GtfA Interacting with GtfB is Required for PsrP Glycosylation in Streptococcus pneumoniae

Abstract

Background: Streptococcus pneumoniae can adhere to lung cells via many adhesins, including pneumococcal serine-rich repeat protein (PsrP). Glycosyltransferase A (GtfA) and glycoslytransferase B (GtfB) were found to initiate PsrP glycosylation in Streptococcus pneumoniae serotype 4, isolate TIGR4. Objectives: To test whether GtfA and GtfB are conserved among S. pneumoniae isolates presenting with different serotypes and are involved in the glycosylation of PsrP via formation of the glycosyltransferase complex GtfA-GtfB. Methods: Serotyping of S. pneumoniae was performed using polymerase chain reaction (PCR) assays. The evolutionary divergences of GtfA and GtfB among different isolates were estimated using MEGA4 software. The contribution of GtfA and GtfB to the glycosylation of PsrP was identified using an in vivo glycosylation system in Escherichia coli. Interaction of GtfA with GtfB was assessed using a glutathione S-transferase (GST) pull-down assay and yeast two-hybrid experiment. Results: Nine different S. pneumoniae serotypes were found, of which the most frequent was 19F. The pneumococcal isolate TIGR4 and 22 other clinical isolates were included to estimate evolutionary divergence. The average homologies of the GtfA and GtfB sequences were 99.59% and 98.53%, respectively. Glycosyltransferase A and GtfB were shown to be required for the transfer of N-acetylglucosamine moieties to PsrP1-374. Glutathione S-transferase pull-down assays and yeast two-hybrid experiments showed that GtfA directly interacted with GtfB. Conclusions: Glycosyltransferase A and GtfB are conserved in S. pneumoniae isolates. Glycosyltransferase A interacts with GtfB, forming the glycosyltransferase complex GtfA-GtfB, which is required for PsrP glycosylation.