UC San Diego

Group B Streptococcus (GBS) asymptomatically colonizes the lower gastrointestinal and urogenital tracts in 25-35% of the human population. However, in immune compromised individuals and infants, GBS is able to cause severe invasive disease and death. In newborns, GBS is the leading cause of sepsis and meningitis. GBS is surrounded by a thick capsular polysaccharide (CPS) layer. Nine antigenically distinct serotypes have been documented, all of which display a terminal [alpha]2-3 linked sialic acid (Sia) residue. This residue is key to GBS evasion of host immune response, and as such, is a key virulence factor for the bacterium. Terminal Sias are commonly expressed on the surface of mammalian cells and in this context serve to dampen immune response against self. This is accomplished by binding to factor H, which regulates the alternative complement pathway, as well as through interactions with Siglecs, Sia binding receptors with immunomodulatory capabilities, on the surface of immune cells. Sialic acid on the surface of GBS similarly engages these immune-suppressive mechanisms in a highly effective form of molecularly mimicry. It has only recently been discovered that GBS biochemically modifies terminal Sia residues by adding an O-acetylationetyl group to the C-7 position of the Sia moiety. Acetylation is incomplete, ranging from 5 to 40% of total surface Sia residues, and is characteristic of different serotype strains. This level is controlled by the balance in activities of the NeuD acetyltransferase and NeuA acetylesterase. Both enzymes are dual functional, being required for surface sialylation as well as acetylation. In this work we develop methodology using genetic techniques to manipulate O-acetylationetylation (O-acetylation) without impacting overall surface sialylation. Using isogenic strains with artificially high and low O-acetylation we proceed to analyze the impact of O-acetylation on various biological interactions and ultimate consequence for GBS virulence. We find that O-acetylation protects GBS surface Sias from removal by various microbial sialidases, presenting a possible benefit to in the context of colonization. Looking at Sia-dependent immune interactions, we discover that GBS O-acetylation does not impact alternative complement pathway activation, with both OAchigh and OAclow strains showing identical Sia-dependent deposition of the active opsonin C3b. O-acetylation does impair interactions with Siglec-9, thereby impairing the ability of GBS to downregulate neutrophil responses. In this regard,we document increased elastase secretion, oxidative burst and bacterial killing by neutrophils in response to the OAchigh strain. In the more complex context of ex-vivo human whole blood, these differences are enhanced, with the OAchigh strain being killed more rapidly. The OAchigh strain also shows greatly attenuated virulence in a mouse IP model of infection. Thus, in the context of invasion O- acetylation appears to be a cost to bacterial survival. Finally we document O-acetylation levels in 100 clinical and colonizing type Ia and type III GBS isolates. We find that type Ia strains consistently show very low levels of O-acetylation (5%) while type III strains display much higher O-acetylation (30%). This is correlated with the NeuD allele, possessing high or low enzymatic activity. We hypothesize that the distinct O-acetylation levels in type Ia and type III strains represent a balance between opposing forcing of selection, which differ in these different genetic backgrounds. Further studies will be necessary to elucidate these unique constraints, and their implications in disease manifestation