Unique genomic arrangements in an invasive serotype M23 strain of Streptococcus pyogenes identify genes that induce hypervirulence

Abstract

The first genome sequence of a group A Streptococcus pyogenes serotype M23 (emm23) strain (M23ND), isolated from an invasive human infection, has been completed. The genome of this opacity factor-negative (SOF(-)) strain is composed of a circular chromosome of 1,846,477 bp. Gene profiling showed that this strain contained six phage-encoded and 24 chromosomally inherited well-known virulence factors, as well as 11 pseudogenes. The bacterium has acquired four large prophage elements, ΦM23ND.1 to ΦM23ND.4, harboring genes encoding streptococcal superantigen (ssa), streptococcal pyrogenic exotoxins (speC, speH, and speI), and DNases (spd1 and spd3), with phage integrase genes being present at one flank of each phage insertion, suggesting that the phages were integrated by horizontal gene transfer. Comparative analyses revealed unique large-scale genomic rearrangements that result in genomic rearrangements that differ from those of previously sequenced GAS strains. These rearrangements resulted in an imbalanced genomic architecture and translocations of chromosomal virulence genes. The covS sensor in M23ND was identified as a pseudogene, resulting in the attenuation of speB function and increased expression of the genes for the chromosomal virulence factors multiple-gene activator (mga), M protein (emm23), C5a peptidase (scpA), fibronectin-binding proteins (sfbI and fbp54), streptolysin O (slo), hyaluronic acid capsule (hasA), streptokinase (ska), and DNases (spd and spd3), which were verified by PCR. These genes are responsible for facilitating host epithelial cell binding and and/or immune evasion, thus further contributing to the virulence of M23ND. In conclusion, strain M23ND has become highly pathogenic as the result of a combination of multiple genetic factors, particularly gene composition and mutations, prophage integrations, unique genomic rearrangements, and regulated expression of critical virulence factors.

FIG 1

Circular representation of the 1,846,477-bp genome of S. pyogenes strain M23ND. Data are shown from the outermost to the innermost circles. Circles 1 and 2 display annotated coding sequences for the reverse (pink) and forward (blue) DNA strands, respectively. Circle 3 shows the locations of four phage elements, ΦM23ND.1 to ΦM23ND.4 (red boxes), and short mobile elements (black lines). Circle 4 illustrates the virulence genes identified in M23ND (orange). Circles 5 and 6 represent the locations of tRNA (turquoise) and rRNA (brown) genes, respectively. The genome contains 57 tRNA genes and 5 rRNA operons. Circles 6 and 7 display the GC content and GC skew ([G − C]/[G + C]). The triangles on the outermost circumference indicate the positions of the replication origin (ori; green triangle) at bp 0 and the replication terminus (ter; red triangle) at bp 702443. The genome contains 1,851 ORFs, 231 of which are present in phage regions. Approximately 1,389 genes have assigned functions.

FIG 2

Global comparisons of fully sequenced S. pyogenes genomes. The sequences of the 20 previously sequenced GAS genomes were obtained from the NCBI database and were correlated with a basic local alignment with S. pyogenes strain M23ND (innermost circle). The areas of similarity and divergence within the sequences are contrasted, with areas with white gaps indicating the regions of the highest variance. The profiles of M1 strain 5005 and M1 strain A20 are similar to the profile of M1 strain 476 and are therefore not included. Phage proteins and short mobile genetic elements are indicated by black and red arrows, respectively, in the outer circle. The strains whose sequences were compared to the sequence of M23ND, which is positioned on the central ring, are M1 strain SF370, M1 strain 476, M2 strain 10270, M3 strain 315, M3 strain SSI-1, M4 strain 10750, M5 strain Manfredo, M6 strain 10394, M12 strain 9429, M12 strain 2096, M12 strain HKU16, M14 strain HSC5, M18 strain 8232, M28 strain 6180, M49 strain NZ131, M53 strain Alab49, M59 strain 1882, and M59 strain 15252. Numbers in the innermost circle are positions. M, million bases.

