The degree of microbiome complexity influences the epithelial response to infection

Abstract

Background: The human microflora is known to be extremely complex, yet most pathogenesis research is conducted in mono-species models of infection. Consequently, it remains unclear whether the level of complexity of a host's indigenous flora can affect the virulence potential of pathogenic species. Furthermore, it remains unclear whether the colonization by commensal species affects a host cell's response to pathogenic species beyond the direct physical saturation of surface receptors, the sequestration of nutrients, the modulation of the physico-chemical environment in the oral cavity, or the production of bacteriocins. Using oral epithelial cells as a model, we hypothesized that the virulence of pathogenic species may vary depending on the complexity of the flora that interacts with host cells.

Results: This is the first report that determines the global epithelial transcriptional response to co-culture with defined complex microbiota. In our model, human immortalized gingival keratinocytes (HIGK) were infected with mono- and mixed cultures of commensal and pathogenic species. The global transcriptional response of infected cells was validated and confirmed phenotypically. In our model, commensal species were able to modulate the expression of host genes with a broad diversity of physiological functions and antagonize the effect of pathogenic species at the cellular level. Unexpectedly, the inhibitory effect of commensal species was not correlated with its ability to inhibit adhesion or invasion by pathogenic species.

Conclusion: Studying the global transcriptome of epithelial cells to single and complex microbial challenges offers clues towards a better understanding of how bacteria-bacteria interactions and bacteria-host interactions impact the overall host response. This work provides evidence that the degree of complexity of a mixed microbiota does influence the transcriptional response to infection of host epithelial cells, and challenges the current dogma regarding the potential versus the actual pathogenicity of bacterial species. These findings support the concept that members of the commensal oral flora have evolved cellular mechanisms that directly modulate the host cell's response to pathogenic species and dampen their relative pathogenicity.

Figure 1

HIGK Gene expression upon P. gingivalis and S. gordonii single- and mixed infections. Hierarchical clustering was performed on variance-normalized signal of gene expression data from uninfected HIGK cells (CTRL) and from cells in co-culture with either organism or a mixture of both for 2 h before RNA isolation and purification. Probe set signal intensities were variance-normalized, mean-centered across samples, and subjected to hierarchical cluster analysis. Heat map and dendrogram were constructed from 6066 probe sets that were differentially expressed among the treatment conditions at a level of significance of p < 0.05. The degree of similarity between the transcriptional profiles of each sample is expressed by Pearson's correlation coefficient distance metric, according to the adjacent scale. The expression state of each data point is represented as standard deviations from the mean expression level for that gene in all samples. Red indicates a relative increase, green indicates a relative decrease, and black indicates no relative change of mRNA transcripts for a given gene.

Figure 2

Impact of a mixed microbial challenge on cell cycle control in HIGK. Transcriptional regulation of the KEGG cell cycle pathway modulated by infection with P. gingivalis alone (A) or a mixture of P. gingivalis and S. gordonii (B). Genes represented by red boxes indicate up-regulated transcript levels compared to uninfected controls, where as those genes depicted in blue represent down-regulated transcript levels.

Figure 3

Infection with S. gordonii antagonizes the effect of P. gingivalis on the cell cycle. HIGK (uninfected in A) were simultaneously grown in the presence of BrdU and infected with P. gingivalis labeled with CellTracker Green BIODIPY shown in blue (B), CellTracker Blue CMAC labeled S. gordonii shown in pink (C), or a combination of P. gingivalis and S. gordonii for 4 h (D). HIGK cells were treated with antibiotic to kill extracellular bacteria and were cultured for an additional 20 h prior to harvesting for FACS analysis. The cell cycle positions and active DNA synthetic activities of cells were determined by analyzing the correlated expression of total DNA (7-AAD) and incorporated BrdU levels. Uninfected control HIGK were included for comparison of baseline cycling patterns. Data represent duplicate experiments. Region gate 1, HIGK were apoptotic (defined as sub G₀/G₁); region 2, G₀/G₁; region 3, S phase; region 4, G₂+M. Panel E and F demonstrate the gated HIGK populations used throughout all the analyses for CellTracker Green labeled P. gingivalis and CellTracker Blue CMAC labeled S. gordonii, respectively. Panel G depicts quantitative analysis of HIGK cell cycling in response to mono- and mixed infections. Results are the mean of three experiments. Statistical analysis was conducted using an ANOVA with Bonferroni's Multiple Comparison Test. * Pg vs. Sg sub G₀/G₁, Pg vs. Sg G₀/G₁, Pg vs. Sg G₂+M, Pg vs. Sg S phase, p value < 0.05. # Pg vs. Sg+Pg sub G₀/G₁, G₀/G₁, G₂+M, S phase, p values < 0.001. Error bars represent the mean ± SD.

Figure 4

Accelerated HIGK proliferation upon infection with P. gingivalis is inhibited in co-culture with S. gordonii. A) Total interaction and invasion levels determined by measuring the total numbers of P. gingivalis associated with HIGK cells by live counts in mono-(Pg) and in mixed infection (Mixed) with S. gordonii at the highest MOI. Results are the mean of three experiments. B) HIGK cells at a low confluency were co-cultured with single and complex mixtures of bacteria and cultured for up to 144 hours. Labels: Control, uninfected HIGK; Sg, S. gordonii; Pg, P. gingivalis; MIXED, co-infection with S. gordonii and P. gingivalis at different multiplicity of infection (MOI). All cell counts were performed in triplicate and all experiments were repeated twice. * p > 0.05 P. gingivalis vs non-infected controls; #p < 0.05 S. gordonii vs. non-infected controls; ^p < 0.05 S. gordonii co-infection with P. gingivalis (2500:1) vs. non-infected controls (p < 0.05) by ANOVA with Dunnett's Multiple Comparison Test.