Role of the JAK2/STAT3 pathway on infection of Francisella novicida

Abstract

Francisella tularensis is a causative agent of the zoonotic disease tularemia, and is highly pathogenic to humans. The pathogenicity of this bacterium is largely attributed to intracellular growth in host cells. Although several bacterial factors important for the intracellular growth have been elucidated, including the type VI secretion system, the host factors involved in the intracellular growth of F. tularensis are largely unknown. To identify the host factors important for F. tularensis infection, 368 compounds were screened for the negative regulation of F. tularensis subsp. novicida (F. novicida) infection. Consequently, 56 inhibitors were isolated that decreased F. novicida infection. Among those inhibitors, we focused on cucurbitacin I, an inhibitor of the JAK2/ STAT3 pathway. Cucurbitacin I and another JAK2/STAT3 inhibitor, Stattic, decreased the intracellular bacterial number of F. novicida. However, these inhibitors failed to affect the cell attachment or the intrasaccular proliferation of F. novicida. In addition, treatment with these inhibitors destabilized actin filaments. These results suggest that the JAK2/STAT3 pathway plays an important role in internalization of F. novicida into host cells through mechanisms involving actin dynamics, such as phagocytosis.

Fig 1. Suppression of F. novicida infection by JAK2/STAT3 inhibitors.

J774.1 cells treated with 0.01 to 10 μM of cucurbitacin I (A, C) or Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were treated with gentamicin and incubated for 12 h and then observed with confocal microscopy (A, B), or the number of intracellular bacterial was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01, *P < 0.05. Scale bar: 10μm.

Fig 2. Growth of F. novicida in culture medium with inhibitors.

F. novicida was cultured in BHIc with cucurbitacin I (A, C) or Stattic (B, D) and optical density (λ = 595 nm) was measured at the indicated time point (A, B). The number of CFU at 0 and 12 h was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Tukey–Kramer method) (A, C) or Student t-test (B, D) and indicated by asterisks, *P < 0.05.

Fig 3. Intracellular growth of F. novicida cultured with inhibitors.

F. novicida was cultured in BHIc with cucurbitacin I or Stattic. J774.1 cells were infected with inhibitor-treated F. novicida and observed at 12 h post infection. Scale bar: 10μm.

Fig 4. Attachment of F. novicida to cell surface.

J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D) at 10 or 30 min post infection. The data represent the averages and standard deviations of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, *P < 0.05. Scale bar: 10μm.

Fig 5. Internalization and proliferation of F. novicida.

J774.1 cells treated with 1 μM of cucurbitacin I (A, C) or 10 μM of Stattic (B, D) were infected with F. novicida (C, D) or GFP-expressing F. novicida (A, B). Cells were treated with gentamicin and incubated for 1.5 or 12 h, Cells were then observed with confocal microscopy (A, B), or the intracellular bacterial number was counted (C, D). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with Student t-test and indicated by asterisks, **P < 0.01, *P < 0.05. Scale bar: 10μm.

Fig 6. Intracellular proliferation of F. novicida.

J774.1 cells were infected with F. novicida (B, C) or GFP-expressing F. novicida (A). Cells were treated with gentamicin and cultured with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, C) for 12 h. Cells were observed with confocal microscopy(A), or the intracellular bacterial number was counted (B, C). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01. Scale bar: 10μm.

Fig 7. Effects of inhibitors on phagocytosis.

J774.1 cells treated with 1 μM of cucurbitacin I (A, B) or 10 μM of Stattic (A, B) were infected with E. coli (B) or GFP-expressing E.coli (A). Cells were treated with gentamicin and incubated for 3 h. Cells were observed with confocal microscopy (A), or the intracellular bacterial number was counted (B). Data represent the average and standard deviation of three identical experiments. Differences were analyzed with multiple comparison (Dunnett’s test) and indicated by asterisks, **P < 0.01. Scale bar: 10μm.

Fig 8. Effects of inhibitors on actin filaments.

J774.1 cells treated with 1 μM of cucurbitacin I or 10 μM of Stattic were infected with GFP-expressing F. novicida for the indicated time. Cells were stained with phalloidin-rhodamine and observed by confocal microscopy. Scale bar: 10μm.