Dynamic Virus-Bacterium Interactions in a Porcine Precision-Cut Lung Slice Coinfection Model: Swine Influenza Virus Paves the Way for Streptococcus suis Infection in a Two-Step Process

Abstract

Swine influenza virus (SIV) and Streptococcus suis are common pathogens of the respiratory tract in pigs, with both being associated with pneumonia. The interactions of both pathogens and their contribution to copathogenesis are only poorly understood. In the present study, we established a porcine precision-cut lung slice (PCLS) coinfection model and analyzed the effects of a primary SIV infection on secondary infection by S. suis at different time points. We found that SIV promoted adherence, colonization, and invasion of S. suis in a two-step process. First, in the initial stages, these effects were dependent on bacterial encapsulation, as shown by selective adherence of encapsulated, but not unencapsulated, S. suis to SIV-infected cells. Second, at a later stage of infection, SIV promoted S. suis adherence and invasion of deeper tissues by damaging ciliated epithelial cells. This effect was seen with a highly virulent SIV subtype H3N2 strain but not with a low-virulence subtype H1N1 strain, and it was independent of the bacterial capsule, since an unencapsulated S. suis mutant behaved in a way similar to that of the encapsulated wild-type strain. In conclusion, the PCLS coinfection model established here revealed novel insights into the dynamic interactions between SIV and S. suis during infection of the respiratory tract. It showed that at least two different mechanisms contribute to the beneficial effects of SIV for S. suis, including capsule-mediated bacterial attachment to SIV-infected cells and capsule-independent effects involving virus-mediated damage of ciliated epithelial cells.

FIG 1

S. suis adherence to and colonization of porcine PCLS. PCLS were infected with approximately 10⁷ CFU of S. suis strain 10, 10Δsly, or 10cpsΔEF per slide for 4 h, washed thoroughly to remove nonadherent bacteria, and incubated for an additional 20 h. (A and B) Cryosection of PCLS followed by immunostaining was performed 24 h after bacterial infection. Streptococci are labeled in green, and nuclei were stained by DAPI (blue). Bars represent 10 μm. Ciliated cells were stained using anti-β-tubulin antibody (A, red) and mucus-producing cells labeled with anti-Muc5Ac antibody (B, red). (C) Number of bacteria attached to ciliated cells determined by measuring the area of the luminal surface of bronchioli positive for green fluorescent bacteria. Results are expressed as means with SD, and significance, indicated by one (P < 0.05) or two (P < 0.01) asterisks, was determined using one-way ANOVA followed by a Tukey post hoc test.

FIG 2

S. suis adherence to and colonization of H3N2 preinfected porcine PCLS. Porcine PCLS were preinfected with 10⁵ TCID₅₀/ml H3N2 for 1 h, washed, and subsequently infected with S. suis strain 10 or 10cpsΔEF as described for bacterial monoinfection. Slices monoinfected with strain 10, 10cpsΔEF, and H3N2 served as controls. Cryosection of slices followed by immunostaining was performed 24 h after bacterial infection. Streptococci are labeled in green, nucleoproteins of SIV are stained in red, and nuclei are shown in blue (DAPI). (A) Mono- and coinfected cells of the bronchiolar epithelium. Bars represent 10 μm. (B) Mono- and coinfected alveolar cells of PCLS. Bars represent 10 μm.

FIG 3

S. suis adherence to and colonization of H1N1-preinfected porcine PCLS. Porcine PCLS were preinfected with 10⁵ TCID₅₀/ml H1N1 for 1 h, washed, and subsequently infected with S. suis strain 10 or 10cpsΔEF as described for bacterial monoinfection. Slices monoinfected with strain 10, 10cpsΔEF, and H1N1 served as controls. Cryosection of slices followed by immunostaining was performed 48 h after bacterial infection. Streptococci are labeled in green, nucleoproteins of SIV are stained in red, and nuclei are shown in blue (DAPI). (A) Mono- and coinfected cells of the bronchiolar epithelium. Bars represent 10 μm. (B) Mono- and coinfected alveolar cells of PCLS. Bars represent 10 μm.

