Isolation and characterization of a native avirulent strain of Streptococcus suis serotype 2: a perspective for vaccine development

Abstract

Streptococcus suis, an emerging infectious pathogen, is the cause of two large-scale outbreaks of human streptococcal toxic shock syndrome in China, and has attracted much attention from the scientific community. The genetic basis of its pathogenesis remains enigmatic, and no effective prevention measures have been established. To better understand the virulence differentiation of S. suis and develop a promising vaccine, we isolated and sequenced a native avirulent S. suis strain (05HAS68). Animal experiments revealed that 05HAS68 is an avirulent strain and could protect piglets from the attack of virulent strains. Comparative genomics analyses demonstrated the genetic basis for the lack of virulence in 05HAS68, which is characterized by the absence of some important virulence-associated factors and the intact 89K pathogenicity island. Lack of virulence was also illustrated by reduced survival of 05HAS68 compared to a virulent strain in pig whole blood. Further investigations revealed a large-scale genomic rearrangement in 05HAS68, which was proposed to be mediated by transposase genes and/or prophages. This genomic rearrangement may have caused the genomic diversity of S. suis, and resulted in biological discrepancies between 05HAS68 and highly virulent S. suis strains.

Figure 1. SPF-piglet infection experiments.

Each piglet was intravenously injected with a different bacterial suspension avirulent strain (05HAS68), highly virulent strain (05ZYH33 or 98HAH12), or attenuated S. suis strain (ΔsalKR)) at a dose of 10⁸ CFU/piglet. Piglets were given the same dose as a booster at day 14, and then challenged with the highly virulent strains (05ZYH33 or 98HAH12) at day 28 where indicated. Each datum point represents one piglet. Survival time (d) of individual piglet was monitored. (A). Piglets were injected with 05HAS68. (B). Piglets were injected with 05ZYH33. (C). Piglets were injected with 98HAH12. (D and E) Piglets were injected with 05HAS68, and given the same dose for booster at day 14, then challenged with 05ZYH33 (in Panel D) or 98HAH12 (in Panel E). (F). Piglets were injected with ΔsalKR and given the same dose for booster at day 14, then challenged with 05ZYH33.

Figure 2. Schematic circular diagrams of the 05HAS68, 05ZYH33, and 98HAH12 genomes.

(A). Circular map of the 05HAS68 genome completed in the present study. Keys for circular diagrams (from outside to inside): annotated genes are presented as a pair of concentric circles distinguished by different colors in the COG classification (positive strand on the outside, negative strand on the inside); GC content of the 05HAS68 genome; GC skew plot (green for GC skew⁺; purple for GC skew⁻); and scale of the whole genome (bp). Circular maps of the 05ZYH33 (in Panel B) and 98HAH12 genomes (in Panel C) completed previously and used for comparative genomics analyses in this study. (D). Transformed Venn diagram. The genes of 05HAS68, 05ZYH33, and 98HAH12 were compared by BLASTp. Each strain is represented by one color, and the gene numbers are displayed in the same color. Numbers in the intersectional region indicate genes shared by two or three strains. For example, 1751, 1852, and 1829 represents the gene numbers of 05HAS68, 05ZYH33, and 98HAH12 that are shared by these three strains. Moreover, 253, 26, and 32 is the number of the unique genes in 05HAS68, 05ZYH33, and 98HAH12, respectively. Notably, the numbers in the intersections are slightly different due to the fact that there are multiple duplicate gene copies in the bacterial genome.

Figure 3. Mauve comparison diagrams of the 05HAS68, 05ZYH33, and 98HAH12 genomes.

Each colored region is a locally collinear block (LCB). The LCBs below the genome's center line are in reverse complement orientation compared with the 05HAS68 genome. T1 to T8 indicate the positions of transposase-encoded genes. P1 to P4 represent the positions of prophage-related gene clusters.

Figure 4. Whole blood killing and bacterial adherence assays.

(A). Whole blood killing assay. Data are expressed as survival index: (CFU bacteria at end of assay)/(initial bacteria CFU) with the mean ± SD of three independent experiments. (B). Comparison of bacterial adherence capability between 05HAS68 and 05ZYH33. The normalized mean fluorescence intensities (NMFI) of the cells are shown as columns with the mean ± SD of intracellular bacteria/ml. ***P < 0.001.

Figure 5. Phylogenetic tree of S. suis genomes.

Phylogenetic tree showing the relationship of 05HAS68 and other S. suis strains based on alignment of the ITS gene sequences. CA indicates the assumed common ancestor. The bar indicates the relative evolutionary distance.