Abstract
Our recent studies have shown that deficiency of MecA in Streptococcus mutans significantly affects cell division, growth, and biofilm formation. In this study, an in vitro mixed-species model, proteomics, and affinity pull-down assays were used to further characterize the MecA-mediated regulation in S. mutans. The results showed that compared with the wild type, UA159, the mecA mutant significantly reduced its production of glucans and weakened its ability to facilitate mixed-species biofilm formation. Relative to the wild type, the mecA mutant also displayed unique characteristics, including colony morphology, growth rate, and biofilm formation that did not fully resemble any of the clpP, clpX, clpE, clpCE, and clpC individual or combinational mutants. Deletion of mecA was shown to result in alteration of >337 proteins, including down expression of GtfBC&D and adhesin P1. More than 277 proteins were differentially expressed in response to clpP deletion, including increased expression of GtfB. By cross-referencing the two proteomes, a distinctive set of proteins was found to be altered in the mecA mutant, indicating a ClpP-independent role of MecA in the regulation of S. mutans. When analyzed using affinity pull-down, ClpC, ClpX, ClpE, and CcpA were among the members identified in the MecA-associated complex. Further analysis using a bacterial two-hybrid system confirmed CcpA, ClpX, and ClpE as members of the MecA interactome. These results further suggest that MecA in S. mutans is more than an adapter of the Clp-proteolytic machinery, although the mechanism that underlies the Clp-independent regulation and its impact on S. mutans pathophysiology await further investigation. IMPORTANCE: MecA is known as an adaptor protein that works in concerto with ATPase ClpC and protease ClpP in the regulated proteolysis machinery. The results presented here provide further evidence that MecA in S. mutans, a keystone cariogenic bacterium, plays a significant role in its ability to facilitate mixed-species biofilm formation, a trait critical to its cariogenicity. Proteomics analysis, along with affinity pull-down and bacterial two-hybrid system, further confirm that MecA can also regulate S. mutans physiology and biofilm formation through pathways independent of the Clp proteolytic machinery, although how it functions independently of Clp awaits further investigation.