Pneumococcal capsular polysaccharide structure predicts serotype prevalence

Abstract

There are 91 known capsular serotypes of Streptococcus pneumoniae. The nasopharyngeal carriage prevalence of particular serotypes is relatively stable worldwide, but the host and bacterial factors that maintain these patterns are poorly understood. Given the possibility of serotype replacement following vaccination against seven clinically important serotypes, it is increasingly important to understand these factors. We hypothesized that the biochemical structure of the capsular polysaccharides could influence the degree of encapsulation of different serotypes, their susceptibility to killing by neutrophils, and ultimately their success during nasopharyngeal carriage. We sought to measure biological differences among capsular serotypes that may account for epidemiological patterns. Using an in vitro assay with both isogenic capsule-switch variants and clinical carriage isolates, we found an association between increased carriage prevalence and resistance to non-opsonic neutrophil-mediated killing, and serotypes that were resistant to neutrophil-mediated killing tended to be more heavily encapsulated, as determined by FITC-dextran exclusion. Next, we identified a link between polysaccharide structure and carriage prevalence. Significantly, non-vaccine serotypes that have become common in vaccinated populations tend to be those with fewer carbons per repeat unit and low energy expended per repeat unit, suggesting a novel biological principle to explain patterns of serotype replacement. More prevalent serotypes are more heavily encapsulated and more resistant to neutrophil-mediated killing, and these phenotypes are associated with the structure of the capsular polysaccharide, suggesting a direct relationship between polysaccharide biochemistry and the success of a serotype during nasopharyngeal carriage and potentially providing a method for predicting serotype replacement.

Figure 1. Effect of serotype on resistance to non-opsonic killing by neutrophils.

A) Capsule increases resistance of pneumococcus type 6B to non-opsonic surface killing compared to an unencapsulated derivative. Mean+/−SEM from a representative experiment. p<0.001 B) Capsule prevents association of pneumococcus type 6B with human PMNs. Mean+/−range from two independent experiments. MFI: Mean fluorescence intensity of neutrophils. C) Avoidance of neutrophil-mediated killing by the TIGR4 capsule variants is directly correlated with the frequency of each serotype among carriage isolates. Mean survival relative to serotype 9N is shown. ρ = 0.78, p<0.01. D) Susceptibility to neutrophil-mediated killing is a property of capsular type across diverse genetic backgrounds. Linear regression is shown comparing survival from neutrophil-mediated killing between isogenic TIGR4 capsule variants and diverse clinical isolates. Mean+/−range., ρ = 0.49, p<0.05.

Figure 2. Relationship between degree of encapsulation, resistance to neutrophil-mediated killing, and carriage prevalence.

Capsule size, as evaluated by FITC-Dextran exclusion, is related to A) avoidance of neutrophil-mediated killing (ρ = 0.58, p<0.05) and B) carriage prevalence (ρ = 0.68, p<0.01). The mean area of the zone of FITC-dextran exclusion, including the bacterial body and capsule, is shown. 100–250 cells per serotype were measured, and the zone of exclusion for unencapsulated TIGR4 was 286 pixels.

Figure 3. Effect of degree of encapsulation on susceptibility to neutrophil-mediated killing of type 19F.

Growth in fructose leads to A) a decrease in cell-associated type 19F polysaccharide as evaluated by inhibition ELISA and B) decreased resistance to neutrophil-mediated killing. Mean+/−SEM, p<0.001. The capsules of bacteria grown in C) glucose or D) fructose were visualized in India ink.

Figure 4. Relationship between capsule structure (carbon/repeat unit) and carriage prevalence .

Capsule structure is associated with prevalence both A) before introduction of conjugate vaccine (ρ = −0.80, p<0.001, excluding type 3) and B) after introduction of vaccine (2004) among children in Massachusetts (ρ = −0.90, p<0.05, excluding type 3). Vaccine types are denoted by an open circle.