Mechanisms Underlying the Effects of Secretory Protein G22 on Biological Characteristics and Virulence of Streptococcus suis

Abstract

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that seriously harms the swine industry and human health. However, its pathogenic mechanisms are largely unknown, and the few virulence factors reported so far are insufficient to systematically explain its infectious and pathogenic mechanisms. In preliminary research, we identified a gene named G22 encoding a hypothetical secreted protein that may be closely associated with the high-level pathogenicity of S. suis. In this study, we constructed deletion and complementation strains of the G22 gene through homologous recombination and explored its roles in the pathogenicity and susceptibility of S. suis to environmental stresses through in vitro and in vivo experiments. The deletion of G22 clearly influenced the typical capsular structure of SS2 and impaired the bacterium's growth in a medium containing hydrogen peroxide (showing a growth reduction of 32.98% ± 5.23% compared to the wild-type strain SC19, p < 0.001) or with a low pH (with a growth inhibition of 17.44% ± 1.9% relative to the wild-type strain SC19, p < 0.01). ΔG22 also showed reduced survival in whole blood and in RAW 264.7 macrophages (with a survival reduction of 16.44% ± 2.29% compared to the wild-type, p < 0.001). The deletion of G22 also sharply attenuated the virulence of SS2 in a mouse infection model (reducing the mortality rate by 50% ± 0.04%, p < 0.05). We also demonstrated that G22 is required for the adhesion and invasion of SS2 in host cells. An RNA sequencing analysis revealed that 50 genes were differentially expressed in the ΔG22 and wild-type strains: 23 upregulated and 37 downregulated. Many of the genes are involved in carbohydrate metabolism and the synthesis of virulence-associated factors. Several genes associated with the phosphotransferase system were significantly upregulated in strain ΔG22. In summary, G22 plays a role in the morphological development and pathogenesis of the highly virulent SS2 strain SC19.

Keywords: G22; Streptococcus suis; bacterial virulence; gene knockout; transcriptome sequencing.

Figure 1

Identification of G22 gene knockout strain and complementary strain. (A) G22 knockout technology roadmap. G22L: Upstream fragment amplification of G22. G22R: Downstream fragment amplification of G22. W1/W2: Identification outside the homologous region. N1/N2: Identification of the internal fragment of G22. (B) Confirmation of the ∆G22 mutant (M, DNA Marker, Lanes 1, 3, and 5 were amplified with the external primers W1/W2. Lanes 2, 4, and 6 were amplified with the internal primers N1/N2).

Figure 2

(A) Growth characteristics of SC19, ∆G22, and C∆G22. Transmission electron micrographs of SC19 (B), ∆G22 (C), and C∆G22 (D).

Figure 3

Survival capacities of SC19, ∆G22, and C∆G22 under stress imposed by acid (A), H₂O₂ (B), or high temperatures (C). Data represent the mean ± SEM of at least three independent experiments. ‘ns’, ‘**’, ‘***’, and ‘****’ indicate significant difference values with ‘p > 0.05, p < 0.01, p < 0.001, and p < 0.0001’, respectively.

Figure 4

Involvement of G22 in adhesion and invasion of host cells by SS2. (A) Adherence rates of SC19, ∆G22, and C∆G22 in HEp-2 cells. (B) Adherence rates of SC19, ∆G22, and C∆G22 in Caco-2 cells. (C) Invasion rates of SC19, ∆G22, and C∆G22 in HEp-2 cells. (D) Invasion rates of SC19, ∆G22, and C∆G22 in Caco-2 cells. Data represent the mean ± SEM of at least three independent experiments. ‘ns’, ‘**’, ‘***’, and ‘****’ indicate significant difference values with ‘p > 0.05, p < 0.01, p < 0.001, and p < 0.0001’, respectively.

Figure 5

Effects of G22 gene deficiency on pathogenicity phenotype of SS2. (A) Phagocytic rates of SC19, ∆G22, and C∆G22 in Raw 264.7 cells. (B) Growth indices of SC19, ∆G22, and C∆G22 in pig blood. Data represent the mean ± SEM of at least three independent experiments. ‘ns’, ‘**’, and ‘***’ indicate significantly different values with p > 0.05, p < 0.01, and p < 0.001, respectively.

Figure 6

Survival curves of SC19 and ∆G22 in a mouse infection model. Survival was analyzed using the LogRank test. Twenty-four BALB/c mice were randomly divided into three groups, with eight mice in each.

Figure 7

Colonization of various tissues of mice by SC19 and ∆G22. (A) Bacterial burdens in the blood of BALB/c mice at 24 h postinfection. (B) Bacterial burdens in spleen tissues of BALB/c mice at 24 h postinfection. (C) Bacterial burdens in liver tissues of BALB/c mice at 24 h postinfection. (D) Bacterial burdens in lung tissues of BALB/c mice at 24 h postinfection. (E) Bacterial burdens in brain tissues of BALB/c mice at 24 h postinfection. Data represent the mean ± SEM of at least three independent experiments. ‘*’, ‘**’, and ‘***’ indicate significantly different values with p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 8

Transcriptomic profiles of strains ∆G22 and SC19. (A) Volcano plot of differentially expressed genes (upregulated genes are shown in red, and downregulated genes are shown in green). (B) KEGG metabolic pathways of differentially expressed genes.