Deletion of the Zinc Transporter Lipoprotein AdcAII Causes Hyperencapsulation of Streptococcus pneumoniae Associated with Distinct Alleles of the Type I Restriction-Modification System

The capsule is the dominant virulence factor, yet how variation in capsule thickness is regulated is poorly understood. Here, we describe an unexpected relationship between mutation of , which encodes a zinc uptake lipoprotein, and capsule thickness. Partial deletion of in three of five capsular serotypes frequently resulted in a mucoid phenotype that biochemical analysis and electron microscopy of the D39 mutants confirmed was caused by markedly increased capsule thickness. Compared to D39, the hyperencapsulated mutant strain was more resistant to complement-mediated neutrophil killing and was hypervirulent in mouse models of invasive infection. Transcriptome analysis of D39 and the mutant identified major differences in transcription of the Sp_0505-0508 locus, which encodes an SpnD39III (ST5556II) type I restriction-modification system and allelic variation of which correlates with capsule thickness. A PCR assay demonstrated close linkage of the SpnD39IIIC and F alleles with the hyperencapsulated strains. However, transformation of with fixed SpnD39III alleles associated with normal capsule thickness did not revert the hyperencapsulated phenotype. Half of hyperencapsulated strains contained the same single nucleotide polymorphism in the capsule locus gene , which is required for the initiation of capsule synthesis. These results provide further evidence for the importance of the SpnD39III (ST5556II) type I restriction-modification system for modulating capsule thickness and identified an unexpected linkage between capsule thickness and mutation of Further investigation will be needed to characterize how mutation of affects SpnD39III (ST5556II) allele dominance and results in the hyperencapsulated phenotype. The capsule affects multiple interactions with the host including contributing to colonization and immune evasion. During infection, the capsule thickness varies, but the mechanisms regulating this are poorly understood. We have identified an unsuspected relationship between mutation of , a gene that encodes a zinc uptake lipoprotein, and capsule thickness. Mutation of resulted in a striking hyperencapsulated phenotype, increased resistance to complement-mediated neutrophil killing, and increased virulence in mouse models of infection. Transcriptome and PCR analysis linked the hyperencapsulated phenotype of the strain to specific alleles of the SpnD39III (ST5556II) type I restriction-modification system, a system which has previously been shown to aff