Analysis of the in vitro transcriptional response of human pharyngeal epithelial cells to adherent Streptococcus pneumoniae: evidence for a distinct response to encapsulated strains

Abstract

Infection of the human host by Streptococcus pneumoniae begins with colonization of the nasopharynx, which is mediated by the adherence of bacteria to the respiratory epithelium. Several studies have indicated an important role for the pneumococcal capsule in this process. Here, we used microarrays to characterize the in vitro transcriptional response of human pharyngeal epithelial Detroit 562 cells to the adherence of serotype 2 encapsulated strain D39, serotype 19F encapsulated strain G54, serotype 4 encapsulated strain TIGR4, and their nonencapsulated derivatives (Deltacps). In total, 322 genes were found to be upregulated in response to adherent pneumococci. Twenty-two genes were commonly induced, including those encoding several cytokines (e.g., interleukin 1beta [IL-1beta] and IL-6), chemokines (e.g., IL-8 and CXCL1/2), and transcriptional regulators (e.g., FOS), consistent with an innate immune response mediated by Toll-like receptor signaling. Interestingly, 85% of genes were induced specifically by one or more encapsulated strains, suggestive of a capsule-dependent response. Importantly, purified capsular polysaccharides alone had no effect. Over a third of these loci encoded products predicted to be involved in transcriptional regulation and signal transduction, in particular mitogen-activated protein kinase signaling pathways. Real-time PCR of a subset of 10 genes confirmed the microarray data and showed a time-dependent upregulation of, especially, innate immunity genes. The downregulation of epithelial genes was most pronounced upon adherence of D39Deltacps, as 68% of the 161 genes identified were repressed only by this nonencapsulated strain. In conclusion, we identified a subset of host genes specifically induced by encapsulated strains during in vitro adherence and have demonstrated the complexity of interactions occurring during the initial stages of pneumococcal infection.

FIG. 1.

In vitro adherence of S. pneumoniae. Adherence of WT S. pneumoniae strains D39, G54, and TIGR4 and their isogenic nonencapsulated derivatives (Δcps) to Detroit 562 human pharyngeal epithelial cells. Data were obtained from three independent experiments and are presented as the means ± standard errors of the means. D39 Δcps low, low-dose D39Δcps.

FIG. 2.

Distribution of genes upregulated in epithelial cells in response to adherent pneumococci. (A) Distribution of epithelial genes induced by adherent cells of serotype 2 encapsulated WT D39 and its nonencapsulated derivative D39Δcps. (B) Distribution of epithelial genes induced by adherent cells of serotype 19F encapsulated WT G54 and its nonencapsulated derivative G54Δcps. (C) Distribution of epithelial genes induced by adherent cells of serotype 4 encapsulated WT TIGR4 and its nonencapsulated derivative TIGR4Δcps. (D) Distribution of epithelial genes induced by the three WT S. pneumoniae strains. The number of genes in each area within the Venn diagrams is indicated.

FIG. 3.

Validation of microarray data by real-time PCR. Ratios of transcript abundances in infected versus uninfected cells obtained by microarray analysis (x axis) or real-time PCR (y axis).

FIG. 4.

Real-time PCR analysis of gene expression over time. Expression of specific genes (indicated at the top of each graph) was measured by real-time PCR after predetermined time points of adherence (x axis). Log₂ ratios of infected cells/control cells are the averages of the results of two separate experiments. The dashed lines indicate a twofold change in expression relative to expression in uninfected control cells.

FIG. 5.

Cytotoxicity of epithelial cells upon adherence of pneumococci. Pneumococcal strains were allowed to adhere to Detroit 562 cells for time periods indicated on the x axis, after which cytotoxicity was assessed by LDH release. Error bars represent the standard errors of the means.

FIG. 6.

Distribution of genes downregulated in pharyngeal epithelial Detroit 562 cells. Distribution of epithelial genes repressed by adherent cells of WT strains D39, G54, and TIGR4 and isogenic nonencapsulated derivatives D39Δcps and G54Δcps. The number of genes in each area within the Venn diagrams is indicated. The ellipse represents genes repressed in response to G54Δcps. No genes were found to be repressed in response to TIGR4Δcps.