FIG 4

Quantification of adherent and invasive S. suis in porcine PCLS preinfected with SIV. Porcine PCLS were preinfected with 10⁵ TCID₅₀/ml H3N2 or H1N1 for 1 h, washed, and subsequently infected with S. suis strain 10 or 10cpsΔEF as described for bacterial monoinfection. Slices monoinfected with strains 10 and 10cpsΔEF served as a control. The number of bacteria attached to ciliated cells was determined by measuring the area of the luminal surface of bronchioli positive for green fluorescent bacteria based on immunofluorescent overview images (see Fig. S3 to S5 in the supplemental material). (A) Determination of adherent bacteria 24 h after bacterial infection on H3N2-preinfected PCLS. (B) Determination of adherent bacteria 48 h after bacterial infection on H1N1-preinfected PCLS. (C) Determination of adherent and invasive bacteria 72 h after bacterial infection on H3N2- and H1N1-preinfected PCLS. Results are expressed as means with SD, and significance, indicated by one (P < 0.05), two (P < 0.01), or three (P < 0.001) asterisks, was determined by one-way-ANOVA followed by a Tukey post hoc test.

FIG 5

Ciliary activity of SIV-preinfected porcine PCLS coinfected with S. suis. PCLS were preinfected with 10⁵ TCID₅₀/ml of either H3N2 or H1N1 for 1 h, washed, and subsequently infected with S. suis strain 10 or 10cpsΔEF as described for bacterial monoinfection. Slices monoinfected with only a viral or bacterial strain served as controls. Ciliary activity was estimated by analyzing ciliary beating of mono-, co-, and uninfected PCLS (Ctr) under the light microscope at 24, 48, and 72 h after bacterial infection (hpi). Results are expressed as means with SD. Significant differences are indicated for Ctr versus infected slices (**, P < 0.01) and (****, P < 0.001), for strain 10 versus H3N2 plus strain 10 (++, P < 0.01; +++, P < 0.001), for 10cpsΔEF versus H3N2 plus 10cpsΔEF (#, P < 0.05; ###, P < 0.001), and for H1N1 versus H3N2 (††, P < 0.01; †††, P < 0.001) and were determined by one-way-ANOVA followed by a Tukey post hoc test.

FIG 6

Costaining of ciliated epithelial cells and S. suis in porcine PCLS preinfected with SIV and coinfected with S. suis at early stage of infection. Porcine PCLS were preinfected with 10⁵ TCID₅₀/ml H3N2 or H1N1 for 1 h, washed, and subsequently infected with S. suis strain 10 or 10cpsΔEF as described for bacterial monoinfection. Slices monoinfected with strains 10 and 10cpsΔEF served as a control. Streptococci are shown in green, ciliated cells were stained using anti-β-tubulin antibody (red), and nuclei were labeled by DAPI (blue). Bars represent 10 μm. (A) Immunostained cryosections of adherent bacteria of PCLS slices preinfected with H3N2 24 h after bacterial infection. (B) Immunostained cryosections of adherent bacteria of PCLS slices preinfected with H1N1 48 h after bacterial infection.

FIG 7

Invasion and damage of the bronchiolar epithelium of porcine PCLS preinfected with SIV and coinfected with S. suis at late stage of infection. Porcine PCLS were preinfected with 10⁵ TCID₅₀/ml H3N2 or H1N1 for 1 h, washed, and subsequently infected with S. suis strain 10 (A) or 10cpsΔEF (B) as described for bacterial monoinfection. Slices monoinfected with strain 10 and 10cpsΔEF only served as a control. Cryosection of slices followed by immunostaining was performed 72 h after bacterial infection. Maximum intensity projections of confocal stacks showing streptococci are in green, ciliated cells were stained using anti-β-tubulin antibody (red), and nuclei were labeled by DAPI (blue). Bars represent 25 μm.