Inhibitory effect of capsule on natural transformation of <i>Streptococcus pneumoniae</i>

The capsule of <i>Streptococcus pneumoniae</i> (<i>Spn</i>) is highly heterogeneous based on the expression of distinct polysaccharides. <i>Spn</i> transformation, controlled by the Com regulon, has been predominantly studied using unencapsulated laboratory strains. However, genomic studies revealed different rates of recombination events in clinical isolates of different serotypes. As these isolates were genetically distinct beyond capsule-encoding genes, the exact relationship between transformation and capsule remains unclear. Herein, we compared the transformability of a collection of isogenic capsule-switch strains. Strains with different capsule types and amounts significantly differed in their transformation frequency, with the unencapsulated strain having a higher frequency compared to encapsulated strains. A GFP-reporter of each strain monitoring the expression of a Com regulon-controlled gene showed similar kinetics, indicating differences in transformability were due to processes downstream of competence activation. The Com pilus, induced by competence, binds and takes in the donor DNA and is the central component of the transformation apparatus. The surface exposure of Com pilus significantly differed among serotypes with highly transformable strains having more cells binding ComGC antibody. Furthermore, electron microscopy demonstrated that transformability correlated with the proportion of cells bearing a Com pilus, which was affected by both the presence of capsule and serotype. Additionally, the unencapsulated strain displayed longer pili than encapsulated strains. Examination of capsule porosity revealed that serotypes with higher transformation frequencies had more porous capsules. Together, these results indicate that the capsule interferes with the assembly of Com pilus, thereby inhibiting the natural transformation of <i>Spn</i>.IMPORTANCEThe capsule is a major virulence factor of <i>Streptococcus pneumoniae</i> (<i>Spn</i>), providing a physical shield and exhibiting extensive diversity across at least 100 serotypes. Although natural transformation of <i>Spn</i> has predominantly been characterized in unencapsulated laboratory strains, clinical encapsulated isolates also exhibit transformability and demonstrate varied recombination rates during host carriage. We utilized otherwise genetically identical capsule-switch strains to isolate the effect of capsule on transformation. We demonstrate serotype- and quantity-dependent inhibition of transformation by the capsule, mediated through hindrance with the transformation pilus assembly and function. This study challenges the paradigm that unencapsulated laboratory strains fully recapitulate natural transformation dynamics. By redefining the capsule as a multifunctional modulator of <i>Spn</i> biology, balancing virulence and adaptability, our findings advance our understanding of pneumococcal evolution.