Influenza viral neuraminidase primes bacterial coinfection through TGF-β-mediated expression of host cell receptors

Abstract

Influenza infection predisposes the host to secondary bacterial pneumonia, which is a major cause of mortality during influenza epidemics. The molecular mechanisms underlying the bacterial coinfection remain elusive. Neuraminidase (NA) of influenza A virus (IAV) enhances bacterial adherence and also activates TGF-β. Because TGF-β can up-regulate host adhesion molecules such as fibronectin and integrins for bacterial binding, we hypothesized that activated TGF-β during IAV infection contributes to secondary bacterial infection by up-regulating these host adhesion molecules. Flow cytometric analyses of a human lung epithelial cell line indicated that the expression of fibronectin and α5 integrin was up-regulated after IAV infection or treatment with recombinant NA and was reversed through the inhibition of TGF-β signaling. IAV-promoted adherence of group A Streptococcus (GAS) and other coinfective pathogens that require fibronectin for binding was prevented significantly by the inhibition of TGF-β. However, IAV did not promote the adherence of Lactococcus lactis unless this bacterium expressed the fibronectin-binding protein of GAS. Mouse experiments showed that IAV infection enhanced GAS colonization in the lungs of wild-type animals but not in the lungs of mice deficient in TGF-β signaling. Taken together, these results reveal a previously unrecognized mechanism: IAV NA enhances the expression of cellular adhesins through the activation of TGF-β, leading to increased bacterial loading in the lungs. Our results suggest that TGF-β and cellular adhesins may be potential pharmaceutical targets for the prevention of coinfection.

Keywords: TGF-beta; bacterial adherence; coinfection; fibronectin binding protein; influenza A virus.

Fig. 1.

IAV enhanced TGF-β1 activity and GAS adherence to human lung epithelial cells. (A) Cell-culture supernatant was collected after A549 cells were infected with PR8 (5,000 TCID₅₀) for 2 h. Active TGF-β in the supernatant was measured by Mv1Lu luciferase assay, and the results were extrapolated from a standard curve run in parallel. (B) A549 cells were infected with PR8 as described in Materials and Methods followed by an adhesion assay with GAS strain 90226 M1 at an MOI of 10. The bacterial adherence to cells without PR8 infection (PBS) was considered as 100%. Data are presented as means ± SE of two to four independent determinants. **P ≤ 0.01.

Fig. 2.

Recombinant viral NA enhanced GAS adherence in a TGF-β signaling-dependent manner. Adherence assays were performed with the GAS M1 strain on A549 cells infected with PR8 as in Fig. 1B or treated with recombinant NA at 5 U (A) or indicated doses (B) for 18 h. For the adherence inhibition assay, cells were infected with PR8 or were treated with recombinant NA at 10 U in the presence of the TGF-β inhibitor SB 431542 (50 μM) followed by an adherence assay (C). Data are presented as means ± SEM of two or three independent experiments (n = 4–6). *P ≤ 0.05, **P ≤ 0.01.

Fig. 3.

Expression of cellular adhesion molecules was enhanced by PR8 or NA and was prevented by TGF-β1 and NA inhibitors. (A and B) Cells were infected with PR8 in the presence of DMSO (carrier control) (PR8/DMSO, black lines) or of SB 431542 (PR8/SB, dashed lines). PBS/DMSO is a control without PR8 infection (gray areas). Cells then were stained and analyzed for α5 integrin (A) or Fn (B) by flow cytometry. Ten thousand live cells were acquired; results are expressed as the percentage of cells expressing high levels of α5 integrin or Fn among the total acquired cells. (C) Cells were treated with 10 U of recombinant NA (black line) or same amount of NA buffer (gray area) for indicated times followed by flow cytometric analysis of Fn expression. (D) Cells were treated with PBS/PBS (gray area), NA/PBS (black line), or NA/zanamivir (ZMV) dissolved in PBS (dashed line) and were analyzed as in B. The bar graphs in A, B, and D show summarized data of flow cytometry (n = 4). (E) Cells were treated as in D, and adherence assay was performed as in Fig. 1B. Adherence data are presented as means ± SEM of two independent experiments (n = 6). **P ≤ 0.01, ***P ≤ 0.0001.

Fig. 4.

IAV-mediated increase in bacterial adherence is dependent on Fn-binding protein. Adhesion assays were performed at an MOI of 10 with GAS and K. pneumonia (A) or with L. lactis M1⁺ and L. lactis M1⁻ (B) after A549 cells were infected with PR8. The results are expressed as a percentage of the inoculum. Data are presented as means ± SEM of two independent experiments (n = 4). *P ≤ 0.05.

Fig. 5.

TGF-β inhibitors prevented IAV-enhanced adherence of other coinfective bacterial pathogens. A549 cells were treated with PBS and DMSO, PR8 and DMSO, PR8 and the TGF-β inhibitor SB 431542 (SB), or PR8 and the TGF-β inhibitor SIS3. Adhesion assays then were performed with S. pneumonia (T19F) (A), S. aureus (RN 6390, FnBPA⁺B⁺) (B), or H. influenzae (C) at an MOI of 10. Data are presented as means ± SEM of two or three independent experiments (n = 4–12). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001.

Fig. 6.

(A) Mice deficient in TGF-β signaling were resistant to GAS after PR8 infection. Wild-type C57BL/6 (B6) mice were infected intranasally with PR8 at 500 TCID₅₀ or 10,000 TCID₅₀ or received PBS as control. Three days later the mice were inoculated intranasally with 1 × 10⁷ cfu of GAS. Mice were killed 24 h after the inoculation. Aliquots of lung homogenate were diluted to quantify cfus. Data are presented as means ± SEM of two independent experiments. ***P ≤ 0.0001. (B) Lung homogenate from four randomly selected mice was stained, and 20,000 cells were acquired and analyzed for α5 integrin (Upper) and Fn (Lower) by flow cytometry. Gray areas, black lines, and dashed lines in histograms represent lung cells from mice that received PBS or PR8 at 500 TCID₅₀ or 10,000 TCID₅₀, respectively. Relative numbers of lung cells expressing high levels of α5 integrin or Fn are summarized in the bar graphs (n = 4, **P ≤ 0.01, ***P ≤ 0.0001). (C and D) Smad3^−/− mice and Smad3^+/+ littermates were infected with PR8 at 10,000 TCID₅₀ and were coinfected with GAS as in A. Cfus (*P ≤ 0.05) (C) and the expression of α5 integrin (Left) or Fn (Right) (D) in the lungs were determined as in A and B.

Fig. 7.

IAV facilitates bacterial adherence through the activation of the TGF-β signaling pathway. IAV NA activates latent TGF-β, which turns on the Smad signaling pathway, resulting in the up-regulation of integrins and Fn expression in cells. As bacterial receptors, the overexpression of integrins and Fn increases host susceptibility to bacterial infection.