PMC

Pairwise alignment of the capsule locus of S. pseudopneumoniae strain BHN880 with S. pneumoniae Ambrose (serotype 5) and S. mitis 21/39. Colors and annotations are based on Bentley et al. (). Gray shading indicates the degree of pairwise nucleotide identity. Locus tags of dexB and aliA homologs in BHN880 are E3V35_07635 and E3V35_07740, respectively.

Distribution of known and putative new colonization and virulence factors in the 44 S. pseudopneumoniae genomes. (A) Known pneumococcal surface-exposed proteins, two-component systems (histidine kinase/response regulator pairs), and stand-alone regulators. Asterisks indicate core S. pneumoniae proteins according to Gamez et al. (). (B) Distribution of novel choline-binding proteins, two-component systems, and zinc-metalloproteases. CBP, choline-binding protein; NCSEP, nonclassical surface-exposed protein; TCS, two-component system; HK, histidine kinase; RR, response regulator; Reg., stand-alone regulator; ZMPs, zinc metalloproteases.

Distribution of IPD and ECC genes. (A) Pie charts representing the percentage of core, accessory, and absent genes from S. pneumoniae (n = 39) and S. pseudopneumoniae (n = 44) genomes. Due to the use of draft S. pseudopneumoniae genomes compared with complete S. pneumoniae genomes, genes were considered core in S. pseudopneumoniae when they were found in ≥95% of the genomes. (B) Distribution of the 20 genes absent from S. pseudopneumoniae in the functional categories defined by Orihuela et al. ().

Hemolytic activity of S. pseudopneumoniae. The ability of S. pseudopneumoniae strains BHN880, BHN912, and BHN914 to lyse human blood was tested using a hemolysis assay. S. pneumoniae TIGR4 and TIGR4Δply strains were used as controls. Results are expressed as the percentage of lysis compared to the positive control (100%; indicated by the dashed line) upon incubation with decreasing numbers of CFU/ml. Each data point represents the average from three independent experiments, and standard deviations are indicated.

Phylogenetic and pangenome analysis of S. pseudopneumoniae. (A) Unrooted consensus parsimony phylogenetic tree based on all SNPs (1,230,968) of 147 genomes: LRTI isolates (n = 24) and publicly available S. pseudopneumoniae (n = 38), S. pneumoniae (n = 39), nontypeable S. pneumoniae (n = 8), S. mitis (n = 36), S. oralis (n = 1), and S. infantis (n = 1). Circles indicate LRTI isolates (black), NCBI genomes labeled as S. pseudopneumoniae (open), or nontypeable S. pneumoniae (gray). Background shading delineates clades of different species. The tree was built in kSNP and visualized in MEGA7 (). (B) Pangenome of S. pseudopneumoniae and S. pneumoniae showing the distribution of shared and unique COGs.

Domain prediction of choline-binding proteins and new zinc metalloproteases of S. pseudopneumoniae based on SMART (). (A) Choline-binding proteins. The average percent identity of each CBP in S. pseudopneumoniae and the number of proteins analyzed are indicated. Percent identity with S. pneumoniae (Spn) was calculated using the proteins from IS7493 and S. pneumoniae TIGR4, except in the following cases: NanA (R6) and PspC (allele PspC11.3; AF276622.1). Representations of domains found in each CBP are based on the variant found in IS7493. In the absence of the protein in IS7493, the analysis was based on BHN914 (PspC, Cbp15, Cbp16, Cbp17, Cbp18, and Cbp19), BHN879 (Cbp1), and BHN886 (Cbp19). Nb, number. (B) Zinc metalloproteases ZmpE and ZmpF from S. pseudopneumoniae. ZmpA from S. pneumoniae (Spn ZmpA) is included for comparison. Asterisks indicate the ZMP motif HEMTH….E (). Domain prediction is based on ZmpE from IS7493 and ZmpF from BHN914. Locus tags can be found in .

Phylogenetic distribution of accessory features and allelic variants. (A) Core genome species tree-based SNPs from the 793 single-copy core genes of 44 S. pseudopneumoniae genomes. Circles indicate isolates from LRTI (black), NCBI genomes labeled as S. pseudopneumoniae (open), or nontypeable S. pneumoniae (gray). The tree was built in panX () and visualized in MEGA7 (). Clades are delineated by the background shading. (B) Distribution of accessory features and allelic variants of surface-exposed proteins, regulatory genes, peptide pheromones, genotypic and phenotypic antibiotic resistance, and plasmids. Description of the colors is indicated in the key. Supporting information on allelic variants can be found in to . Roman numerals in the ICE column refer to integration sites (). ICE, Mega-2, and other types of resistance refer to genotypic resistance; penicillin (Pen) and co-trimoxazole (SXT) refer to phenotypic resistance ( and references , , and ). NP, nasopharynx; ND, pseudogenes/truncated; NA, data not available.