Francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication

Abstract

The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth of Francisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly located F. tularensis bacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.

FIG. 1

Intracellular replication of F. tularensis in J774 cells results in a dose-dependent cytopathogenicity in J774 cells. (A) Replication of F. tularensis in J774 cells. F. tularensis infections were performed at an MOI of 500 in triplicate wells, each containing 2 × 10⁵ J774 cells. The number of F. tularensis bacteria in the monolayers was determined by lysis of cells with phosphate-buffered saline–buffered 0.1% sodium deoxycholate solution for 2 min and plating of 10-fold dilutions on agar plates. The number of F. tularensis bacteria recovered at the start of the experiment was 6 × 10⁶ CFU. Data are means ± standard deviations of three wells from one representative experiment of three. (B) Cytopathogenicity of J774 cells was assayed by release of LDH at 24 h and expressed as cytopathogenicity relative to that observed after lysis of the cells. Twenty thousand J774 cells were seeded per well 18 h before use and were infected at the indicated MOI. Supernatants of the infected macrophages were sampled at the indicated time points and assayed for the presence of released LDH (18) using the Cytotox 96 Kit (Promega, Madison, Wis.) according to the manufacturer's instructions. The F. tularensis strain does not have endogenous LDH activity when grown aerobically. Data are means ± standard deviations of four wells from one representative experiment of three.

FIG. 2

F. tularensis infection results in DNA fragmentation, release of nucleosomes, and annexin V staining of J774 cells and requires intracellular multiplication of bacteria. (A) Annexin V and PI staining of infected J774 cells. Cells were infected at an MOI of 500 and at 18 h of infection were stained with Annexin-V-Fluos (Boehringer, Mannheim, Germany) according to the manufacturer's instructions. Stained cells were analyzed by flow cytometry and data were analyzed by use of CellQuest (Becton-Dickinson, Sunnyvale, Calif.). Data as percentages from one of two similar experiments are shown. The y axis represents PI staining (10,000 cells) and the x axis represents annexin V staining. (B) Fragmentation of DNA from J774 cells was monitored in relation to the duration of the F. tularensis infection. J774 cells (5 × 10⁶ per dish) were infected at an MOI of 500, and at the indicated time points eukaryotic DNA was prepared following a previously described protocol (19). Lane “Stauro” represents DNA from apoptotic J774 cells after staurosporine treatment (4 μM for 6 h) and serves here as a positive control, and lane “Mock” is DNA preparations from noninfected cells at 24 h. (C) Determination of release of nucleosome fragments from J774 cells after F. tularensis infection. The release of cytoplasmic DNA fragments was monitored using the cell death detection enzyme-linked immunosorbent assay (Boehringer) according to the instructions of the manufacturer and as described previously (19). The substrate reaction time was 10 min. Representative results from one of two experiments are shown. Error bars represent standard deviations of triplicate samples. The standard deviation at an MOI of 50 was 0.0. (D) Fragmentation of DNA from J774 cells after F. tularensis infection in the presence (CytD + F.t.) or absence (F.t.) of cytochalasin D (CytD). Cells were treated for 30 min prior to infection with 1 μg of cytochalasin D ml⁻¹ (Sigma) and during the 2 h of incubation with F. tularensis. Cells were infected and the eukaryotic DNA was prepared and analyzed as described for panel B. The “Mock” lane represents preparations from noninfected cells. DNA was prepared at 24 h. A 100-bp DNA molecular size marker (Bio-Rad) was included as a standard (lane M, arrow points to the position of 100 bp). (E) Fragmentation of DNA from J774 cells after F. tularensis infection in the presence (F.t. + Cp) or absence (F.t.) of ciprofloxacin. Cells were infected and the eukaryotic DNA was prepared and analyzed as for panel B. Ciprofloxacin was used at a concentration of 20 μg ml⁻¹ and was added at 2 h after the addition of F. tularensis. DNA was prepared after 24 h of infection.