Study on function and interaction of GTF and ORF4 coded by fap1-orf4 gene locus of Streptococcus parasanguis

Objective: To investigate whether glucosyltransferase(GTF) and open reading frame 4 (ORF4) coded by fap1-orf4 gene locus of Streptococcus parasanguis was involved in the regulation of Fap1 glycosylation, and mature and to determine whether there was interaction between GTF and ORF4. Methods: A gene replacement strategy was adapted to construct gtf and orf4 alleic replace mutant of 5. parasanguis. Complementation assay and Western blot were used to test fap1 expression levels. Yeast two-hybrid analysis and GST pull down assays were adapted to determine the interaction between GTF and ORF4. Results: (1) Compared with wild S. parasanguis, mature Fap1 (M _r about 220 × 10 ³) disappeared and were substituted with high molecular weight Fap1 (M _r about 360 × 10 ³) in gtf or orf4 alleic replace mutants of S. parasanguis. Complementation assay showed that pVPT-GFP-gtf and pVPT-GFP-or/4 restored mature fap1 expression in gtf or or/4 alleic replace mutants, respectively. (2) With Yeast two-hybrid analysis, the cotransformants, AH109/pAD-Gtf + pBD-orf4 and AH109/pAD-orf4 + pBD-gtf growed on SD-LTHA selective agar plate after streaked, reversely, the cotransformants, AH109/pAD + pBD-or/4, AH109/pAD + pBD-gtf, AH109/pBD + pAD-orf4, AH109/pBD + pAD-gtf did not grow on SD-LTHA selective agar plate, furthermore, the cotransformants , AH109/pAD + pBD-or/4 and AH 109/pAD-orf4 + pBD-gtf showed blue during X-α-gal assay. (3) CST pull down assay confirmed the direct interaction between GTF and ORF4. Conclusion: There is interaction between GTF and ORF4 coded by fap1-orf4 gene locus of S. parasanguis and the formation of the GTF and ORF4 complex was required for the glycosylation and mature of Fap1 in S. parasanguis.