Discovery of a Streptococcus pneumoniae serotype 33F capsular polysaccharide locus that lacks wcjE and contains a wcyO pseudogene

Abstract

As part of large on-going vaccine impact studies in Fiji and Mongolia, we identified 25/2750 (0.9%) of nasopharyngeal swabs by microarray that were positive for Streptococcus pneumoniae contained pneumococci with a divergent 33F capsular polysaccharide locus (designated '33F-1'). We investigated the 33F-1 capsular polysaccharide locus to better understand the genetic variation and its potential impact on serotyping results. Whole genome sequencing was conducted on ten 33F-1 pneumococcal isolates. Initially, sequence reads were used for molecular serotyping by PneumoCaT. Phenotypic typing of 33F-1 isolates was then performed using the Quellung reaction and latex agglutination. Genome assemblies were used in phylogenetic analyses of each gene in the capsular locus to investigate genetic divergence. All ten pneumococcal isolates with the 33F-1 cps locus typed as 33F by Quellung and latex agglutination. Unlike the reference 33F capsule locus sequence, DNA microarray and PneumoCaT analyses found that 33F-1 pneumococci lack the wcjE gene, and instead contain wcyO with a frameshift mutation. Phylogenetic analyses found the wzg, wzh, wzd, wze, wchA, wciG and glf genes in the 33F-1 cps locus had higher DNA sequence similarity to homologues from other serotypes than to the 33F reference sequence. We have discovered a novel genetic variant of serotype 33F, which lacks wcjE and contains a wcyO pseudogene. This finding adds to the understanding of molecular epidemiology of pneumococcal serotype diversity, which is poorly understood in low and middle-income countries.

Fig 1. Comparison of the wcyO open reading frames of 33F-1 sequences to a representative serotype 34 sequence.

Only a selected portion of the DNA sequence is shown. Numbers refer to the position number in the serotype 34 sequence with an in-frame wcyO gene.

Fig 2. Maximum likelihood phylogenetic trees of 33F-1 cps genes (wzg-wciD) with homologues from all other serotypes.

As all genes except wchA were identical in all 33F-1 isolates only one sequence is included as a representative. Un-collapsed trees are provided in S5 Fig. Tree for wciC is not shown as this gene is only present in serotypes 33F, 33A and 37, which all have over 98% DNA sequence identity to the 33F-1 sequence. DNA sequences were aligned using MUSCLE and trees were constructed using the Tamura-Nei model in MEGA 7. Only bootstrap values above 50% are shown.

Fig 3. Maximum likelihood phylogenetic trees of 33F-1 cps genes (wciE-wcyO) with homologues from all other serotypes.

As all genes except wcyO were identical in all 33F-1 isolates only one sequence is included as a representative. Un-collapsed trees are provided in S5 Fig. DNA sequences were aligned using MUSCLE and trees were constructed using the Tamura-Nei model in MEGA 7. Only bootstrap values above 50% are shown.