Molecular and genetic characterization of CPS by Streptococcus parasanguinis

Production of polysaccharide capsule is critical for Streptococcus pneumoniae virulence.  S. pneumoniae produces over 90 structurally distinct capsular polysaccharides (CPS) (serotypes). Comparative genetic and phylogenetic analysis of CPS revealed strong homology among available capsular biosynthetic loci in diverse streptococci. Objectives: To identify and characterize CPS genetic locus in an oral streptococcus, S. parasanguinis FW213.  Methods: Based on conserved genetic organization of CPS loci in streptococcal and staphylococcal genomes, partially sequenced S. parasanguinis FW213 genome was searched for the presence of CPS-like locus. The production of CPS by FW213 was tested with serotype-specific antibodies from S. pneumoniae by ELISA and western blotting assays. GC-MS was used to determine glycan composition of purified CPS. Homologous genes responsible for synthesis of CPS were inactivated by allelic replacement mutagenesis. The mutated alleles were complemented to define the function of each gene in CPS production. FW213 CPS serotype was switched by introduction of serotype-specific genes of S. pneumoniae. Results: FW213 possesses a putative WZY-dependent CPS biosynthesis pathway, which produces 19B-like CPS as determined immunological assays using 19B-serotype specific antibody of S. pneumoniae and glycan composition analysis by GC-MS.  Inactivation of the first glycosyltransferase gene cpsE abolished CPS production, altered bacterial morphology and led to production of long-chain streptococci and bacterial aggregation.    Interestingly, the 19B-like FW213 CPS can be transformed into three other 19 serotypes of S. pneumoniae: 19A, 19F and 19C upon introduction of respective serotype-specific genes of S. pneumoniae.  Conclusions:  S. parasanguinis FW213 produces a 19B-like CPS which may be a prototype CPS of S. pneumoniae as this serotype of S. parasanguinis can be transformed into other prevalent subtypes of S. pneumoniae. Oral streptococci may provide a genetic repertoire for capsule evolution by S. pneumoniae. 

This study was supported by NIH/NIDCR R01DE011000, R01DE017954 and R01AI31473.