The Streptococcus pyogenes capsule is required for adhesion of bacteria to virus-infected alveolar epithelial cells and lethal bacterial-viral superinfection

Abstract

An apparent worldwide resurgence of invasive group A Streptococcus (GAS) infections remains unexplained. However, we recently demonstrated in mice that when an otherwise nonlethal intranasal GAS infection is preceded by a nonlethal influenza A virus (IAV) infection, induction of lethal invasive GAS infections is often the result. In the present study, we established several isogenic mutants from a GAS isolate and evaluated several virulence factors as candidates responsible for the induction of invasive GAS infections. Disruption of the synthesis of the capsule, Mga, streptolysin O, streptolysin S, or streptococcal pyrogenic exotoxin B of GAS significantly reduced mortality among mice superinfected with IAV and a mutant. In addition, the number of GAS organisms adhering to IAV-infected alveolar epithelial cells was markedly reduced with the capsule-depleted mutant, although this was not the case with the other mutants. Wild-type GAS was found to bind directly to IAV particles, whereas the nonencapsulated mutant showed much less ability to bind. These results suggest that the capsule plays a key role in the invasion of host tissues by GAS following superinfection with IAV and GAS.

FIG. 1.

Targeted mutagenesis of slo in GAS strain SSI-9. (A) pIN151 contains an internal fragment of slo and a kanamycin resistance gene (aphA3). TR-10 was produced by a single-crossover recombination. The speB (TR-11) and sagA (TR-17) mutants were constructed in a similar fashion. (B) The hasA, slo, speB, and sagA mutants were subjected to PCR with a targeted gene-specific forward primer and an aphA3- or aad9-specific reverse primer. (C) Strain SSI-9 and its mutants (10⁷ CFU per 50 μl) were inoculated into 5 ml of THY broth and incubated at 37°C. Growth was determined by measuring the optical density at 600 nm (OD₆₀₀).

FIG. 2.

Mortality among mice superinfected with IAV and the indicated GAS test strain. Groups of 30 mice were intranasally infected first with IAV (100 FFU) in 25 μl of PBS on day −2 and then with SSI-9 or one of its mutant strains (10⁷ CFU) in 25 μl of PBS on day 0. Mortality was assessed daily for 10 days after GAS infection. Fisher's exact test was used to determine significant differences in mortality among the groups: P = 0.0012, P < 0.0001, P = 0.0127, P = 0.0470, and P = 0.0061, respectively, for TR-5, TR-8, TR-10, TR-11, and TR-17 versus SSI-9.

FIG. 3.

Effect of capsule removal on the number of GAS organisms in the internal organs of superinfected mice. (A) Ten mice were intranasally infected first with IAV (100 FFU) on day −2 and then with GAS strains (10⁷ CFU) on day 0. The number of GAS organisms in the lungs was assessed on day 1. Circles under the broken line indicate that GAS was not detectable. Horizontal bars indicate the median values. Wilcoxon two-sample rank tests were used to evaluate the differences between groups: P = 0.1403, P = 0.0003, P = 0.1032, P = 0.2718, and P = 0.0880, respectively, for TR-5, TR-8, TR-10, TR-11, and or TR-17 versus SSI-9. (B) The numbers of GAS organisms in the lungs, livers, spleens, and kidneys of mice superinfected with IAV and SSI-9, or TR-8 were determined on days 1 and 3. Circles under the broken line indicate that GAS was undetectable. Horizontal bars indicate the median values. Mann-Whitney U tests were used to evaluate differences between groups *, P < 0.01; **, P < 0.05.

FIG. 4.

Association of GAS with IAV-infected alveolar epithelial cells. Mice were infected with SSI-9 or superinfected with IAV and SSI-9 or TR-8 as described in the legend to Fig. 2. Lungs were harvested 48 h after GAS infection, and sections were stained with Alexa Fluor 568-conjugated anti-HA MAb or rabbit polyclonal anti-GAS antibody and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G.

FIG. 5.

Expression of capsule by GAS is required for maximal adhesion to and invasion of cultured IAV-infected A549 epithelial cells. A549 cells were infected with IAV for 17 h. After subsequent infection with SSI-9 or TR-8 for 2 h, the number of GAS organisms adhering to or invading the epithelial cells was assessed. Data are shown as means ± standard errors of the means from 12 wells (four independent experiments performed in triplicate). Mann-Whitney U tests were used to evaluate the differences between groups. *, P < 0.01.

FIG. 6.

Direct binding of GAS to IAV particles and effect of the GAS capsule and sialic acid on direct binding. (A) Wild-type SSI-9 or hasA-inactivated TR-8 mutant bacteria were added to IAV-coated wells (4 μg of IAV per ml, suspended in PBS containing 0.2% BSA), after which GAS binding to IAV particles was quantitated. Data are shown as means ± standard errors of the means from nine wells (three independent experiments performed in triplicate). Mann-Whitney U tests were used to evaluate differences between groups. *, P < 0.01 versus binding of SSI-9 to uncoated wells. (B) GAS (SSI-9) and GBS (COH1; type III) bacteria were cultured in THY broth, capsular polysaccharides from 100 mg (dry weight) of GAS and GBS were harvested, and the amount of sialic acid was estimated. (C) SSI-9 organisms were pretreated with or without neuraminidase, and the direct binding assay was performed.