Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes

Abstract

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Figure 1. Representation of the Wzx/Wzy-Dependent Pathway for Biosynthesis of CPS 9A

Pictured is a hypothetical model for capsule biosynthesis in S. pneumoniae based on a mixture of experimental evidence and speculation. For a recent review, see Yother [15]. (1) Non-housekeeping nucleotide sugar biosynthesis. (2) The initial transferase (WchA in this case) links the initial sugar as a sugar phosphate (Glc-P) to a membrane-associated lipid carrier (widely assumed to be undecaprenyl phosphate). (3) Glycosyl transferases sequentially link further sugars to generate repeat unit. (4) Wzx flippase transports the repeat unit across the cytoplasmic membrane. (5) Wzy polymerase links individual repeat units to form lipid-linked CPS. (6) Wzd/Wze complex translocates mature CPS to the cell surface and may be responsible for the attachment to peptidoglycan. The complex of WchA, Wzy, Wzx, Wzd, and Wze shown in the membrane is based on that in Figure 2 of Whitfield and Paiment [47] for the related Escherichia coli Type 1 capsule.

Figure 2. Capsule Biosynthesis Genes and Repeat-Unit Polysaccharide Structures

Shown are the cps gene clusters for cases discussed in the text, together with the polysaccharide structure of the encoded repeat unit where known [31] (the full set is shown in Figure S1). Genes are represented on the forward and reverse strands by boxes coloured according to the gene key, with gene designations indicated above each box. Grey blocks indicate regions of sequence similarity between gene clusters. Repeat-unit structures are displayed with the linkage to undecaprenyl pyrophosphate at the right-hand side (not necessarily the case for the published structures [31]), so residue numbers are counted from right to left. Monosaccharides are represented as shapes coloured according to the structure key. Housekeeping sugars are coloured grey. Non-housekeeping sugar colours correspond to the associated sugar biosynthesis gene colours. Glycerol, choline, and acetate are indicated as text. Also shown are the nature of linkages with the associated gene, and the linkages between repeat units created by the Wzy polymerase. Gene designations are in parentheses where their substrate specificity is unclear.