Protective potential of outer membrane vesicles derived from a virulent strain of Francisella tularensis

Abstract

Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.

Keywords: FSC200; Francisella tularensis; host-pathogen interaction; outer membrane vesicles; vaccination.

Figure 1

Bacterial proliferation in OMV-treated macrophages. BMMs were treated with OMVs or IFN-γ for 24 h and then infected with F. tularensis subsp. holarctica FSC200. The cells were lysed at 1, 5, and 8 h post-infection and the number of intracellular bacteria was enumerated by CFU plating. The data are expressed as mean ± SEM (n = 3), results shown are representative of five independent experiments. Statistical significance was determined by Holm-Sidak’s multiple comparisons test (**p < 0.01, ***p < 0.001).

Figure 2

Western blot analysis of BMMs treated with OMVs, LPS from E. coli, IL-4, or infected by F. tularensis subsp. holarctica FSC200. Untreated macrophages were taken as control. Results are representative of two independent experiments.

Figure 3

Cytokine production in macrophages treated sequentially with a combination of OMVs and infection by Francisella. (A) Experimental scheme: BMMs were pre-treated with OMVs or F. tularensis FSC200 for 4 h and the supernatant was collected for cytokine detection (t = 0 h). Sequentially BMMs were infected with FSC200 or treated with OMVs and incubated further 4 or 24 h for cytokine detection. Three independent controls were used—BMMs treated with OMVs only, BMMs infected only, and mock-treated BMMs. (B) Selected cytokines were determined in the culture supernatants by ELISA. The data are the mean ± SEM (n = 3), results shown are representatives of two independent experiments. Significance is shown for comparison versus the group treated only with OMVs in the respective time intervals (*p ≤ 0.05), two-way ANOVA followed by Dunnett’s multiple comparison post hoc test. The figure was partly generated using Servier Medical Art provided by Servier, licensed under a Creative Commons Attribution 3.0 Unported License.

Figure 4

Protective effect of OMVs against infection of F. tularensis subsp. holarctica FSC200. (A) Vaccination and sera collection scheme: BALB/c mice were immunized i.n. or i.p. with 15 μg of isolated OMVs or mock and after 14 days they were boosted with the same. Six weeks after the first immunization the mice were challenged with 100 CFU of FSC200 per mouse and observed for survival. Sera were taken for the detection of cytokines, antibody isotypes, and immunoreactive proteins 14 and 42 days after immunization. (B) Survival curves after i.n. and i.p. administration, (*p < 0.05, ***p < 0.0001) significantly longer survival according to Mantel-Cox test. The figure was partly generated using Servier Medical Art provided by Servier, licensed under a Creative Commons Attribution 3.0 Unported License.

Figure 5

Cytokine levels in murine sera 14 and 42 days after i.n. immunization with OMVs. The immunization and sera collection scheme is depicted in Figure 4A. Cytokine levels were determined by a fluorescence-based multiplex Quantibody ELISA microarray chip. The data are shown as individual concentrations and mean (n = 6). Significance was estimated from the Mann–Whitney test (*p ≤ 0.05).

Figure 6

Antibody isotype levels in murine sera 14 and 42 days after i.n. immunization with OMVs. The immunization and sera collection scheme is depicted in Figure 4A. Antibody isotype levels were determined by fluorescence-based multiplex Quantibody Mouse Immunoglobulin Isotype Array. The data are shown as individual concentrations and mean (n = 6 in OMVs vaccinated and 3 in control). Significance was estimated from an unpaired t-test with Welch’s correction (*p ≤ 0.05).