Direct interactions with influenza promote bacterial adherence during respiratory infections

Abstract

Epidemiological observations and animal models have long shown synergy between influenza virus infections and bacterial infections. Influenza virus infection leads to an increase in both the susceptibility to secondary bacterial infections and the severity of the bacterial infections, primarily pneumonias caused by Streptococcus pneumoniae or Staphylococcus aureus. We show that, in addition to the widely described immune modulation and tissue-remodelling mechanisms of bacterial-viral synergy, the virus interacts directly with the bacterial surface. Similar to the recent observation of direct interactions between enteric bacteria and enteric viruses, we observed a direct interaction between influenza virus on the surface of Gram-positive, S. pneumoniae and S. aureus, and Gram-negative, Moraxella catarrhalis and non-typeable Haemophilus influenzae, bacterial colonizers and pathogens in the respiratory tract. Pre-incubation of influenza virus with bacteria, followed by the removal of unbound virus, increased bacterial adherence to respiratory epithelial cells in culture. This result was recapitulated in vivo, with higher bacterial burdens in murine tissues when infected with pneumococci pre-incubated with influenza virus versus control bacteria without virus. These observations support an additional mechanism of bacteria-influenza virus synergy at the earliest steps of pathogenesis.

Fig. 1 |. Influenza co-sediments with S. pneumoniae and is bound to the bacterial surface.

a, The amount of the influenza virus strain PR8 that co-sedimented with wild-type pneumococcus strain BHN97 (serotype 19F) was quantified by qRT–PCR and TCID₅₀. b, Percentage of influenza virus strains PR8 and A/Sydney/5/1997 (Syd97) that co-sedimented with S. pneumoniae wild-type (WT) and mutant (ΔStrA and Δcapsule) BHN97 strains (BHN97 grown in CY supplemented with ethanolamine (EA) instead of choline to remove choline binding proteins) killed pneumococci, pneumococci treated with neuraminidase or when bound at different pHs. Mann-Whitney tests were used for statistical comparisons; the P value comparing binding of PR8 to WT with binding to inert beads is shown. c, Percentage of virus that co-sedimented with varying amounts of added BHN97. The data represent the mean ± s.d. from 3–6 biological replicates. d-f, Representative images of S. pneumoniae without virus (d; confocal microscopy), the S. pneumoniae-IAV complex (e; confocal microscopy) and pneumococcus (green) with influenza virus (red) bound to the bacterial cell surface (f; structured illumination microscopy). The images are each representative of at least five independent fields of view from at least two biological replicates. Scale bars, 1 μm (d,e) and 0.5 μm (f).

Fig. 2 |. Quatification of binding by flow cytometry.

a, Representative flow plots from three biological replicates of pneumococci alone or mixed with mRuby2–HA–PR8. b, Percentage of the BHN97 and D39 strains associated with mRuby2–HA–PR8. The data represent the mean ± s.d. from three biological replicates. The P values were determined by unpaired Student’s t-tests comparing the same strain with or without virus. See Supplementary Fig. 3 for gating strategy.

Fig. 3 |. Surface-bound influenza enhances the adherence of S. pneumoniae.

a,b, Relative pneumococcal adherence by the strains TIGR4 (serotype 4), D39 (serotype 2) and BHN97 (serotype 19F) to A549 (a) and Detroit 562 (b) cells with S. pneumoniae alone, S. pneumoniae pre-incubated with A/California/4/2009 influenza virus (+ IAV complex) and S. pneumoniae simultaneously administered with influenza virus (+ IAV). c, Relative pneumococcal adherence by strain BHN97 (serotype 19F) plus PR8 to A549 or Detroit 562 cells. Dotted lines indicate 100% adherence. The data represent the mean ± s.d. from at least three independent experiments with triplicate technical replicates in each experiment. The P values were determined by Mann-Whitney tests with pairwise comparisons to the control/no virus samples.

Fig. 4 |. Influenza binds to the surface of multiple Gram-positive and Gram-negative bacterial respiratory pathogens.

a,b, Amount of IAV strain PR8 that co-sedimented with representative Gram-positive and Gram-negative bacterial species of the respiratory tract measured by TCID₅₀ (a) or flow cytometry (b). The data represent the mean ± s.d. from 3–6 biological replicates. The P values were determined by Mann–Whitney test comparisons to binding to inert beads in a and unpaired Student’s t-tests comparing the same strain with and without virus in b. c-k, Representative confocal (c,d,f,g,i,j) and structured illumination (e,h,k) microscopy images of bacteria (green) with influenza virus (red) localized on the bacterial surface of M. catarrhalis (c–e), NTHi (f–h) and S. aureus (i–k). No virus was added to c,f,i. For the NTHi images, the red channel was cut off at median intensity of unstained NTHi to account for autofluorescence and MitoDeepRed was falsely coloured as green to match the WGA-488 staining of other species. The images are each representative of at least five independent fields of view from at least two biological replicates. Scale bars, 1 μm (c,d,f,g,i,j) and 0.5 μm (e,h,k).

Fig. 5 |. Surface-bound influenza enhances the adherence of multiple bacterial species to mammalian respiratory cells.

a–c, Relative adherence of the bacteria S. aureus (a), M. catarrhalis (b) and NTHi (c)—alone, pre-incubated with A/California/4/2009 influenza virus (strain + IAV complex) or simultaneously administered with influenza (strain + IAV)—to Detroit 562 cells. Dotted lines indicate 100% adherence. The data represent the mean ± s.d. from at least three independent experiments with triplicate technical replicates in each experiment. The P values were determined by Mann–Whitney pairwise comparisons to the control with no virus added.

Fig. 6 |. Surface-bound influenza enhances the initial colonization of nasal passages and translocation into the middle ear, and lethal disease.

a–d, Bacterial titres of the pneumococcal strain BHN97 (serotype 19F) in the nasal passages (a,b) or ears (c,d) of mice 24 h post-infection with bacteria alone, bacteria co-incubated with wild-type or temperature-sensitive (TS) PR8 (BHN97 + PR8 complex), and bacteria with PR8 without pre-mixing (BHN97 + PR8). Each point represents one sample and the bar is the median (n = 19 or 20 nasal passages per group for a, 10 nasal passages per group for b, 30 animals (60 ears) per group in c and 10 animals (20 ears) per group in d). The P values were determined by Mann–Whitney comparisons to bacteria alone. e, Survival following lethal challenge with D39, D39 premixed with PR8 (D39 + PR8 complex), D39 with PR8 without pre-mixing (D39 + PR8) and PR8 alone (n = 15 animals per group). The P values were determined by Mantel–Cox comparisons to D39 alone. n.s., not significant.