Characterization of a Type VI Secretion System vgrG2 Gene in the Pathogenicity of Burkholderia thailandensis BPM

Abstract

Burkholderia thailandensis is a clinically underestimated conditional pathogen in the genus Burkholderia, the pathogenicity of the infection caused by B. thailandensis remains poorly understood. According to previous studies, Type-VI secretion system (T6SS) is a protein secreting device widely existing in Gram-negative bacilli. Valine-glycine repeat protein G (VgrG) is not only an important component of T6SS, but also a virulence factor of many Gram-negative bacilli. In one of our previous studies, a unique T6SS vgrG gene (vgrG2 gene) was present in a virulent B. thailandensis strain BPM (BPM), but not in the relatively avirulent B. thailandensis strain E264 (E264). Meanwhile, transcriptome analysis of BPM and E264 showed that the vgrG2 gene was strongly expressed in BPM, but not in E264. Therefore, we identified the function of the vgrG2 gene by constructing the mutant and complemented strains in this study. In vitro, the vgrG2 gene was observed to be involved in the interactions with host cells. The animal model experiment showed that the deletion of vgrG2 gene significantly led to the decrease in the lethality of BPM and impaired its ability to trigger host immune response. In conclusion, our study provides a new perspective for studying the pathogenicity of B. thailandensis and lays the foundation for discovering the potential T6SS effectors.

Keywords: BPM; T6SS; pathogenicity; vgrG2 gene; virulence factor.

FIGURE 1

Heatmap of vgrG gene expression in BPM and E264. Colors in the heatmap represent gene expression levels among samples. The BPM and E264 results came from three repeated samples.

FIGURE 2

Survival rate of BALB/c mice infected with BPM, mutant and complemented strains. The mortality of BALB/c mice after the intraperitoneal injection of all strains was observed over 7 days. Data points represent the percentage of BALB/c mouse survival in each group (n = 10 mice per strain and 1 × 10⁷CFU per mouse). After infection for 7 days, the survival rate of BALB/c mice infected with PBS and ΔvgrG was significantly lower than that of BALB/c mice infected with BPM *(P < 0.05).

FIGURE 3

Pathological characterization of lungs and liver tissues of BALB/c mice infected with BPM, mutant and complemented strains. Lungs and liver tissues of BALB/c mice infected with BPM, mutant, complemented strains and PBS (control) were prepared for light microscopy analysis and examined for differences in pathological changes (hematoxylin and eosin staining; original magnification × 200).

FIGURE 4

Serum levels of cytokines in BLAB/c mice 16 h after infection with BPM, mutant and complemented strains. Serum IL-1β, IL-6, and TNF-α levels in BALB/c mice 16 h after infection with the BPM, mutant and complement strains. All data are from three independent experiments. Significant differences between groups are indicated: *(P < 0.05) and ***(P < 0.001).

FIGURE 5

Whole-blood bactericidal experiments of BPM, mutant and complemented strains. Survival rates of the BPM, mutant and complemented strains in human whole blood. The survival rates are expressed relative to those of BPM (100%). Means and SDs of three independent experiments performed in triplicate were calculated. Significant differences between groups are indicated: **(P < 0.01).

FIGURE 6

Interaction between bacteria and RAW264.7 cells. (A–C) indicate the invasion ability, intracellular survival ability, and the cytotoxicity, respectively, of the BPM, mutant and complemented strains in RAW264.7 cells. Rates of invasion, survival and cytotoxicity are expressed relative to those of BPM (100%). The means and SDs of three independent experiments in conducted triplicate were calculated. Significant differences between groups are indicated: *(P < 0.05) and **(P < 0.01).