Roles of the spiA gene from Salmonella enteritidis in biofilm formation and virulence

Abstract

Salmonella enteritidis has emerged as one of the most important food-borne pathogens for humans, and the formation of biofilms by this species may improve its resistance to disadvantageous conditions. The spiA gene of Salmonella typhimurium is essential for its virulence in host cells. However, the roles of the spiA gene in biofilm formation and virulence of S. enteritidis remain unclear. In this study we constructed a spiA gene mutant with a suicide plasmid. Phenotypic and biological analysis revealed that the mutant was similar to the wild-type strain in growth rate, morphology, and adherence to and invasion of epithelial cells. However, the mutant showed reduced biofilm formation in a quantitative microtitre assay and by scanning electron microscopy, and significantly decreased curli production and intracellular proliferation of macrophages during the biofilm phase. In addition, the spiA mutant was attenuated in a mouse model in both the exponential growth and biofilm phases. These data indicate that the spiA gene is involved in both biofilm formation and virulence of S. enteritidis.

Fig. 1.

Growth curves and protein patterns of the wild-type strain, C50041, and the spiA mutant, ΔspiA. (a) Cultures of C50041 (⧫) and ΔspiA (▪) were monitored spectrophotometrically at 600 nm hourly for 7 h. (b) Proteins (40 µg samples) prepared from the supernatants of C50041 (lane 1) and ΔspiA (lane 2) cultures were subjected to SDS-PAGE. Prestained protein markers are indicated on the left (lane M).

Fig. 2.

Determination of biofilm formation and its components in the wild-type strain (C50041), the spiA mutant (ΔspiA) and the spiA-complemented strain (ΔspiAR). (a) A₅₇₀ values of crystal-violet-stained C50041, ΔspiA and ΔspiAR biofilms grown in 96-well plates. (b) Overnight broth cultures were diluted 1 : 10 in cell-culture flasks. After incubation at 28 °C for 48 h, the broth was removed and the biofilm cells were stained with crystal violet. (c) Scanning electron micrographs of cells of the wild-type strain C50041 and ΔspiA grown on polystyrene coverslips at 28 °C for 48 h. (d, e) Morphotypes of C50041 and ΔspiA grown on plates of LB agar lacking NaCl and supplemented with Congo red and Coomassie brilliant blue G (d) or with Calcofluor (e). (f) Biofilm cells of strains C50041, ΔspiA and ΔspiAR with the same optical densities (OD₆₀₀ 3.0) were collected and treated. Curli proteins were examined by SDS-PAGE with 15 % separating gel (top) and confirmed by Western blotting (bottom).

Fig. 3.

Distribution of bacteria in BALB/c mice challenged with exponential-phase cells (a) or biofilm-phase cells (b) of the wild-type strain C50041 and the spiA mutant ΔspiA. Six-week-old BALB/c mice were intraperitoneally injected with 0.2 ml of bacterial suspension. The numbers of bacteria present in the blood, liver, spleen and lungs of the mice were measured 6 and 48 h post-challenge. Significant differences in the bacterial counts of organs were determined by Student’s t-test. *, P<0.05.