Aggregatibacteraphrophilus T6SS Effectors in Host-Bacterial Interactions

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.