A Novel Protein, RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Abstract

ABSTRACT Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that Δ rafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 Δ rafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 Δ rafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the Δ rafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, Δ rafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.