Position of O-Acetylation within the Capsular Repeat Unit Impacts the Biological Properties of Pneumococcal Serotypes 33A and 33F

Abstract

Streptococcus pneumoniae (pneumococcus) produces many capsule types that differ in their abilities to evade host immune recognition. To explain these serotypedependent protective capacities, many studies have investigated capsular thickness or the interaction of the capsule with complement proteins, but the effects of small chemical modifications of the capsule on its function have not been studied. One small chemical modification found frequently among pneumococcal capsules is O-acetylation. Pneumococcal serotype 33A has two membrane-bound O-acetyltransferase genes, wciG and wcjE. A 33A wcjE-deficient variant, 33F, occurs naturally and is increasing in prevalence in the wake of widespread conjugate vaccine use, but no wciG-deficient variants have been reported. To study the biological consequence of the loss of O-acetylation, we created wciG-deficient variants in both serotypes 33A and 33F, which we named 33X1 (ΔwciG) and 33X2 (ΔwciG ΔwcjE). Serotypes 33X1 and 33X2 express novel capsule types based on serological and biochemical analyses. We found that loss of WcjE-mediated O-acetylation appears not to affect cell wall shielding, since serotypes 33A and 33F exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to nasopharyngeal cells, though serotype 33F survived short-term drying better than serotype 33A. Loss of WciG-mediated O-acetylation in serotypes 33X1 and 33X2, however, resulted in a phenotype resembling that of nonencapsulated strains: increased cell wall accessibility, increased nonspecific opsonophagocytic killing, enhanced biofilm formation, and increased adhesion to nasopharyngeal cells. We conclude that WciG-mediated, but not WcjE-mediated, O-acetylation is important for producing protective capsules in 33A and that small chemical changes to the capsule can drastically affect its biological properties.KEYWORDS O-acetyltransferase, O-acetylation, capsular diversity, pneumococcal vaccine, capsular polysaccharide, serotype S treptococcus pneumoniae (pneumococcus) is an important human pathogen that can colonize the nasopharynx asymptomatically and disseminate opportunistically to cause pneumonia, meningitis, septicemia, and otitis media. The pneumococcus has a polysaccharide capsule, and 98 distinct capsular types (serotypes) have been identified based on chemical differences in their capsular repeat units (1-3). These chemical differences are mediated by genetic changes in the capsular polysaccharide synthesis (cps) operon (4) and result in different reactivities with anti-capsule antibodies. The capsule is essential for pneumococcal virulence (5), since it shelters pneumococci from complement fixation and opsonophagocytosis (6). However, differences in capsule type have been shown to affect complement fixation and virulence (7).

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

“…Taken together, however, these data suggest that O-acetylation has little effect on the biological properties of serotype 15B and 15C capsule. These data reinforce our recent observation that the effects of O-acetylation are polysaccharide context dependent (12), and these data are consistent with epidemiological studies, which found that both serotypes 15B and 15C are commonly found in the nasopharynx (46).…”

“…rpsL (Sm r ) was amplified from strain BLS101 (12,56) and transformed as described above with selection for Sm resistance into serotype 15B clinical isolate 0556-97 to create strain BLS141 or into serotype 15C clinical isolate 3031-06 to create strain BLS142. We detected no differences in the O-acetylation (fs15b) or capsular amount (pool H) between the parental clinical isolates (0556-97 and 3031-06) and the transformed reference strains (BLS141 and BLS142, respectively) by inhibition ELISA (see Fig.…”

“…Many bacteria have membrane-bound O-acetyltransferases (MOATs) that are thought to attach O-acetyl groups to the capsular repeat unit at the end of the capsular biosynthetic process (22)(23)(24). Accordingly, the loss of MOAT-mediated O-acetylation may not disrupt global capsule biosynthesis (12,(25)(26)(27).…”

“…Pneumococcal capsular types differ in their shielding abilities; therefore, some capsule types are more virulent than others (9, 10). Our previous studies have indicated that small chemical modifications of the capsular repeat unit result in differential shielding of closely related serotypes (11,12). …”

Bacterial polysaccharides as major surface antigens: interest in O-acetyl substitutions

“…In particular, S. pneumoniae types 33A and 33F were shown to exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to 10 nasopharyngeal cells, though type 33F survived short-term drying better than type 33A. 75 To our knowledge, the evolutionary relationship between the two serotypes is not yet identified.…”