Phylogenetic analysis and accessory genome diversity reveal insight into the evolutionary history of Streptococcus dysgalactiae

Abstract

Streptococcus dysgalactiae (SD) is capable of infecting both humans and animals and causing a wide range of invasive and non-invasive infections. With two subspecies, the taxonomic status of subspecies of SD remains controversial. Subspecies equisimilis (SDSE) is an important human pathogen, while subspecies dysgalactiae (SDSD) has been considered a strictly animal pathogen; however, occasional human infections by this subspecies have been reported in the last few years. Moreover, the differences between the adaptation of SDSD within humans and other animals are still unknown. In this work, we provide a phylogenomic analysis based on the single-copy core genome of 106 isolates from both the subspecies and different infected hosts (animal and human hosts). The accessory genome of this species was also analyzed for screening of genes that could be specifically involved with adaptation to different hosts. Additionally, we searched putatively adaptive traits among prophage regions to infer the importance of transduction in the adaptation of SD to different hosts. Core genome phylogenetic relationships segregate all human SDSE in a single cluster separated from animal SD isolates. The subgroup of bovine SDSD evolved from this later clade and harbors a specialized accessory genome characterized by the presence of specific virulence determinants (e.g., cspZ) and carbohydrate metabolic functions (e.g., fructose operon). Together, our results indicate a host-specific SD and the existence of an SDSD group that causes human-animal cluster infections may be due to opportunistic infections, and that the exact incidence of SDSD human infections may be underestimated due to failures in identification based on the hemolytic patterns. However, more detailed research into the isolation of human SD is needed to assess whether it is a carrier phenomenon or whether the species can be permanently integrated into the human microbiome, making it ready to cause opportunistic infections.

Keywords: SDSD and SDSE; Streptococcus dysgalactiae; accessory genome; core-genome; prophages regions.

FIGURE 1

Distribution of the core and accessory genes in different functional categories investigated in S. dysgalactiae genomes.

FIGURE 2

Phylogenetic analysis of the S. dysgalactiae single-copy core genome. (A) The tree was rooted on the largest branch as the closest outgroup species. (B) Phylogenetic unrooted tree relationships. The evolutionary history was inferred using the maximum likelihood analysis of the alignment of 1,134 single-copy core protein-coding sequences shared by 106 S. dysgalactiae strains. Bootstrap support values were calculated from 1,000 replicates. A phylogenetic tree was generated using IQ-TREE v2.0.3.

FIGURE 3

The heatmap chart generated from distances calculated based on the ANI values of S. dysgalactiae core genome. The colors in the heatmap represent pairwise ANI values, with a gradient from dark green (low identity) to light green (high identity). The dendrogram directly reflects the degree of identity between genomes increasing in similarity from dark to green. Heatmap and dendrogram of ANI values were performed using the CLC Genomic Workbench.

FIGURE 4

Metagenomics analysis of the accessory genome of S. dysgalactiae. CCMetagen graph shows the percentage of accessory genes shared with other bacteria. The alignment of accessory genes against the GenBank database using BLAST was performed to trace homolog genes using the CCMetagen analysis tool. The figure and legend were automatically generated based on the nomenclature deposited in the NCBI.

FIGURE 5

Virulence and carbohydrate metabolism profiles of the 106 S. dysgalactiae isolates. (A) Phylogenetic analysis of the S. dysgalactiae single-copy core genome as in Figure 2. (B) Graphic representation of the presence (gray) and absence (white) of the identified relevant gene clusters in the S. dysgalactiae genomes in this study. sagA to sagI – Streptolysin (SLS) operon; vpr gene – C5a peptidase; M protein-like (SDSD); cpsZ gene – Emm-like cell surface protein; sdrE2 – adhesin; HrtAB – Heme efflux system; lacA to G, lacX and lacR genes – lactose metabolization operon; fruA to D – fructose metabolism operon; Sorbitol operon – Sorbitol/glucitol metabolism operon. The complete set of data is shown in Supplementary Table S9.

FIGURE 6

(A–C) Graphical representation of the organization of the region flanking the gene that codes for the C5a peptidase. The fhs1 and cls genes encoding formate–tetrahydrofolate ligase and cardiolipin synthetase, respectively, shared an identity greater than 95% among S. dysgalactiae strains. The gene identified as vpr (C5a peptidase precursor ScpZ) in strain SDSENCTC4669 (CDS: VDZ40442.1) is shared among SDSD animals SDSE strains, while scp gene (C5a peptidase) of the SDSENCTC9413 strain (CDS: VTT04007.1) is shared among human SDSE strains. (D) BLASTp alignment of predicted proteins VPR and SCP suggests that scp is a fragment of the vpr gene and reveals that the region coding for the C5a peptidase is absent from the scp gene.