FIG 3

DNA characterizations of genomic sequences of S. pyogenes. (A) A circular visualization of the locations of phage elements across M23ND (red) in comparison with the locations in the 20 previously fully sequenced GAS genomes available through NCBI (blue). The integration sites of phages are generally clustered at several regions, but the locations of orthologous phages are nonconserved. For example, prophage ΦM23ND.1 is closely similar to ΦManfredo.4 (M5), ΦMGAS10394.3 (M6), ΦMGAS1882.2 (M18), and several other prophages, but these prophages are inserted at distinct sites, suggesting that phage recombination via horizontal gene transfer plays an important role in genetic diversity. Black triangles on the circumference, positions of the replication origin (ori) at bp 0 and terminus (ter) at bp 702433. Inversions around ori (white triangle) are clearly observed, especially for srv, and inversions around ter (white triangle) are readily seen in the cases of sen, sagA, and fbp (fbp54). ICE, integrating conjugative elements. (B) A circular visualization of the comparative locations of the virulence factors of interest within the genomes of the 21 fully sequenced GAS genomes. Elongated bars, regions in which each gene can be found across all of the 21 fully sequenced and assembled GAS genomes, unless the gene appears elsewhere, e.g., covRS for SSI-1, Manfredo, HKU16, and M23ND or sen, srv, and sagA for M23ND.

FIG 4

Comparisons of the whole genome of S. pyogenes M23ND with the whole genomes of two phylogenetic neighbors, M5 strain Manfredo and M18 strain 8232. Red and blue lines, forward and reverse alignments, respectively; colored boxes, prophage elements; arrows, replication terminus in each genome. Large segmental inversions and translocations are observed in M23ND relative to the genomic architectures of its neighbors. These rearrangements result in an imbalanced global genomic architecture, where the replication terminus (ter) and three prophage insertions were located within the same replichore of the chromosome. M23ND also contains an additional short inversion in the final 100 kb of the sequence. Prophage elements are located on the breakpoints of the rearrangements or within the rearrangements themselves.

FIG 5

Profiling of phage-carried virulence factors across S. pyogenes genomes. Specific genes are represented by colored triangles, and the length of each gene is scaled by the triangle size. M23ND contains six known phage-carried virulence factors incorporated into four prophage regions, namely, speC, spd1, ssa, spd3, speI, and speH. Profiling of these six virulence factors, together with others across the 21 GAS sequences, showed that they are randomly distributed throughout the chromosome.

FIG 6

Profiling of chromosomally inherited virulence factors across S. pyogenes genomes. Genes are represented by colored triangles, and the length of each gene is scaled by the triangle width. These genes are present in almost all of the 21 fully sequenced GAS genomes, except for fibronectin-binding protein (sfbI), streptococcal inhibitor of complement (sic), and pyrogenic exotoxin J (speJ), which are carried by 10, 4, and 7 GAS strains, respectively. The overall genomic locations of chromosomal virulence factors are conserved across different GAS strains, with the exception of M23ND, M5 strain Manfredo, M12 strain HKU16, and M3 strain SSI-1, where the gene locations are obscured by large-scale genomic arrangements, including translocations and inversions. A total of 26 known virulence factors present in M23ND include endoS, hasA, hylA, ideS, prtS, scpA, ska, smeZ, spd, speB, speG, speJ, and spyA (A) and cfa, dltA, dltC, eno, graB, lmb, nga, plr, sagA, sclA, sfbI, sic, and slo (B).

FIG 7

Profiling of six regulatory genes across S. pyogenes genomes. Genes are represented by colored triangles, and the length of each gene is scaled by the triangle width. All 21 currently available genome sequences were analyzed. The genes examined included emm, mga, covR, covS, rgg, and srv. The results for the M-protein gene, emm, have been artificially shifted in order to avoid overlap with those for mga. The locations of regulatory genes are highly conserved across all GAS strains, except for srv in M23ND. This gene is displaced from a position of approximately Mbp 1.5 to approximately Mbp 1.2, induced by the translocation of a large fragment within the genome. Similar translocations are evident for other genes in M23ND, e.g., sagA and sfbI.

FIG 8

Gene expression in full-length and truncated CovS M23ND strains. mRNAs isolated from the two strains (CovS⁺ [S⁺] and CovS⁻ [S⁻] strains) during LP (L; A₆₀₀ = 0.6) and SP (S; A₆₀₀ > 1.0) were analyzed for virulence factor gene expression using qRT-PCR. Primers specific for each gene (see Table S1 in the supplemental material) were used to measure the levels of transcription relative to the level of transcription of the GAPDH housekeeping gene. (A) Relative expression of the gene for extracellular cysteine protease, speB. (B) Gene expression in the multigene regulon (Mga) of GAS, which, in the case of M23ND, contains a minimal gene content, viz., the genes for M protein (emm23) and C5a peptidase (scpA). (C) qRT-PCR analyses of cell surface and secreted virulence factors involved in fibronectin binding (sfbI and fbp54), hyaluronic acid capsule biosynthesis (hasA), DNase activity (spd and spd3), and host cell lysis (slo). (D) qRT-PCR analysis of host-plasminogen activator sk2a, showing very similar expression in both strains and throughout both LP and SP growth.