoparticles. (A) Schu S4-specific IgG titers from serum collected 40 days postvaccination. (B) At 42 days postvaccination, mice were challenged with 25 CFU Schu S4 and morbidity and mortality levels were assessed. (C and D) Mice received a prime [mixture of 10 μg Schu S4 membranes and 5 μg poly(I·C) or the same amount of Schu S4 membrane and poly(I·C) in PLGA nanoparticles] and after 40 days received a boost (2,000 CFU LVS i.p.). (C) Schu S4-specific IgG titers from serum 37 days postboost. (D) Morbidity and mortality levels of mice were assessed. Numbers of surviving mice are indicated. *, P < 0.05; ***, P < 0.001 (determined by one-way ANOVA).

FIG 4

Western blot of Schu S4 outer membrane proteins probed with serum from a Schu S4 whole-membrane-PLGA-vaccinated mouse boosted with LVS obtained prior to challenge. The bands extracted for mass spectrometric analyses are indicated.