Features of sepsis caused by pulmonary infection with Francisella tularensis Type A strain

Abstract

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F. tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen.

Fig. 1

(A). Disease progression in the mice infected intranasally with indicated doses of SchuS4 was recorded daily. The infected mice showed disease symptoms by 3 days p.i., became moribund and succumbed to infection by 4-5 days p.i.. (B). Pulmonary infection with SchuS4 resulted in bacteremia and dissemination of bacteria to systemic organs. C57BL/6 mice were infected intranasally with 3 × 10² CFU of Type A strain SchuS4. At indicated times post-infection, blood, liver, spleen and lung tissues were recovered and total bacterial burdens in each organ were determined by dilution plating onto chocolate II agar. The data shown is average of 3-5 mice in two independent experiments.

Fig. 2

Histopathological analysis of SchuS4 infected tissues. C57BL/6 mice were infected intranasally with 3 × 10² CFU of Type A strain SchuS4. At 96 h p.i., liver, spleen and lungs were harvested, sectioned, and stained with Hematoxylin & Eosin. The figure shows representative sections of lung, liver and spleen from mock control (A, B and C respectively) and SchuS4 infected (D, E and F respectively) mice. Data are from one representative experiment of three performed (n= 3 for each group per experiment). H&E staining, Bar = 100μm.

Fig. 3

Mice infected with SchuS4 display extensive apoptosis in lungs, liver and spleen. Organs from mice infected intranasally with 3 × 10² CFU of Type A strain SchuS4 were harvested 96h p.i., embedded in OCT and sectioned as described in Materials and Methods. In-situ terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP)-biotin nick end labeling (TUNEL) was used for detection of DNA fragmentation of nuclei (green) in mock control (A, B and C) or in SchuS4 infected mice (D, E and F). The same sections were co-stained for detection of bacteria (red) using a rabbit anti-F. tularensis serum as primary antibody followed by goat anti-rabbit antibody conjugated with Rhodamine-red (G, H and I). Images J, K and L show merged TUNEL and bacterial staining. Nuclei (blue) were stained with 4’6’ diamidino-2-phenylindol-dilactate (DAPI). Bar = 100μm.

Fig. 4

Lethal infection with SchuS4 results in hypercytokinemia. The lungs from mock control and SchuS4 infected mice were harvested at 24 h, 72 h and 96 h p.i., homogenized in PBS with protease inhibitors and analyzed for rodent multi-analyte profile (Rules-Based Medicine, Austin, TX). Cytokines and chemokines described as markers of sepsis are depicted. Results shown are average of three infected and mock control mice from 2-3 independent experiments. Statistical analysis of the data comparing mock control with infected samples was performed by ANOVA with Dunnett's multiple comparison post-test, * p<0.05; ** p<0.005; *** p<0.0005.

Fig. 5

Lethal infection with SchuS4 results in coagulopathy, cardiovascular injury and acute phase response associated with severe sepsis. The lungs from mock control and SchuS4 infected mice were harvested at 24 h, 72 h and 96 h p.i., homogenized in PBS with protease inhibitors and analyzed for rodent multi-analyte profile (Rules-Based Medicine, Austin, TX). Results shown are average of three infected and mock control mice from 3 independent experiments. FGF-6, fibroblast growth factor-9; FGF-basic, basic fibroblast growth factor ; SAP, Serum Amyloid Protein; SGOT, Serum glutamic oxaloacetic transaminase; TPO, Thrombopoietin; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor. Statistical analysis of the data comparing mock control with infected samples was performed by ANOVA with Dunnett's multiple comparison post-test, * p<0.05; ** p<0.005; *** p<0.0005.

Fig. 6

Lethal pulmonary infection with SchuS4 causes upregulation and release of alarmins in systemic organs. (A) Immunofluorescence staining of S100A9, an alarmin, in the lung, liver and spleen of mock, and SchuS4 infected mice. The organs from mock control and SchuS4 infected mice were harvested at 96 h p.i., sectioned and stained for S100A9 (red) using a goat anti-S100A9 antibody followed by Cy3-conjugated rabbit anti-goat antibody. Nuclei (blue) were stained with 4’6’ diamidino-2-phenylindol-dilactate (DAPI). Bar = 100μm. Images G’-I’ represent higher magnification of S100A9 positive lesion areas in lungs (bar = 50 μm). Asterisks in these images show S100A9 staining in cell free areas depicted by absence of nuclear staining. Images are from one representative experiment of two performed (n=3 mice per group). (B). Kinetics of S100A9 upregulation in lungs, liver and spleen of mock control and SchuS4 infected mice determined by Western blotting. Thirty micrograms tissue homogenates from mock control and SchuS4 infected mice harvested at indicated times p.i. were run on SDS-polyacrylamide gels, transferred to PVDF membranes and probed with anti-S100A9 antibody as described in Materials and Methods. The data shown is representative of two independent experiments with 3 animals for each experimental group. (C). Detection of HMGB1 by Western blot in sera of mock control and SchuS4 infected mice harvested at indicated times p.i.. Equal volumes of sera from mock control and SchuS4 infected mice were run on SDS-polyacrylamide gels, transferred to PVDF membranes and probed with anti-HMGB1 antibody as described in Materials and Methods. The data shown is representative of two independent experiments with 3 animals for each experimental group.