Aggregatibacteraphrophilus T6SS Effectors in Host-Bacterial Interactions

Abstract

Aggregatibacter aphrophilus is the only known oral bacterium with a functional type VI secretion system (T6SS) that acts against Aggregatibacter actinomycetemcomitans. Bacteria use the T6SS to deliver toxic effectors into bacterial or eukaryotic cells during interbacterial competition or host colonization. To date, the T6SS of A. aphrophilus has not been demonstrated to participate in antieukaryotic activity, nor have the cytotoxic effectors involved been identified. Here, we identified 2 T6SS effectors in A. aphrophilus, a glycosyl hydrolase (Glh) and a phospholipase D (Tle5), which, upon inactivation of their respective genes, together resulted in abolished T6SS activity against A. actinomycetemcomitans in multispecies biofilms. Next, we probed the role of the 2 T6SS effectors in host-cell interactions using gingival keratinocytes. Interestingly, although neither of the effectors appeared to contribute to the acute inflammatory response directly, the co-presence of both species reduced the inflammatory effect, likely due to the T6SS-dependent elimination of A. actinomycetemcomitans, hence decreasing the bacterial abundance. This reduction was not observed using A. aphrophilus mutants lacking the effectors or the T6SS "tube" core protein, hemolysin co-regulated protein (Hcp). Here, we show that the T6SS effectors in A. aphrophilus have distinct functions in eukaryotic versus bacterial cell interactions. Hence, these T6SS effectors may represent novel mechanisms of interaction between bacteria and the oral-mucosal barrier, offering potential therapeutic targets for managing periodontal pathogens.

Keywords: Aggregatibacter actinomycetemcomitans; biofilm; dental plaque; phospholipase D; type VI secretion system; type VI secretion system effectors.

Figure 1.

Quantitative composition of multiple-species biofilms. The relative cell abundances of all species in the multispecies biofilms containing either A. aphrophilus HK 83 wild type or Δhcp (A), Δtle5 (B), Δglh (C), or Δtle5 Δglh (D) were measured. Numbers of each strain were counted by colony-forming units (CFUs), and data were plotted on a logarithmic scale. Asterisks (****) indicate significant differences (P ≤ 0.0001) between the groups. A. a., A. actinomycetemcomitans.

Figure 2.

Overview of human proteins quantified from each sample. (A) Heat map displaying the normalized abundance of quantified proteins. The colors in the map represent the value of the hyperbolic arcsine transformed normalized abundance plus 1 for individual proteins (represented by a single row) within each experimental sample (represented by a single column). Expression values are shown on a color scale, with higher values in red and lower values in blue. The 8 groups of the immortalized epithelial cell line, either challenged with or without the bacterial strains, are as follows: control without bacterial challenge (control, n = 5), A. aphrophilus strain HK83 (HK83, n = 6), A. aphrophilus strain HK83 Δhcp mutant (HK83 Δhcp, n = 6), A. aphrophilus strain HK83 Δtle5 Δglh mutant (HK83 Δtle5 Δglh, n = 6), and A. actinomycetemcomitans strain JP2 (JP2, n = 6). These are color coded. (B) Principal component analysis (PCA) of the proteome, clustered based on the inverse hyperbolic sine transformation of the spectrum counts for the identified proteins across the 8 groups for all human proteins.

Figure 3.

Regulated human proteins in bacterial-challenged epithelial cells compared with unchallenged cells. (A) Number of regulated proteins in each comparison. (B) Results of the “acute inflammatory response” based on overrepresentation analysis (ORA) enrichment across different comparisons (Supplementary Table 5). (C) Protein interactions between regulated proteins contributing to the enrichment of the “acute inflammatory response,” “response to interleukin-1,” and “immune response–regulating signaling pathway.” These interactions were established using STRING 10.5 (Supplementary Table 6), with the highest confidence score (0.9) for proteins involved in the corresponding pathways (highlighted with different circles). (D) Expression levels of proteins contributing to the “acute inflammatory response,” response to interleukin-1,” and “immune signaling pathways.” Proteins contributing to the enrichment of these pathways are marked with brown circles, red boxes, and blue boxes, respectively. Protein abundance is shown as hyperbolic arcsine-transformed normalized abundances. Different letters (a, b, or c) on the right of the box plot indicate significant differences (P < 0.05, fold change >2) between the compared groups based on the Progenesis QI exported result (Supplementary Table 4).

Figure 4.

Regulated proteins in epithelial cells challenged by wild-type A. aphrophilus and its mutants, alone and in combination. (A) Number of regulated human proteins in each comparison. (B) Acute inflammatory response results based on overrepresentation analysis (ORA) enrichment across different comparisons (Supplementary Table 5). (C) Response to interleukin-1 results based on ORA enrichment across different comparisons.