Mutational analysis of the C-terminal anchoring domains of Streptococcus mutans P1 antigen: Role of the LPXTGX motif in P1 association with the cell wall

The salivary agglutinin-interacting adhesin P1 of Streptococcus mutans is a nchored to the cell wall via the carboxy (C) terminus, which contains a wal l-associated domain, a conserved LPXTGX motif, a hydrophobic domain, and a charged tail. To further investigate the role of the C-terminal anchoring r egions in cell wall sorting and anchoring, mutational analysis was performe d on P1 in this study. Three truncated P1 mutants and seven site-directed m utants were generated by a polymerase chain reaction-based technique. The m utated P1 genes were returned to the P1-negative S. mutans SM3352 for expre ssion and localization studies by ELISA and Western immunoblotting. The res ults showed that P1 mutants with deletion of the hydrophobic domain and cha rged tail, or deletion of the charged tail alone resulted in the secretion of P1 to the culture medium. Results from cellular fractionation experiment s with the truncated mutants showed that P1 was not trapped in the membrane or cytoplasm. The site-directed mutants showed normal distribution of P1 t o the cell surface as compared to the wild-type. However, when cell walls p repared from the site-directed mutants were boiled with SDS, P1 could be re moved readily from the mutants with Thr residue in the LPNTGV motif, altere d to either Ser (T1531S) or Phe (T1531F); the mutant with Thr and Gly resid ues altered to two Phe residues (TG(1531-1532)FF), and the LPNTGV-deleted m utant (LPNTGV(-)). In contrast, the wild-type P1 and the other three site-d irected P1 mutants (P1529V, N1530I, and G(1532)F) could not be removed by b oiling SDS. When the cell wall P1s from the wild-type, mutants P1529V, N153 0I, and G(1532)F were reacted with an antibody directed against the hydroph obic domain and charged tail, no reaction was detected. However, P1s from m utants T1531S, T1531F, TG(1531-1532)FF, and LPNTGV(-) were recognized by th e antibody, indicating that the inability of these mutated P1s to firmly li nk to the cell wall was the result of failure in proteolytic cleavage of th e hydrophobic domain and charged tail. In summary, the results suggest that the charged tail plays a decisive role in sorting P1 to the cell surface, while the LPXTGX motif determines the nature of P1-cell wall association. T he Thr residue of the LPXTGX motif is required for enzymatic processing to link P1 to the cell wall, presumably via a covalent bond.