Interaction between Gap1 and SecA2 mediates biogenesis of glycoprotein Fap1

Fap1, a serine-rich glycoprotein from Streptococcus parasanguinis, is required for bacterial adhesion and biofilm formation. Fap1 biogenesis is mediated by a 7-gene cluster located immediately downstream of the fap1 locus. Structural homology analysis revealed that a glycosylation associated protein Gap1 is a putative glycosyltransferase. Mutagenesis of gap1 resulted in alternation of Fap1 glycosylation, suggesting Gap1 is important for Fap1 glycosylation. Interestingly, although SecA2 is involved in secretion of mature Fap1, its mutant also exhibited a phenotype similar to that of Gap1, implying that SecA2 may play a role in Fap1 glycosylation. Objectives: To investigate the molecular mechanism how SecA2 is involved in Fap1 glycosylation, we hypothesize that SecA2 interacts with Gap1, thereby modulating Fap1 glycosylation and biogenesis. Methods: In this study, a gap1 and secA2 double mutant was constructed using allelic replacement mutagenesis strategy, and Western blotting using Fap1-specific antibodies was performed to detect Fap1 expression. The plasmid-borne Gap1 was transformed into wild type and secA2 mutant respectively to examine Gap1 subcellular localization in the presence and absence of the SecA2 protein. GST pull-down experiment was used to determine the direct interaction between Gap1 and SecA2 in vitro. Co-immunoprecipitation (Co-IP)was used to investigate in vivo interaction between Gap1 and SecA2. Results: gap1 and secA2 single mutants as well as their double mutant displayed a similar phenotype. Subcellular localization analysis revealed that the expression level of Gap1 in the cell membranes of the secA2 mutant decreased compared with the same fraction prepared from the wild type strain, suggesting the SecA2 deficiency affects Gap1 localization. In vitro GST pull-down assays and in vivo Co-IP analyses demonstrated that Gap1 interacted with SecA2. Conclusions: Our data suggested that Gap1 and SecA2 interact with each other, the SecA2 deficiency affects Gap1 subcellular localization, thereby affecting Fap1 glycosylation and biogenesis. Supported by NIH/NIDCR R01DE11000 and K22014726.